112 results on '"Anjan Thakurta"'
Search Results
2. Whole-genome analysis identifies novel drivers and high-risk double-hit events in relapsed/refractory myeloma
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Naser Ansari-Pour, Mehmet Samur, Erin Flynt, Sarah Gooding, Fadi Towfic, Nicholas Stong, Maria Ortiz Estevez, Konstantinos Mavrommatis, Brian Walker, Gareth Morgan, Nikhil Munshi, Herve Avet-Loiseau, and Anjan Thakurta
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Large-scale analyses of genomic data from patients with newly diagnosed multiple myeloma (ndMM) have been undertaken, however, large-scale analysis of relapsed/refractory MM (rrMM) has not been performed. We hypothesize that somatic variants chronicle the therapeutic exposures and clonal structure of myeloma from ndMM to rrMM stages. We generated whole-genome sequencing (WGS) data from 418 tumors (386 patients) derived from 6 rrMM clinical trials and compared them with WGS from 198 unrelated patients with ndMM in a population-based case-control fashion. We identified significantly enriched events at the rrMM stage, including drivers (DUOX2, EZH2, TP53), biallelic inactivation (TP53), noncoding mutations in bona fide drivers (TP53BP1, BLM), copy number aberrations (CNAs; 1qGain, 17pLOH), and double-hit events (Amp1q-ISS3, 1qGain-17p loss-of-heterozygosity). Mutational signature analysis identified a subclonal defective mismatch repair signature enriched in rrMM and highly active in high mutation burden tumors, a likely feature of therapy-associated expanding subclones. Further analysis focused on the association of genomic aberrations enriched at different stages of resistance to immunomodulatory agent (IMiD)–based therapy. This analysis revealed that TP53, DUOX2, 1qGain, and 17p loss-of-heterozygosity increased in prevalence from ndMM to lenalidomide resistant (LENR) to pomalidomide resistant (POMR) stages, whereas enrichment of MAML3 along with immunoglobulin lambda (IGL) and MYC translocations distinguished POM from the LEN subgroup. Genomic drivers associated with rrMM are those that confer clonal selective advantage under therapeutic pressure. Their role in therapy evasion should be further evaluated in longitudinal patient samples, to confirm these associations with the evolution of clinical resistance and to identify molecular subsets of rrMM for the development of targeted therapies.
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- 2023
3. Loss of COP9 signalosome genes at 2q37 is associated with IMiD resistance in multiple myeloma
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Sarah Gooding, Naser Ansari-Pour, Mohammad Kazeroun, Kubra Karagoz, Ann Polonskaia, Mirian Salazar, Evie Fitzsimons, Korsuk Sirinukunwattana, Selina Chavda, Maria Ortiz Estevez, Fadi Towfic, Erin Flynt, William Pierceall, Daniel Royston, Kwee Yong, Karthik Ramasamy, Paresh Vyas, and Anjan Thakurta
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Ubiquitin-Protein Ligases ,Immunology ,Humans ,Cell Biology ,Hematology ,RNA, Small Interfering ,Multiple Myeloma ,Lenalidomide ,Biochemistry ,Adaptor Proteins, Signal Transducing ,Peptide Hydrolases - Abstract
The acquisition of a multidrug refractory state is a major cause of mortality in myeloma. Myeloma drugs that target the cereblon (CRBN) protein include widely used immunomodulatory drugs (IMiDs), and newer CRBN E3 ligase modulator drugs (CELMoDs), in clinical trials. CRBN genetic disruption causes resistance and poor outcomes with IMiDs. Here, we investigate alternative genomic associations of IMiD resistance, using large whole-genome sequencing patient datasets (n = 522 cases) at newly diagnosed, lenalidomide (LEN)-refractory and lenalidomide-then-pomalidomide (LEN-then-POM)-refractory timepoints. Selecting gene targets reproducibly identified by published CRISPR/shRNA IMiD resistance screens, we found little evidence of genetic disruption by mutation associated with IMiD resistance. However, we identified a chromosome region, 2q37, containing COP9 signalosome members COPS7B and COPS8, copy loss of which significantly enriches between newly diagnosed (incidence 5.5%), LEN-refractory (10.0%), and LEN-then-POM-refractory states (16.4%), and may adversely affect outcomes when clonal fraction is high. In a separate dataset (50 patients) with sequential samples taken throughout treatment, we identified acquisition of 2q37 loss in 16% cases with IMiD exposure, but none in cases without IMiD exposure. The COP9 signalosome is essential for maintenance of the CUL4-DDB1-CRBN E3 ubiquitin ligase. This region may represent a novel marker of IMiD resistance with clinical utility.
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- 2022
4. Prognostic impact of NPM1 and FLT3 mutations in patients with AML in first remission treated with oral azacitidine
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Hartmut Döhner, Andrew H. Wei, Gail J. Roboz, Pau Montesinos, Felicitas R. Thol, Farhad Ravandi, Hervé Dombret, Kimmo Porkka, Irwindeep Sandhu, Barry Skikne, Wendy L. See, Manuel Ugidos, Alberto Risueño, Esther T. Chan, Anjan Thakurta, C.L. Beach, and Daniel Lopes de Menezes
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Neoplasm, Residual ,Remission Induction ,Immunology ,Nuclear Proteins ,Cell Biology ,Hematology ,Protein-Tyrosine Kinases ,Prognosis ,Biochemistry ,Leukemia, Myeloid, Acute ,fms-Like Tyrosine Kinase 3 ,Recurrence ,Mutation ,Azacitidine ,Humans ,Nucleophosmin - Abstract
The randomized, placebo-controlled, phase 3 QUAZAR AML-001 trial (ClinicalTrials.gov identifier: NCT01757535) evaluated oral azacitidine (Oral-AZA) in patients with acute myeloid leukemia (AML) in first remission after intensive chemotherapy (IC) who were not candidates for hematopoietic stem cell transplantation. Eligible patients were randomized 1:1 to Oral-AZA 300 mg or placebo for 14 days per 28-day cycle. We evaluated relapse-free survival (RFS) and overall survival (OS) in patient subgroups defined by NPM1 and FLT3 mutational status at AML diagnosis and whether survival outcomes in these subgroups were influenced by presence of post-IC measurable residual disease (MRD). Gene mutations at diagnosis were collected from patient case report forms; MRD was determined centrally by multiparameter flow cytometry. Overall, 469 of 472 randomized patients (99.4%) had available mutational data; 137 patients (29.2%) had NPM1 mutations (NPM1mut), 66 patients (14.1%) had FLT3 mutations (FLT3mut; with internal tandem duplications [ITD], tyrosine kinase domain mutations [TKDmut], or both), and 30 patients (6.4%) had NPM1mut and FLT3-ITD at diagnosis. Among patients with NPM1mut, OS and RFS were improved with Oral-AZA by 37% (hazard ratio [HR], 0.63; 95% confidence interval [CI], 0.41-0.98) and 45% (HR, 0.55; 95% CI, 0.35-0.84), respectively, vs placebo. Median OS was improved numerically with Oral-AZA among patients with NPM1mut whether without MRD (48.6 months vs 31.4 months with placebo) or with MRD (46.1 months vs 10.0 months with placebo) post-IC. Among patients with FLT3mut, Oral-AZA improved OS and RFS by 37% (HR, 0.63; 95% CI, 0.35-1.12) and 49% (HR, 0.51; 95% CI, 0.27-0.95), respectively, vs placebo. Median OS with Oral-AZA vs placebo was 28.2 months vs 16.2 months, respectively, for patients with FLT3mut and without MRD and 24.0 months vs 8.0 months for patients with FLT3mut and MRD. In multivariate analyses, Oral-AZA significantly improved survival independent of NPM1 or FLT3 mutational status, cytogenetic risk, or post-IC MRD status.
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- 2022
5. Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma
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Keiji Kurata, Anna James-Bott, Mark A. Tye, Leona Yamamoto, Mehmet K. Samur, Yu-Tzu Tai, James Dunford, Catrine Johansson, Filiz Senbabaoglu, Martin Philpott, Charlotte Palmer, Karthik Ramasamy, Sarah Gooding, Mihaela Smilova, Giorgia Gaeta, Manman Guo, John C. Christianson, N. Connor Payne, Kritika Singh, Kubra Karagoz, Matthew E. Stokes, Maria Ortiz, Patrick Hagner, Anjan Thakurta, Adam Cribbs, Ralph Mazitschek, Teru Hideshima, Kenneth C. Anderson, and Udo Oppermann
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Oncology ,Hematology - Abstract
Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.
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- 2023
6. Oral azacitidine modulates the bone marrow microenvironment in patients with acute myeloid leukaemia in remission: A subanalysis from the <scp>QUAZAR AML</scp> ‐001 trial
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Daniel L. Menezes, Wendy L. See, Alberto Risueño, Kao Tai Tsai, Jae K. Lee, Johnny Ma, Rida Khan, Thomas Prebet, Barry Skikne, C. L. Beach, Anjan Thakurta, and Anita Gandhi
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Hematology - Published
- 2023
7. Correction: Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma
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Keiji Kurata, Anna James-Bott, Mark A. Tye, Leona Yamamoto, Mehmet K. Samur, Yu-Tzu Tai, James Dunford, Catrine Johansson, Filiz Senbabaoglu, Martin Philpott, Charlotte Palmer, Karthik Ramasamy, Sarah Gooding, Mihaela Smilova, Giorgia Gaeta, Manman Guo, John C. Christianson, N. Connor Payne, Kritika Singh, Kubra Karagoz, Matthew E. Stokes, Maria Ortiz, Patrick Hagner, Anjan Thakurta, Adam Cribbs, Ralph Mazitschek, Teru Hideshima, Kenneth C. Anderson, and Udo Oppermann
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Oncology ,Hematology - Published
- 2023
8. High-Dose Melphalan Treatment Significantly Increases Mutational Burden at Relapse in Multiple Myeloma
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Mehmet Kemal Samur, Marco Roncador, Anil Aktas Samur, Mariateresa Fulciniti, Abdul Hamid Bazarbachi, Raphael Szalat, Masood A. Shammas, Adam S. Sperling, Paul G. Richardson, Florence Magrangeas, Stephane Minvielle, Aurore Perrot, Jill Corre, Philippe Moreau, Anjan Thakurta, Giovanni Parmigiani, Kenneth C. Anderson, Hervé Avet-Loiseau, and Nikhil C. Munshi
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
High-dose melphalan (HDM) improves progression-free survival in multiple myeloma (MM), yet melphalan is a DNA-damaging alkylating agent; therefore, we assessed its mutational effect on surviving myeloma cells by analyzing paired MM samples collected at diagnosis and relapse in the IFM 2009 study. We performed deep whole-genome sequencing on samples from 68 patients, 43 of whom were treated with RVD (lenalidomide, bortezomib, and dexamethasone) and 25 with RVD + HDM. Although the number of mutations was similar at diagnosis in both groups (7137 vs 7230; P = .67), the HDM group had significantly more mutations at relapse (9242 vs 13 383, P = .005). No change in the frequency of copy number alterations or structural variants was observed. The newly acquired mutations were typically associated with DNA damage and double-stranded breaks and were predominantly on the transcribed strand. A machine learning model, using this unique pattern, predicted patients who would receive HDM with high sensitivity, specificity, and positive prediction value. Clonal evolution analysis showed that all patients treated with HDM had clonal selection, whereas a static progression was observed with RVD. A significantly higher percentage of mutations were subclonal in the HDM cohort. Intriguingly, patients treated with HDM who achieved complete remission (CR) had significantly more mutations at relapse yet had similar survival rates as those treated with RVD who achieved CR. This similarity could have been due to HDM relapse samples having significantly more neoantigens. Overall, our study identifies increased genomic changes associated with HDM and provides rationale to further understand clonal complexity.
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- 2022
9. Suppression of Myeloid Cell-Derived Proinflammatory Cytokines with Celmod Agents: Implications for CRS with T-Cell Engagers (TCEs)
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Danny V Jeyaraju, Maryam Alapa, Aimee O'Donohue, Ross La Motte-Mohs, Isaac W. Boss, Patrick Hagner, Michael Pourdehnad, Michael R. Burgess, William E. Pierceall, and Anjan Thakurta
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
10. Multiple cereblon genetic changes are associated with acquired resistance to lenalidomide or pomalidomide in multiple myeloma
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Dan Rozelle, Fadi Towfic, Erin Flynt, Maria Ortiz Estevez, Paula Dhiman, Naser Ansari-Pour, Marissa Hirst, Anjan Thakurta, Kao-Tai Tsai, Karthik Ramasamy, Paola Neri, Nizar J. Bahlis, Paresh Vyas, Philip P Chamberlain, and Sarah Gooding
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Ubiquitin-Protein Ligases ,Immunology ,Antineoplastic Agents ,Drug resistance ,Biochemistry ,Exon ,medicine ,Humans ,Lenalidomide ,Multiple myeloma ,Adaptor Proteins, Signal Transducing ,Lymphoid Neoplasia ,biology ,business.industry ,Point mutation ,Cereblon ,Genetic Variation ,Cell Biology ,Hematology ,Pomalidomide ,medicine.disease ,Thalidomide ,Ubiquitin ligase ,Drug Resistance, Neoplasm ,biology.protein ,Cancer research ,Multiple Myeloma ,business ,medicine.drug - Abstract
Emergence of drug resistance to all available therapies is the major challenge to improving survival in myeloma. Cereblon (CRBN) is the essential binding protein of the widely used immunomodulatory drugs (IMiDs) and novel CRBN E3 ligase modulator drugs (CELMoDs) in myeloma, as well as certain proteolysis targeting chimeras (PROTACs), in development for a range of diseases. Using whole-genome sequencing (WGS) data from 455 patients and RNA sequencing (RNASeq) data from 655 patients, including newly diagnosed (WGS, n = 198; RNASeq, n = 437), lenalidomide (LEN)-refractory (WGS, n = 203; RNASeq, n = 176), and pomalidomide (POM)-refractory cohorts (WGS, n = 54; RNASeq, n = 42), we found incremental increases in the frequency of 3 CRBN aberrations, namely point mutations, copy losses/structural variations, and a specific variant transcript (exon 10 spliced), with progressive IMiD exposure, until almost one-third of patients had CBRN alterations by the time they were POM refractory. We found all 3 CRBN aberrations were associated with inferior outcomes to POM in those already refractory to LEN, including those with gene copy losses and structural variations, a finding not previously described. This represents the first comprehensive analysis and largest data set of CBRN alterations in myeloma patients as they progress through therapy. It will help inform patient selection for sequential therapies with CRBN-targeting drugs.
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- 2021
11. P-093: Iberdomide induces activation and proliferation of innate and adaptive immune cell subsets in the tumor microenvironment of relapsed/refractory myeloma patients
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Oliver Van Oekelen, Michael Amatangelo, Manman Guo, Bhaskar Updahyaya, Adam Cribbs, Geoffrey Kelly, Manishkumar Patel, Seunghee Kim-Schulze, Erin Flynt, Alessandro Lagana, Sarah Gooding, Sundar Jagannath, William Pierceall, Anjan Thakurta, Udo Oppermann, and Samir Parekh
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Cancer Research ,Oncology ,Hematology - Published
- 2022
12. Differential RNA splicing as a potentially important driver mechanism in multiple myeloma
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Brian A Walker, Gareth J. Morgan, Eileen M Boyle, Christopher P. Wardell, Cody Ashby, Michael A Bauer, Anjan Thakurta, Erin Flynt, and Maria Ortiz
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Spliceosome ,RNA Splicing ,Computational biology ,Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Gene expression ,Humans ,splice ,Gene ,030304 developmental biology ,0303 health sciences ,Serine-Arginine Splicing Factors ,Alternative splicing ,Wild type ,RNA ,Hematology ,Phosphoproteins ,Alternative Splicing ,030220 oncology & carcinogenesis ,Mutation ,RNA splicing ,Spliceosomes ,RNA Splicing Factors ,Multiple Myeloma - Abstract
Disruption of the normal splicing patterns of RNA is a major factor in the pathogenesis of a number of diseases. Increasingly research has shown the strong influence that splicing patterns can have on cancer progression. Multiple Myeloma is a molecularly heterogeneous disease classified by the presence of key translocations, gene expression profiles and mutations but the splicing patterns in MM remains largely unexplored. We take a multifaceted approach to define the extent and impact of alternative splicing in MM. We look at the spliceosome component, SF3B1, with hotspot mutations (K700E and K666T/Q) shown to result in an increase in alternative splicing in other cancers. We discovered a number of differentially spliced genes in comparison of the SF3B1 mutant and wild type samples that included, MZB1, DYNLL1, TMEM14C and splicing related genes DHX9, CLASRP, and SNRPE. We identified a broader role for abnormal splicing showing clear differences in the extent of novel splice variants in the different translocation groups. We show that a high number of novel splice loci is associated with adverse survival and an ultra-high risk group. The enumeration of patterns of alternative splicing has the potential to refine MM classification and to aid in the risk stratification of patients.
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- 2020
13. Multiple Myeloma DREAM Challenge reveals epigenetic regulator PHF19 as marker of aggressive disease
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Brian S. White, Mehmet Kemal Samur, Alexander V. Ratushny, Michael Mason, Gareth J. Morgan, Justin Guinney, Yi Cui, Pieter Sonneveld, Bailiang Li, Carolina Schinke, Fadi Towfic, Samuel A. Danziger, Hervé Avet-Loiseau, Matthew Trotter, Elias Chaibub Neto, Hongyue Y Dai, Valeriy V. Lyzogubov, Christine Eng, Jonathan Göke, Anjan Thakurta, Hongjie Chen, Andrew Dervan, William S. Dalton, Daniel Auclair, Douglas Bassett, Nikhil C. Munshi, Kenneth H. Shain, Fred K. Gruber, Boris Hayete, Brian A Walker, Aditya Pratapa, Frank Schmitz, Hartmut Goldschmidt, Thomas Yu, Dirk Hose, Erin Flynt, Kamlesh Bisht, Maria Ortiz, Yuanfang Guan, Konstantinos Mavrommatis, Dan Rozelle, Computer Science, and Basic (bio-) Medical Sciences
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Cancer Research ,Clinical Trials as Topic/statistics & numerical data ,Databases, Factual ,Transcription Factors/genetics ,Regulator ,Datasets as Topic ,Myeloma ,Multiple Myeloma/genetics ,Computational biology ,Aggressive disease ,Article ,Epigenesis, Genetic ,03 medical and health sciences ,0302 clinical medicine ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Humans ,Medicine ,Epigenetics ,Staging system ,Gene ,Multiple myeloma ,030304 developmental biology ,Epigenesis ,Regulation of gene expression ,Clinical Trials as Topic ,0303 health sciences ,Models, Statistical ,biology ,business.industry ,Hematology ,medicine.disease ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Histone ,cell proliferation ,Risk factors ,Oncology ,Expression (architecture) ,030220 oncology & carcinogenesis ,biology.protein ,cell cycle ,Biomarkers, Tumor/genetics ,Multiple Myeloma ,business ,DNA-Binding Proteins/genetics ,Transcription Factors - Abstract
While the past decade has seen meaningful improvements in clinical outcomes for multiple myeloma patients, a subset of patients do not benefit from current therapeutics for unclear reasons. Many gene expression-based models of risk have been developed, but each model uses a different combination of genes and often involve assaying many genes making them difficult to implement. We organized the Multiple Myeloma DREAM Challenge, a crowdsourced effort to develop models of rapid progression in newly diagnosed myeloma patients and to benchmark these against previously published models. This effort lead to more robust predictors and found that incorporating specific demographic and clinical features improved gene expression-based models of high risk. Furthermore, post challenge analysis identified a novel expression-based risk marker and histone modifier,PHF19, which featured prominently in several independent models. Lastly, we show that a simple four feature predictor composed of age, International Staging System stage (ISS), and expression ofPHF19andMMSETperforms similarly to more complex models with many more gene expression features included.Key pointsMost comprehensive and unbiased assessment of prognostic biomarkers in MM resulting in a robust and parsimonious model.Identification ofPHF19as the expression based biomarker most strongly associated with rapid progression in MM patients.
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- 2020
14. Iberdomide (CC-220) is a potent cereblon E3 ligase modulator with antitumor and immunostimulatory activities in lenalidomide- and pomalidomide-resistant multiple myeloma cells with dysregulated CRBN
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Ann Polonskaia, Jian Kang, Maria Ortiz, Suzana Couto, Yan Ren, William E. Pierceall, Michael Amatangelo, J Erin Flynt, Mark A. Katz, Chad C. Bjorklund, Hsiling Chiu, Anjan Thakurta, Fadi Towfic, and Maria Wang
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Cancer Research ,Letter ,Morpholines ,Ubiquitin-Protein Ligases ,Phthalimides ,Myeloma ,Drug resistance ,Heterocyclic Compounds, 4 or More Rings ,Antineoplastic Combined Chemotherapy Protocols ,Tumor Cells, Cultured ,medicine ,Humans ,Lenalidomide ,Piperidones ,Multiple myeloma ,Adaptor Proteins, Signal Transducing ,Clinical Trials as Topic ,biology ,business.industry ,Cereblon ,Signal transducing adaptor protein ,Hematology ,medicine.disease ,Pomalidomide ,Thalidomide ,Ubiquitin ligase ,Cancer therapeutic resistance ,Oncology ,Drug Resistance, Neoplasm ,Leukocytes, Mononuclear ,biology.protein ,Cancer research ,Multiple Myeloma ,business ,medicine.drug - Published
- 2019
15. Overcoming IMiD resistance in T-cell lymphomas through potent degradation of ZFP91 and IKZF1
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Wenchao Wu, Geoffrey M. Nelson, Raphael Koch, Katherine A. Donovan, Radosław P. Nowak, Tayla B. Heavican-Foral, Ajit J. Nirmal, Huiyun Liu, Lei Yang, Jessica Duffy, Foster Powers, Kristen E. Stevenson, Marcus Kenneth Jones, Samuel Y. Ng, Gongwei Wu, Salvia Jain, Ran Xu, Sam Amaka, Christopher Trevisani, Nicholas L. Donaldson, Patrick R. Hagner, Laurence de Leval, Philippe Gaulard, Javeed Iqbal, Anjan Thakurta, Eric S. Fischer, Karen Adelman, and David M. Weinstock
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Lymphoid Neoplasia ,Ubiquitin-Protein Ligases ,Immunology ,Ubiquitination ,Cell Biology ,Hematology ,Lymphoma, T-Cell ,Biochemistry ,Thalidomide ,Immunomodulating Agents ,Ikaros Transcription Factor ,Drug Resistance, Neoplasm ,Humans ,Immunologic Factors ,Multiple Myeloma ,Lenalidomide - Abstract
Immunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here, we show that 2 factors determine resistance to IMiDs among TCLs. First, limited CRBN expression reduces IMiD activity in TCLs but can be overcome by newer-generation degrader CC-92480. Using mass spectrometry, we show that CC-92480 selectively degrades IKZF1 and ZFP91 in TCL cells with greater potency than pomalidomide. As a result, CC-92480 is highly active against multiple TCL subtypes and showed greater efficacy than pomalidomide across 4 in vivo TCL models. Second, we demonstrate that ZFP91 functions as a bona fide transcription factor that coregulates cell survival with IKZF1 in IMiD-resistant TCLs. By activating keynote genes from WNT, NF-kB, and MAP kinase signaling, ZFP91 directly promotes resistance to IKZF1 loss. Moreover, lenalidomide-sensitive TCLs can acquire stable resistance via ZFP91 rewiring, which involves casein kinase 2–mediated c-Jun inactivation. Overall, these findings identify a critical transcription factor network within TCLs and provide clinical proof of concept for the novel therapy using next-generation degraders.
- Published
- 2021
16. Cereblon pathway biomarkers and immune profiles in patients with myeloma receiving post-ASCT lenalidomide maintenance (LEOPARD)
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Andrew Spencer, Anjan Thakurta, Tiffany Khong, Anna Kalff, Samir Parekh, Maria Wang, William E. Pierceall, Samuel E Norton, John V. Reynolds, Manman Guo, Yan Ren, Nola Kennedy, Malarmathy Ramachandran, Anthony P. Schwarer, Patricia Walker, Udo Oppermann, Andrew W. Roberts, Robin Filshie, Mary Young, and Philip Campbell
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Oncology ,Cancer Research ,medicine.medical_specialty ,Transplantation, Autologous ,Maintenance Chemotherapy ,Autologous stem-cell transplantation ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Humans ,Lenalidomide ,Multiple myeloma ,Hematology ,business.industry ,Bortezomib ,Cereblon ,Hematopoietic Stem Cell Transplantation ,medicine.disease ,Thalidomide ,Prednisolone ,business ,Multiple Myeloma ,Biomarkers ,medicine.drug - Abstract
LEOPARD was a single arm, phase II study of lenalidomide (LEN) and alternate day prednisolone maintenance in patients with newly diagnosed multiple myeloma (MM) following autologous stem cell transplantation (ASCT). Sixty patients were enrolled. Estimated median potential follow-up was 44 m, median PFS was 38.3 m, median OS was not reached (landmark 36 m OS: 71.4%). Correlative immunohistochemistry performed on pre-ASCT trephines demonstrated high MM tumor cereblon (total/cytoplasmic) was associated with superior OS (p = .045, p = .031, respectively), whereas high c-Myc was associated with inferior PFS (p = .04). Patients with high cereblon (total/nuclear) were more likely to improve depth of response, whereas patients with high c-Myc were less likely, suggesting alternative/more effective post-ASCT strategies for patients with high c-Myc need identification. Peripheral blood immune profiling (mass cytometry) informed a more sustained response to LEN maintenance, demonstrating enrichment of activated/cytotoxic NK cells and cytotoxic T cells in patients with durable responses, contrasting with enrichment of B-regs in early relapsers.
- Published
- 2021
17. P-065: Development of a mass cytometry-based toolkit to investigate myeloma therapeutic responses ex vivo
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Sarah Gooding, Manman Guo, Oliver Van Oekelen, Kinda Al-Hourani, Edmund Watson, Charlotte Palmer, Martin Philpott, Warren Baker, David Ahern, Bhaskar Updahyaya, Seunghee Kim-Schulze, Erin Flynt, William Pierceall, Karthik Ramasamy, Adam Cribbs, Samir Parekh, Anjan Thakurta, and Udo Oppermann
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Cancer Research ,Oncology ,Hematology - Published
- 2022
18. P-231: Biomarker analysis to support dose optimization of iberdomide (IBER) as monotherapy and in combination with standard of care treatments for multiple myeloma from a phase 1/2 trial
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Michael Amatangelo, Yiming Cheng, William Pierceall, Niels W.C.J. van de Donk, Sagar Lonial, Maria Wang, Joshua Emerson, Kevin Hong, Paulo Maciag, Teresa Peluso, Anita Gandhi, and Anjan Thakurta
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Cancer Research ,Oncology ,Hematology - Published
- 2022
19. Oral azacitidine (CC-486) in combination with lenalidomide and dexamethasone in advanced, lenalidomide-refractory multiple myeloma (ROAR study)
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Roberto Guzman, Andrew Spencer, Yan Ren, Maria Wang, Tiffany Khong, Suzana Couto, Anna Kalff, Krystal Bergin, John V. Reynolds, Sridurga Mithraprabhu, Anjan Thakurta, and Kathryn M. Bowen
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Male ,Proteomics ,Antimetabolites, Antineoplastic ,Cancer Research ,Maximum Tolerated Dose ,Ubiquitin-Protein Ligases ,Administration, Oral ,Dexamethasone ,Drug Administration Schedule ,Oral Azacitidine ,Epigenesis, Genetic ,03 medical and health sciences ,0302 clinical medicine ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Humans ,Lenalidomide ,Adaptor Proteins, Signal Transducing ,Aged ,Aged, 80 and over ,Dose-Response Relationship, Drug ,business.industry ,Cereblon ,Refractory Multiple Myeloma ,Hematology ,DNA Methylation ,Middle Aged ,Prognosis ,Progression-Free Survival ,Gene Expression Regulation, Neoplastic ,Oncology ,Hypomethylating agent ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Azacitidine ,Cancer research ,Female ,Neoplasm Recurrence, Local ,Multiple Myeloma ,business ,030215 immunology ,medicine.drug - Abstract
In preclinical studies, oral azacitidine (CC-486), a hypomethylating agent, has been shown to have a direct anti-MM effect and in vitro anti-MM synergism when combined with lenalidomide (LEN). We present a phase 1b, single center, 3 × 3 dose escalation study with planned expansion at maximum tolerated dose (MTD), which assessed the safety and efficacy of combining CC-486 with LEN (25 mg d1-21/28) and dexamethasone (DEX) (40 mg weekly) in patients with relapsed/refractory MM who had previously failed LEN. Twenty-four patients were enrolled. The MTD of CC-486 was 150 mg d1-21; recommended expansion dose was 100 mg d1-21. Adverse events were predictable and manageable. ORR was 37.5%; clinical benefit rate was 50%. Median OS was 10.3 m; median PFS was 2.6 m. Correlative proteomics demonstrated that higher MM tumor cell cereblon expression (pretreatment, C1D5) was associated with superior PFS/OS. CC-486, LEN and DEX produced meaningful clinical responses in heavily treated LEN refractory MM patients. Proteomics may have utility in predicting clinical outcomes.
- Published
- 2019
20. A high-risk, Double-Hit, group of newly diagnosed myeloma identified by genomic analysis
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Hongwei Wang, Matthew Trotter, John C. Obenauer, Michael A Bauer, Kenneth C. Anderson, Brian A Walker, Zhinuan Yu, Pingping Qu, Zhihong Yang, T. Cody Ashby, Erin Flynt, Nikhil C. Munshi, Mariateresa Fulciniti, Mehmet Kemal Samur, A. Keith Stewart, Konstantinos Mavrommatis, Gareth J. Morgan, Dan Rozelle, Antje Hoering, Daniel Auclair, Brian G.M. Durie, Pieter Sonneveld, Hermann Einsele, Adam Rosenthal, Maria Ortiz, Marc S. Raab, Niccolo Bolli, Graham Jackson, Faith E. Davies, Fadi Towfic, Hartmut Goldschmidt, Jonathan J Keats, Christopher P. Wardell, Raphael Szalat, Hervé Avet-Loiseau, Anjan Thakurta, Jesús F. San Miguel, Sagar Lonial, P. Moreau, and Hematology
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0301 basic medicine ,Oncology ,Cancer Research ,medicine.medical_specialty ,Double-Hit ,Population ,Recursive partitioning ,risk stratification ,Article ,03 medical and health sciences ,0302 clinical medicine ,Risk Factors ,high-risk ,Internal medicine ,Biomarkers, Tumor ,medicine ,Humans ,Progression-free survival ,education ,Survival rate ,Exome ,Multiple myeloma ,Chromosome Aberrations ,education.field_of_study ,CKS1B ,Genome, Human ,Proportional hazards model ,business.industry ,High-Throughput Nucleotide Sequencing ,Genomics ,Hematology ,Prognosis ,medicine.disease ,personalised treatment ,Survival Rate ,multiple myeloma ,Editorial ,030104 developmental biology ,030220 oncology & carcinogenesis ,business ,Ciencias de la Salud::Oncología [Materias Investigacion] - Abstract
Patients with newly diagnosed multiple myeloma (NDMM) with high-risk disease are in need of new treatment strategies to improve the outcomes. Multiple clinical, cytogenetic, or gene expression features have been used to identify high-risk patients, each of which has significant weaknesses. Inclusion of molecular features into risk stratification could resolve the current challenges. In a genome-wide analysis of the largest set of molecular and clinical data established to date from NDMM, as part of the Myeloma Genome Project, we have defined DNA drivers of aggressive clinical behavior. Whole-genome and exome data from 1273 NDMM patients identified genetic factors that contribute significantly to progression free survival (PFS) and overall survival (OS) (cumulative R2 = 18.4% and 25.2%, respectively). Integrating DNA drivers and clinical data into a Cox model using 784 patients with ISS, age, PFS, OS, and genomic data, the model has a cumlative R2 of 34.3% for PFS and 46.5% for OS. A high-risk subgroup was defined by recursive partitioning using either a) bi-allelic TP53 inactivation or b) amplification (≥4 copies) of CKS1B (1q21) on the background of International Staging System III, comprising 6.1% of the population (median PFS = 15.4 months; OS = 20.7 months) that was validated in an independent dataset. Double-Hit patients have a dire prognosis despite modern therapies and should be considered for novel therapeutic approaches.
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- 2019
21. A phase II study of pomalidomide, daily oral cyclophosphamide, and dexamethasone in relapsed/refractory multiple myeloma
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Oliver Van Oekelen, Joshua Richter, Erika Florendo, Chun Ip, Kenneth Lau, Alessandro Laganà, Deepu Madduri, Seunghee Kim-Schulze, William E. Pierceall, Violetta V. Leshchenko, Anjan Thakurta, Sundar Jagannath, Ines Stefania Mancia, Joanne Thomas, Samir Parekh, Hearn Jay Cho, Ajai Chari, Katarzyna Zarychta, Lisa La, David Melnekoff, Suzana Couto, Daniel Verina, Elaine Chan, Nivetha Vishnuvardhan, Maria Wang, and Gina Strumolo
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Oncology ,Cancer Research ,medicine.medical_specialty ,Cyclophosphamide ,Phases of clinical research ,Dexamethasone ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Tumor Microenvironment ,Humans ,Multiple myeloma ,business.industry ,Refractory Multiple Myeloma ,Hematology ,medicine.disease ,Pomalidomide ,Thalidomide ,030220 oncology & carcinogenesis ,Relapsed refractory ,Oral cyclophosphamide ,business ,Multiple Myeloma ,030215 immunology ,medicine.drug - Abstract
Relapsed/refractory multiple myeloma patients treated with pomalidomide and dexamethasone have an overall response rate (ORR) of ∼30% and median progression-free survival (PFS) of 4-5 months. Previous studies explored addition of weekly cyclophosphamide, but we hypothesized that daily dosing allows for better synergy. We report the open-label, single-center phase II study of pomalidomide, daily cyclophosphamide and weekly dexamethasone (PCD). Thirty-three patients were evaluable for efficacy and underwent 28-day cycles of pomalidomide (4 mg/day, D1-21), cyclophosphamide (50 mg b.i.d., D1-21) and weekly dexamethasone. All were lenalidomide-refractory and 55% were refractory to lenalidomide and proteasome inhibitor. ORR was 73%; median PFS and overall survival were 13.3 months and 57.2 months respectively. Grade 3/4 toxicities were primarily hematologic but manageable with dose reductions. Early disease progression correlated with MYC expression and flow cytometry demonstrates an activated microenvironment post-PCD. Addition of metronomic cyclophosphamide to pomalidomide and dexamethasone is a cost-effective, oral regimen with encouraging PFS.
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- 2020
22. Low Exposure Extended Dosing Mimicking Clinical Exposures of the Oral Formulation of Azacitidine Results in a Sustained Hypomethylation and Targets Leukemic Stem Cells
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Jammula Sriganesh, Charalampos Kyriakopoulos, Patrick Hagner, Alberto Risueño, Gaurav Jain, Danny V Jeyaraju, Anjan Thakurta, Amit Anand, Daniel Menezes, Christopher Hartl, Aarif Ahsan, Chao Wang, Madhusudhan Reddy, Maryam Alapa, Nathalie Lailler, Prakash Subramanyam, Ann Polonskaia, and Kaushik Ghosh
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business.industry ,Immunology ,Azacitidine ,Cancer research ,Low exposure ,Medicine ,Cell Biology ,Hematology ,Dosing ,Stem cell ,business ,Biochemistry ,medicine.drug - Abstract
Acute Myeloid Leukemia (AML) is characterized by uncontrolled proliferation of incompletely differentiated myeloid stem/ progenitor cells. Despite recent advances in therapy, high rates of clinical relapse, even in patients who achieve complete remission, remains a critically unmet need. One of the strategies to prolong remission post-induction in AML is to employ effective maintenance therapies. Oral Azacitidine (Oral-AZA; CC-486) is the first and only currently approved maintenance therapy in AML. However, the mechanism of action of Oral-AZA and whether it is differentiated from the Injectable-AZA used in AML induction therapy, remains unclear. In this work, we explore the mechanism of Oral-AZA by in vitro modelling of the relative clinical exposures of Oral-AZA vs Injectable-AZA in AML cell line models. Following Vu et al (Nat. Commun. 2020), we used the Injectable-AZA concentration (1 μM aZA) as a single dose (HELD - High Exposure Limited Duration). A fractionated dose of 0.2 μM each day over 5 days (LEED - Low Exposure Extended Duration) was used to model Oral-AZA. Azacitidine incorporates into RNA and DNA of AML cells leading to hypomethylation driven gene expression changes. We found that HELD but not LEED dosing produced an acute anti-proliferative effect in sensitive AML cell lines suggesting potential for a non-hypomethylation mediated/integrated stress response (ISR) driven mechanism. This ISR effect was rescued by co-treatment with ISRIB, an ISR inhibitor. With HELD, we observed robust ATF4 activation as early as 6 hours that was sustained up to 24 hours. In contrast, LEED induced modest and transient ATF4 activation. Thus, an Injectable-AZA-like regimen activated the ISR pathway more robustly than an Oral-AZA-like regimen. Interestingly, decitabine, a DNA-only incorporating cytidine analog did not activate ISR. In AML cell lines, the target of Azacitidine, DNMT1 was depleted (about 90% compared to control) within 24 hours in both HELD and LEED regimens. However, LEED produced a more sustained DNMT1 loss, up to 7 days whereas in HELD, DNMT1 protein levels recovered 96 hours post-dosing. Given the relative level and duration of DNMT1 loss, we hypothesized that LEED would lead to a more durable hypomethylation. We performed whole genome bisulfite sequencing (WGBS) in 3 AML cell lines (OCI-AML2, MV-4-11 and SKM1) at 48- and 96-hours post-initiation of HELD and LEED dosing. Consistent with the DNMT1 depletion kinetics, at 48 hours we observed almost 75% global DNA hypomethylation with both HELD and LEED treatments. At 96 hours, HELD demonstrated a recovery effect and reverted to 50% hypomethylation. In contrast, LEED showed up to 85% hypomethylated sites, demonstrating the durability of the hypomethylation mediated by an Oral-AZA-like (LEED) regimen. Next, we explored the effect of Oral-AZA-like dosing in the differentiation of leukemic stem cells (LSCs) towards a more mature phenotype. In an in vitro LSC model (OCI-AML-20) with flow cytometry analysis, LEED dosing resulted in a greater depletion (2-fold more) of LSCs (CD34+/38- or 38 low) and concomitant enrichment of cells with more differentiated phenotype (CD34+/38+) compared to HELD dosing. To further validate these observations at the transcriptomic level, we performed single cell RNAseq in OCI-AML-20 cells at different timepoints (3, 5 and 7 days). Data were analyzed using a classifier to identify leukemic myeloid cell lineages (Van Galen et al., Cell 2019). Compared to untreated cells, at day 7, both LEED and HELD dosing resulted in an increase of GMP and promonocytes and those differences were more pronounced with Oral-AZA-like (50% GMP and 20% promonocytic) than the Injectable-AZA-like regimen (28% GMP and 12.5% promonocytic). Taken together, our in vitro modeling of the clinically relevant exposures of Oral-AZA vs Injectable-AZA by using HELD and LEED dosing strategies show a shift of the molecular mechanism between the clinical entities. Our work demonstrates that an Injectable-AZA-like regimen mediates cytotoxicity through an early ISR driven effect. Oral-AZA mechanism leads to a more sustained pharmacodynamic DNA hypomethylation effect that in turn could be linked to a differentiation inducing effect on the LSC population. Disclosures Jeyaraju: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Alapa: Bristol Myers Squibb: Current Employment. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Risueño: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Wang: Bristol Myers Squibb: Current Employment. Subramanyam: Bristol Myers Squibb: Current Employment. Sriganesh: Bristol Myers Squibb: Current Employment. Anand: Bristol Myers Squibb: Current Employment. Jain: Bristol Myers Squibb: Current Employment. Reddy: Bristol Myers Squibb: Current Employment. Ghosh: Bristol Myers Squibb: Current Employment. Kyriakopoulos: Bristol Myers Squibb: Current Employment. Lailler: Rancho Biosciences: Current Employment. Hartl: Rancho Biosciences: Current Employment. Lopes de Menezes: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.
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- 2021
23. Location of the t(4;14) Translocation Breakpoint Identifies a Subset of Newly-Diagnosed Multiple Myeloma Patients with Poor Prognosis
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William E. Pierceall, Fadi Towfic, Erin Flynt, Maria Ortiz, Anjan Thakurta, and Nicholas Stong
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Oncology ,Poor prognosis ,medicine.medical_specialty ,business.industry ,Immunology ,Translocation Breakpoint ,Cell Biology ,Hematology ,Newly diagnosed ,medicine.disease ,Biochemistry ,Internal medicine ,Medicine ,business ,Multiple myeloma - Abstract
Introduction: The recombination of chromosomes 4 and 14 (t(4;14)) is a primary, predominantly clonal event in newly diagnosed multiple myeloma (ndMM) that is present in ~15% of patients. The translocation results in enhancer regions from the immunoglobulin heavy chain locus upregulating the expression of NSD2 and FGFR3 genes implicated in the disease biology of this subset of MM patients (Chesi et al. Blood. 1998, Keats et al, Leuk Lymph. 2006). The presence of t(4;14) translocation is a considered a biomarker of aggressive disease and is part of the Revised International Staging System (R-ISS) for clinical risk stratification. However, historically only ~40% of t(4;14) patients are high-risk based on the GEP70 gene expression signature. (Weinhold et al. Leukemia. 2016) Our previous analysis of a large cohort of ndMM patients described the genomic features of t(4;14) vs ndMM overall population demonstrating that only ~25% of t(4;14) patients died within 24 months of diagnosis and described biomarkers in this high-risk subset. This analysis identified both known and novel aberrations in ndMM, including some that were associated with high-risk t(4;14) (Ortiz et al Blood. 2019; 134 (Suppl_1):366). In this updated analysis, we provide a more robust analysis of the t(4;14) dataset and demonstrate the prognostic value of the NSD2 breakpoint location. Methods: We generated a large genomic dataset from t(4;14) ndMM patients with whole genome sequencing (WGS) and RNA-seq from a TOUL dataset (t(4;14) N=114) patients treated in routine practice), the IFM2009 trial (N=19), and the Myeloma Genome Project (MGP) (N=34) for discovery and validation. Gene expression, copy number aberration, single nucleotide variant and translocations were derived from RNAseq and WGS profiling of biopsies from patients aged less than 75 years who received transplant, and integrated with clinical information (including age, OS). Cytogenetic assessments from WGS were made by MANTA and used to identify translocation DNA breakpoint location. Results: In all datasets, three DNA breakpoint locations were identified, and based on their position with respect to the NSD2 gene named "no-disruption" (upstream of NSD2 gene), "early-disruption" (in the 5' UTR of NSD2 gene) and "late-disruption" (in the coding region of NSD2 gene). Using paired RNA-seq data, we identified IGH-NSD2 RNA fusion transcripts relative to the breakpoints that corresponded with previously described NSD2 isoforms. "No-disruption" and "early-disruption" breakpoints predominantly produced a fusion transcript (MB4-1) that retained the full coding sequence of the gene, while the "late-disruption" produced truncated fusion transcripts (MB4-2/3). We conducted survival analysis in our datasets based on both DNA breakpoint location and RNA fusion transcripts. This analysis demonstrated a significant difference in outcome between the patient samples with "no-disruption" and the "late-disruption" breakpoints that associated with good and poor OS, respectively (OS pval < 3e-4) in the discovery TOUL dataset. Patients with "late-disruption" had a median OS of 28.64 mo vs 59.18 mo for "early disruption" and 82.26 mo for those with "no disruption" (Figure). This association was replicated in an independent dataset (MGP N=33, replication pval Conclusion: From a large genomic dataset, we were able to discover and validate a clear association between the translocation breakpoints and survival outcome in t(4:14) ndMM patients. While prospective validation is needed before clinical application of our finding, molecular identification of high-risk t(4;14) patients using DNA breakpoint location may enable proper risk classification for this patient group at diagnosis, and would provide improved opportunities for risk-adjusted therapy and identification of a therapeutic target for this high-risk subpopulation. Ongoing work on mutations, copy number, and differential gene expression analyses between translocation breakpoint sub-groups and will be presented. Figure 1 Figure 1. Disclosures Stong: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ortiz: Bristol Myers Squibb: Current Employment. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierceall: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flynt: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.
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- 2021
24. Discovery of Prolyl-tRNA Synthetase As a Novel Target in Multiple Myeloma
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Keiji Kurata, Anjan Thakurta, Catrine Johansson, Sarah Gooding, Ralph Mazitschek, Mark A Tye, Udo Oppermann, N Connor Payne, Teru Hideshima, Anna-James Bott, Yu-Tzu Tai, Martin Philpott, Leona Yamamoto, Karthik Ramasamy, Filiz Senbabaoglu, James E. Dunford, Adam P. Cribbs, Mehmet Kemal Samur, Patrick Hagner, and Kenneth C. Anderson
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Biochemistry ,Chemistry ,Immunology ,medicine ,Cell Biology ,Hematology ,Prolyl tRNA synthetase ,medicine.disease ,health care economics and organizations ,Multiple myeloma - Abstract
Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that charge tRNAs with their cognate amino acids. Recent studies have revealed that the expression of aaRSs is dynamically altered in response to cellular stresses, and that specific isoforms play pivotal roles in carcinogenesis and tumor progression. Despite emerging interest in aaRSs in cancer, the biologic sequelae of aaRSs in multiple myeloma (MM) have not yet been elucidated. We first examined RNA expression levels of aaRSs in tumor cells from MM patients. Gene expression analyses revealed that most aaRSs were upregulated in MM patients compared to healthy donors. Importantly, expression of 4 aaRSs, including glutamyl-prolyl-tRNA synthetase (EPRS), correlated with worse clinical prognosis: high EPRS expression correlated with disease progression (GSE6477; P< 0.001) and shorter survival (CoMMpass; P = 0.007). Consistent with clinical data, ERPS knockdown in MM cells (AMO1, RPMI 8226, and MM.1S) triggered significant cell growth inhibition, suggesting that EPRS is a growth and survival factor in MM cells. Halofuginone (HF) is an inhibitor of the prolyl-tRNA-synthetase (PRS) activity of EPRS and potently inhibits MM growth. However, HF is proline (Pro) competitive and increased levels of Pro abrogates the effect of HF, including the Pro-rich tumor microenvironment. To overcome this limitation, we developed a novel ATP competitive and Pro non-competitive PRS inhibitor, NCP26. NCP26 potently decreased growth in MM cell lines, including drug (Dox, Dex, PI, IMiDs) resistant isogenic derivatives, and patient MM cells (EC50 ≈ 500 nM), without significant toxicity to peripheral blood mononuclear cells. Neither anti-apoptotic cytokines (i.e., IL-6) nor co-culture with bone marrow (BM) stromal cells abrogated NCP26-induced MM cell death. Importantly, unlike HF, NCP26-induced cell growth inhibition was not altered by an excess of exogenous Pro. Since aaRSs inhibition results in accumulation of uncharged tRNAs sensed by the amino acid response (AAR), we next examined whether NCP26 activates the AAR pathway in MM cells. As expected, NCP26 increased both p-GCN2 and p-eIF2α, associated with upregulation of ATF4 and CHOP expression. Moreover, NCP26 treatment induced G0/G1 cell cycle arrest followed by apoptotic cell death, evidenced by cleavage of caspases and PARP. We also evaluated in vivo efficacy of NCP26 in the AMO1 xenograft mouse model: NCP26 (10 mg/kg, q.d.) significantly inhibited tumor growth (P = 0.005) and prolonged host survival (P < 0.001) compared to control, without affecting body weight. To further characterize the biological impact of NCP26 in MM cells, we performed single-cell RNA-seq using BM samples from MM patients. While both HF and NCP26 treatment reduced MM, NCP26 triggered more sustained and robust activation of integrated stress response, evidenced by induction of GCN2, CHOP, and IRE1, compared to HF. Gene set enrichment analysis also revealed significant alteration in chromatin processes, besides proteostasis, such as translation, signal peptide, and stress response, after NCP26 treatment. Since previous studies suggested that translation of genes bearing high Pro-Pro (PP) motifs is more susceptible to EPRS inhibition than non-PP-bearing genes, we next performed a quantitative proteomic analysis in AMO1 cells treated with NCP26: of 7,366 proteins, 84 were upregulated and 363 were downregulated after 6-hour treatment. We also performed in silico bioinformatic analysis to uncover PP-bearing genes, and incorporated CRISPR-screening data to identify genetic dependencies in MM. By overlapping the three datasets, we identified a group of 55 PP motif-containing proteins which play essential roles in MM cell pathogenesis, including MYC, PIM2, and CCND1/2. We also confirmed that PP motif-containing proteins were significantly downregulated by NCP26 treatment in MM cells both in vitro and in vivo. In conclusion, our results identify EPRS as a biomarker of poor prognosis and a novel therapeutic target in MM. NCP26, a novel ATP-competitive and Pro non-competitive EPRS inhibitor, induces MM cytotoxicity both in vitro and in vivo by triggering AAR and EPRS-Pro-rich proteins pathways. Our data provide the preclinical rationale for development of NCP26-based clinical candidates to improve patient outcomes in MM. Disclosures Gooding: Bristol Myers Squibb: Research Funding. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Celgene (BMS): Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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- 2021
25. Presence of Extrachromosomal DNA (ecDNA) Impacts Both Progression Free and Overall Survival and Is an Independent Poor Prognostic Marker in Multiple Myeloma
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Parth Shah, Anil Aktas-Samur, Mariateresa Fulciniti, Raphael Szalat, Masood A. Shammas, Paul G. Richardson, Florence Magrangeas, Stephane Minvielle, Aurore Perrot, Jill Corre, Philippe Moreau, Anjan Thakurta, Kenneth C. Anderson, Herve Avet-Loiseau, Nikhil C. Munshi, and Mehmet K. Samur
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Background Focal amplifications and rearrangements drive tumor growth and evolution in cancer. Focally amplified regions often involve the juxtaposition of rearranged segments of DNA from distinct chromosomal loci into a single amplified region and nearly half of these regions can be explained by circular, extrachromosomal DNA (ecDNA) formation. Cancer-associated ecDNA shows a unique circular placing ecDNA at the interface of cancer genomics and epigenetics. As formation of ecDNA represents a manifestation of genomic instability, we have investigated presence and prognostic impact of ecDNA in multiple myeloma (MM). Methods Whole genome (WGS) and transcriptome (RNAseq) sequencing data from CD138 purified MM cells from 191 uniformly-treated newly diagnosed MM patients were used for this analysis. Copy number variants (CNV), single nucleotide variants (SNV) and structural variants (SV) were identified on all WGS samples using Facets, Mutect2 and Manta. Seed data from these CNV results was passed to the AmpliconArchitect tool to determine presence of focally amplified and rearranged segments of DNA. Seed CNV thresholds were set for a minimum CNV size of 100kb and a copy number of equal or greater to 5. Extrachromosomal calls were then annotated using the Amplicon Classifier to determine the presence of ecDNA. Multivariate survival analysis was performed after segregating samples into the conventional myeloma risk classifications including translocations, copy number alterations, ISS, age and mutations associated with risk. Differential expression analysis was performed on transcriptomic data using DEseq2. Results We identified 6.8% of the newly diagnosed patients with ecDNA, 12.5% with complex non-cyclic DNA amplifications and 10.1% with linear amplifications. ecDNA and complex events were targeting MM dependent genes, including MYC/PVT1, IRF4 as well as known driver genes such as CDYL and TRAF2. We further evaluated association between ecDNA, complex rearrangements, linear amplification and patients with none of these amplification types and found that patients with ecDNA had significantly poor PFS (median PFS 22 months vs. 41 months) and OS (median OS 41 months vs. 105 months). Patients having ecDNA in their MM cells did not show any significant enrichment for known translocations, double hit or TP53 mutations. In a multivariate model including ecDNA and all other known MM risk features, ecDNA was found to be an independent predictor of progression free survival.(HR 2.6, CI: 1.26 -5.6, p=0.0082) and overall survival (HR 7.94 CI:3.5-17.9 p < 0.0001). Patients with ecDNA have higher mutational load probability(8798 vs 6982, effect size = 0.64 , probability is 91.1). However, this was not reflected in heterogeneity by using MATH score. We found that patients with ecDNA are likely to have BRAF mutations (OR= 25.07 [2.57 - 330 95% CI], p value = 0.002), however overall RAS/RAF pathway mutations were similar to other patients. Patients with ecDNA showed fragile DNA with more breaks (median segments 197 vs. 125.5, p value = 0.001). Although ecDNA is defined as copy number gain with fragments having 5 or more copies, overall genomic gain between ecDNA and other patients were similar. However, overall genomic loss in patients with ecDNA were higher than others (7% vs. 4.2%, p = 0.06). By differential gene expression analysis we noted 98 differentially expressed genes in MM cells with ecDNA. The downregulated geneset involved pathways responsible for cell death as well as the RAS pathway. Interestingly, CD38 was upregulated in the ecDNA dataset suggesting greater potential for CD38 targeting therapies in these patients. Conclusions ecDNA, as an unique marker of perturbed genomic integrity, is observed in a subset of patients and is an independent prognostic marker in newly diagnosed MM patients. As patients with ecDNA are not fully captured by other risk features its incorporation in an expanded definition of a high risk group of multiple myeloma should be investigated. Future studies will endeavor to explore the biological mechanism through which ecDNA are formed and influences outcomes in myeloma. Figure 1 Figure 1. Disclosures Richardson: Sanofi: Consultancy; GlaxoSmithKline: Consultancy; Karyopharm: Consultancy, Research Funding; AstraZeneca: Consultancy; AbbVie: Consultancy; Oncopeptides: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Protocol Intelligence: Consultancy; Celgene/BMS: Consultancy, Research Funding; Secura Bio: Consultancy; Regeneron: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Perrot: Abbvie: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Moreau: Abbvie: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Celgene BMS: Honoraria; Oncopeptides: Honoraria. Thakurta: Oxford University: Other: Visiting Professor; BMS: Current Employment, Current equity holder in publicly-traded company. Anderson: Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Pfizer: Consultancy.
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- 2021
26. Super-Enhancer Driven Regulation of CKS1B in Multiple Myeloma: Implications in Mediating Response to BET Inhibitor and Celmod Agent Combination
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Chad C Bjorklund, Erin Flynt, Anjan Thakurta, Nizar J. Bahlis, Chih-Chao Hsu, Fadi Towfic, Maria Ortiz Estevez, Ann Polonskaia, Michael Pourdehnad, and Aarif Ahsan
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BET inhibitor ,Super-enhancer ,CKS1B ,Agent Combination ,Chemistry ,Immunology ,medicine ,Cancer research ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Multiple myeloma - Abstract
Introduction: The Myeloma Genome Project (MGP) characterized the genomic landscape of patients with newly diagnosed multiple myeloma (NDMM) (Walker BA, et al. Blood 2018; 132[6]:587-597). Using a multi-omics unsupervised clustering approach, 12 molecularly-defined disease segments were identified (Ortiz M, et al. Blood 2018; 132[suppl 1]:3165). Here, we performed experimental validation of CDC28 Protein Kinase Regulatory Subunit 1B (CSK1B) that was identified as a putative target from the disease segment with poorest clinical outcome. CKS1B was selected for in-depth validation due to their role in cell cycle pathways associated with high-risk disease, biological mechanisms of chromosome 1q amplification and druggability. Methods: Association of CKS1B with outcomes was analyzed in NDMM patients, across relapses and with clinical outcome datasets from MGP and Mayo clinic. Inducible shRNAs of CKS1B and bromodomain containing protein 4 (BRD4, a member of the BET [bromodomain and extra terminal domain] family) were generated in MM cell lines. BRD4 and Aiolos ChIP-seq datasets were analyzed for binding on CKS1B gene. BRD4 inhibitors JQ1 and CC-90010 were utilized for inhibition studies in MM cell lines. Results: Higher expression of CKS1B was associated with significantly poorer PFS, OS, disease severity and relapse. Knock-down of CKS1B in MM cells led to a significant decrease in proliferation (P Conclusions: In summary, we have identified CKS1B as a key target associated with poor outcome in MM patients. Translational studies suggest a profound downregulation of CKS1B and key pro-survival effector proteins following combination treatment with BRD4inh and IMiD agents/novel CELMoD agents resulting in synergistic anti-tumor effects. These data provide rationale for testing these agents in the clinic for high-risk and IMiD-relapsed patients. Figure: Changes in cell proliferation and protein levels of key signaling mediators were studied in K12PE cell line treated with increasing doses of Lenalidomide, Pomalidomide and CC-92480 in combination with JQ1. Figure 1 Figure 1. Disclosures Ahsan: BMS: Current Employment, Current equity holder in publicly-traded company. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hsu: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bahlis: Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.
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- 2021
27. Prognostic Impact of NPM1 and FLT3 Mutations at Diagnosis and Presence of Measurable Residual Disease (MRD) after Intensive Chemotherapy (IC) for Patients with Acute Myeloid Leukemia (AML) in Remission: Outcomes from the QUAZAR AML-001 Trial of Oral Azacitidine (Oral-AZA) Maintenance
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Alberto Risueño, Manuel Ugidos, C.L. Beach, Wendy L. See, Hartmut Döhner, Irwindeep Sandhu, Daniel Menezes, Kimmo Porkka, Andrew H. Wei, Barry S. Skikne, Hervé Dombret, Esther Chan, Gail J. Roboz, Pau Montesinos, Felicitas Thol, Anjan Thakurta, and Farhad Ravandi
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Oncology ,0303 health sciences ,medicine.medical_specialty ,NPM1 ,business.industry ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Disease ,Intensive chemotherapy ,Biochemistry ,Oral Azacitidine ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Flt3 mutation ,medicine ,business ,030304 developmental biology ,030215 immunology - Abstract
BACKGROUND: Current guidelines for AML ascribe disease-risk partly based on NPM1 and FLT3 mutational status. NPM1 mutations (mut) occur in 25%-30% of patients (pts) with AML and are associated with favorable prognosis in the absence of co-occurring FLT3-ITD. FLT3-ITD alterations are observed in ~15-30% of AML pts and confer poor prognosis, whereas the prognostic implication of FLT3-TKD point mutations (~7% of pts) is less clear. Post-IC, absence of MRD is associated with favorable relapse-free and overall survival (RFS/OS). In the randomized, phase 3 QUAZAR AML-001 trial, Oral-AZA (CC-486) significantly prolonged OS and RFS vs placebo (PBO) in older pts with AML in first remission after IC (Wei, NEJM 2020). It is of high interest to understand the effects of Oral-AZA in pts with NPM1 and/or FLT3 mutations, and whether their outcomes are influenced by post-IC MRD status. OBJECTIVE: Evaluate survival outcomes with Oral-AZA vs PBO in pts with NPM1mut ± FLT3mut at AML diagnosis (Dx), and OS by baseline (BL) MRD status (+/-) in pts with NPM1/FLT3 mutations. METHODS: In QUAZAR AML-001, pts aged ≥55 years with AML and NCCN intermediate or poor-risk cytogenetics at Dx were randomized 1:1 to receive Oral-AZA 300 mg or PBO QD within 4 mo after attaining first CR/CRi with IC (induction ± consolidation). NPM1 and FLT3 statuses (mut or wild-type [wt]) at AML Dx (before IC) were collected from pt diagnostic case report forms. MRD analyses were conducted by MFC (≥0.1% MRD+ cutoff) in bone marrow aspirate samples collected at screening (post-IC; ie, BL). OS and RFS were estimated from the time of randomization using Kaplan-Meier methods. Multivariate (MV) Cox regression analyses of the prognostic effects on OS/RFS were performed, with NPM1 and FLT3 mutational status and cytogenetic risk at Dx; post-IC MRD status (+/-) at BL, and randomized Tx (Oral AZA vs PBO) as variables. RESULTS: Of 472 pts enrolled , 469 (99.4%) had mutational data available at Dx, and the MRD-evaluable cohort comprised 463 pts (98.1%). In all, 137 pts (29%; Oral-AZA n = 66, PBO n = 71) had NPM1mut at AML Dx, and NPM1mut was significantly correlated with MRD- status at BL (post-IC)(P = 0.0178). Among pts with NPM1mut, OS was significantly improved in pts receiving Oral-AZA vs PBO, whether pts were MRD- (median 48.6 vs 26.2 mo, respectively) or MRD+ (median 39.4 vs 10.3 mo) at BL (both P < 0.0001) (Figure). While median OS for NPM1mut pts in the Oral-AZA arm was nominally improved for MRD- pts vs. those MRD+ (48.6 vs. 39.4 mo, respectively), median OS for NPM1mut pts in the PBO arm was substantially influenced by post-IC MRD status (26.2 vs 10.3 mo for MRD- and MRD+ pts, respectively) (Figure). Similarly, median RFS for pts with NPM1mut/MRD- in the Oral-AZA and PBO arms was 24.9 vs 9.9 mo, respectively, and for pts with NPM1mut/MRD+ was 19.4 vs 4.6 mo. In all, 66 pts (14.1%) had FLT3-ITD (n = 46) and/or FLT3-TKD mut (n = 24) at AML Dx; NPM1 and FLT3-ITD status was NPM1mut + FLT3-ITD - in 107 pts, NPM1mut + FLT3-ITD + in 30 pts, and NPM1wt + FLT3-ITD + in 16 pts. In the Oral-AZA arm, median OS in pts with FLT3mut was not meaningfully different from that in pts with FLT3wt (28.2 and 24.7 mo, respectively), but FLT3mut conferred a negative prognosis in the PBO arm (median OS 9.7 mo, vs 15.2 mo for FLT3wt pts). Risk of death was reduced 46% with Oral-AZA vs PBO in pts with FLT3mut (HR 0.54 [95%CI 0.25, 1.14]). When considering MRD status, median OS in FLT3mut/MRD- pts was 28.2 vs. 15.9 mo in the Oral-AZA (n = 14) and PBO (n = 17) arms, respectively, and was 24.0 vs 8.0 mo in FLT3mut/MRD+ pts (Oral-AZA, n = 16; PBO, n = 18). In MV analyses, Oral-AZA significantly improved OS vs PBO when adjusted for other variables (P = 0.035); NPM1 status (P = 0.001), FLT3status (P = 0.035), and cytogenetic risk at Dx (P < 0.001) were each also significantly predictive of OS, as was post-IC MRD status (P < 0.001). All except FLT3 status (P = 0.737) were significantly predictive of RFS. CONCLUSIONS: Oral-AZA prolonged OS and RFS vs PBO in pts with NPM1mut, with improvements beyond the prognostic benefit conferred by MRD-, suggesting that pts with NPM1mut and MRD- can attain substantial OS benefit with Oral-AZA maintenance. An OS benefit was also observed with Oral-AZA vs PBO in pts in FLT3mut at Dx, but outcomes may be confounded by co-occurring NPM1mut, so further investigation is needed. MV analyses confirmed the independent prognostic influence of Oral-AZA, NPM1 and FLT3 mutations at Dx, cytogenetic risk at Dx, and post-IC MRD status on OS. Figure 1 Figure 1. Disclosures Döhner: Janssen: Honoraria; Helsinn: Honoraria; Gilead: Honoraria; GEMoaB: Honoraria; Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Berlin-Chemie: Honoraria; AstraZeneca: Honoraria; Astex Pharmaceuticals: Honoraria; Astellas: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Oxford Biomedica: Honoraria; Pfizer: Research Funding; Roche: Honoraria. Wei: Astellas: Honoraria; Novartis, Celgene, AbbVie, Servier, AstraZeneca, and Amgen: Research Funding; Novartis, Janssen, Amgen, Roche, Pfizer, Abbvie, Servier, BMS, Macrogenics, Agios, Gilead: Membership on an entity's Board of Directors or advisory committees. Roboz: Glaxo SmithKline: Consultancy; Helsinn: Consultancy; AbbVie: Consultancy; Jasper Therapeutics: Consultancy; Bayer: Consultancy; Novartis: Consultancy; Actinium: Consultancy; Agios: Consultancy; Blueprint Medicines: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Jazz: Consultancy; Daiichi Sankyo: Consultancy; Astex: Consultancy; Mesoblast: Consultancy; Amgen: Consultancy; MEI Pharma - IDMC Chair: Consultancy; Bristol Myers Squibb: Consultancy; Janssen: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Janssen: Research Funding; Pfizer: Consultancy; Roche/Genentech: Consultancy. Montesinos: Agios: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero Pharmaceutical: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Forma Therapeutics: Consultancy; Glycomimetics: Consultancy; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Stemline/Menarini: Consultancy. Thol: Abbvie: Honoraria; Astellas: Honoraria; BMS/Celgene: Honoraria, Research Funding; Jazz: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Ravandi: AstraZeneca: Honoraria; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria; Astex: Honoraria, Research Funding; Prelude: Research Funding; Taiho: Honoraria, Research Funding. Dombret: Amgen: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Honoraria, Research Funding; Servier: Research Funding; Abbvie: Honoraria; BMS-Celgene: Honoraria; Daiichi Sankyo: Honoraria. Sandhu: Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Bioverativ: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy; Gilead: Consultancy. Skikne: Bristol Myers Squibb: Current Employment. See: Bristol Myers Squibb: Current Employment. Ugidos: Bristol Myers Squibb: Current Employment. Risueño: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Chan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Beach: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopes de Menezes: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.
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- 2021
28. A Clinically Validated Targeted Capture Panel to Identify Translocations, Copy Number Abnormalities, and Mutations in Multiple Myeloma
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Parvathi Sudha, Evelyn Fitzsimons, Erin Flynt, Naser Ansari-Pour, Mohammad H Kazeroun, Gareth J. Morgan, Timothy Cody Ashby, Patrick Blaney, Karthik Ramasamy, Tasneem Kausar, Outi Salminen, Magdalena Czader, Kwee Yong, Rafat Abonour, Anjan Thakurta, Lin Wang, Aarif Ahsan, Mirian Angulo Salazar, Mohammad Abu Zaid, Akhil Khera, Jon Williams, Sarah Gooding, Frits van Rhee, Gail H. Vance, and Brian A Walker
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business.industry ,Immunology ,Cancer research ,Medicine ,Chromosomal translocation ,Cell Biology ,Hematology ,business ,medicine.disease ,Biochemistry ,health care economics and organizations ,Multiple myeloma - Abstract
Introduction: Multiple myeloma (MM) is a genetically heterogeneous disease where risk stratification and outcomes are associated with translocations involving the immunoglobulin (Ig) loci and MYC, copy number abnormalities including gain(1q), del(1p), and del(17p), as well as mutations. Additionally, MM tumors may harbor rare mutations in genes that are targetable in other tumors, such as in IDH1 and IDH2. Therefore, we designed a comprehensive MM targeted sequencing panel to interrogate the common genomic abnormalities in MM and validated it against known standards. Methods: The targeted panel was designed to include the exons of 228 genes which are either frequently mutated, associated with prognosis or risk stratification, clinically actionable, or sites of important copy number abnormalities. Additional targets were added across the genome to identify hyperdiploidy. These targeted regions encompass the mutation detection part of the panel and involve approximately 990 kb. The Ig loci and region surrounding MYC were tiled to capture translocations and copy number changes. In total, this translocation part of the panel involves approximately 4.7 Mb. The mutation and translocation panels are manufactured separately and combined during the assay resulting in a 5:1 sequencing ratio, respectively, which prevents over-sequencing of the large translocation panel. 100 ng DNA extracted from CD138+ bone marrow cells (n=223) and from non-tumor tissue (peripheral blood or saliva) was processed using the HyperCap workflow (KAPA Biosystems). Of the 223, 48 samples were processed in a clinical diagnostic laboratory. Adapter ligated DNA was hybridized with a mixture of the mutation and translocation panel and purified, amplified libraries were sequenced using 75 bp paired end reads. Sequences were aligned to hg19 and mutations and translocations identified using Strelka and Manta. Copy number was determined using the ratio of non-tumor to tumor reads in each targeted region. Data were validated using clinical FISH (translocations, n=116), MLPA (copy number, n=101), known standards (mutations), ddPCR (mutations), and whole genome sequencing (WGS; translocations and copy number, n=122). Results: Canonical IgH translocations were detected in 43.2% of patients by the panel, and all agreed with WGS. FISH detected one additional "variant" t(4;14), but did not detect 4 translocations detected by both sequencing methods. In the remainder of the samples no canonical IgH translocation was detected, agreeing with FISH results. Non-canonical translocations were detected in 14.5% of samples, 43% of which were to the MYC locus. MYC translocations were detected in 37.3% of samples with copy number abnormalities occurring surrounding MYC in 32.7% of samples. Overall, MYC abnormalities were detected in 46.4% of samples. Copy number was determined by panel sequencing and MLPA for 22 regions that were directly comparable between the technologies in 101 patient samples and 13 myeloma cell lines. The copy number concordance between the technologies was 96.9% and 99.6% in patient samples and cell lines, respectively. For the important prognostic regions, the concordance was R 2=0.962 (CDKN2C), R 2=0.986 (CKS1B), and R 2=0.973 (TP53). Panel copy number data were also compared to WGS data and showed complete concordance across the three prognostic regions, which the exception of 2 samples. In these 2 samples a homozygous deletion was detected by the panel but not by WGS. The deletions were 6.2 and 8.0 kb in size, one encompassing the coding sequencing of TP53 and the other exons 1-4 of TP53. A larger homozygous deletion of 36.3 kb was detected by both sequencing methods. Mutation detection validation was performed using Horizon Discovery samples with known variant allele frequencies (VAF) for common mutations. We were able to determine the sequencing VAF for 74 mutations across 5 samples which had a concordance of R 2=0.9849 between the expected and observed frequencies. The minimum detected VAF was 1.3% at an average depth of 891x. We also performed ddPCR on 6 patient samples with the common KRAS, NRAS and BRAF mutations which resulted in a VAF concordance of R 2=0.9983. Conclusion: We have developed a targeted sequencing panel for MM patient samples that is as robust or better than both FISH and WGS. A full protocol for sample processing and analysis is available, and has been used in a clinical diagnostic laboratory. Disclosures Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abu Zaid: Pieris: Current equity holder in publicly-traded company; Incyte: Research Funding; Pharamcyclic: Research Funding; Syndax: Consultancy, Research Funding. Ramasamy: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Celgene (BMS): Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yong: GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Sanofi: Honoraria, Research Funding; Takeda: Honoraria; Autolus: Research Funding; Janssen: Honoraria, Research Funding. Morgan: Takeda: Honoraria. Abonour: Celgene-BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Jensen: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Flynt: Bristol Myers Squibb: Current Employment. Ansari-Pour: Bristol Myers Squibb: Consultancy. Gooding: Bristol Myers Squibb: Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.
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- 2021
29. CC-92480 Enhances Cell-Autonomous Cytotoxicity through Blockade of G 2/M Transition When Combined with Bortezomib/Dexamethasone in Pre-Clinical Multiple Myeloma
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Michael Pourdehnad, Anjan Thakurta, Patrick Hagner, Michael Amatangelo, Hsiling Chiu, Chad C Bjorklund, Archana Mukhopadhyay, Krista Wollerman, and Jian Kang
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Transition (genetics) ,Chemistry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Blockade ,Cell autonomous ,Cancer research ,medicine ,Cytotoxicity ,Bortezomib/dexamethasone ,Multiple myeloma - Abstract
Background: Pomalidomide (POM) is an established agent in relapsed/refractory (R/R) multiple myeloma (MM). CC-92480, a novel cereblon E3 ligase modulator (CELMoD ®) agent, is being investigated in R/R MM patients in combination with the proteasome inhibitor (PI) bortezomib (BTZ) and steroid dexamethasone (DEX) (NCT03374085/NCT03989414). Previously, we showed mechanistic synergy of POM/BTZ/DEX in MM cell line models (Bjorklund et al). Here we analyzed the cell autonomous cytotoxic activities of CC-92480 or POM alone and in combination with BTZ/DEX to compare and differentiate their mechanisms of action (MOA). Results: Comparative analysis of the anti-proliferative activity against H929 and MM1.S cell lines revealed that CC-92480 demonstrated a more potent inhibition of proliferation by 100-fold lower dose compared to POM. Combination experiments utilizing a titration of POM or CC-92480 in combination with a 1 hr BTZ pulse, to mimic the clinical pharmacokinetics (+/- DEX co-treatment) showed an enhancement of antiproliferative capacity in both doublet and triplet combinations compared to single agents. Combination indices for POM/BTZ/DEX or CC-92480/BTZ/DEX resulted in values 90% for each triplet compared to POM (20%) and CC-92480 (40%). Flow cytometric analysis of Aiolos and Ikaros protein level in MM cells treated with POM/BTZ/DEX or CC-92480/BTZ/DEX resulted in a slight kinetic delay in substrate depletion at early time points (1-4 hr), where the effect is less apparent with CC-92480, and indistinguishable at 24 hr compared to single agent POM or CC-92480 in the clinically relevant concentrations. We performed transcriptomic analyses of H929 cells treated with POM/BTZ/DEX or CC-92480/BTZ/DEX for 24 hrs to identify key pathways responsible for the observed synergistic combination effect. Common pathways dysregulated by POM or CC-92480 included previously identified interferon, protein homeostasis and proliferation gene sets. Gene set enrichment analysis (GSEA) showed many significant pathway differences when comparing the triplets, including general cell cycle progression, cell division and chromatin segregation. Interestingly, genes involved in negative regulation of G 2/M transition were identified as one of the most significant differences between POM/BTZ/DEX and CC-92480/BTZ/DEX. To understand how these pathways contributed to cell cycle effects and apoptosis, we assessed DNA fragmentation by TUNEL in conjunction with cell cycle flow cytometry to examine cell cycle specific apoptotic induction. Temporal assessment (6, 12, 18, 24, and 48 hr treatments) demonstrated accumulation of BrdU incorporation in all cell cycle phases when treated with POM/BTZ/DEX or CC-92480/BTZ/DEX indicating cell death was occurring within all phases. However, there was a marked enhancement of G 2/M BrdU incorporation (80% vs. 40% of G 2/M population) at 18-24 hr when treated with CC-92480/BTZ/DEX compared to POM/BTZ/DEX, or other single agent treatments. Additionally, G 2/M transition-dependent cyclins A and B were shown to be dysregulated by CC-92480. These data indicate that CC-92480 potentiates a G 2/M arrest in combination with BTZ in MM cells. Conclusions: These results demonstrate that CC-92480 alone or in combination with BTZ/DEX elicits a more potent cytotoxic effect on MM cells compared to POM. Importantly, the combination of either POM or CC-92480 with a PI, like BTZ, does not appreciatively affect single agent MOA. We have also identified a key differentiating mechanism of cell autonomous activity for CC-92480 in combination with BTZ/DEX where MM cells enhance apoptotic induction at the G 2/M stage compared to POM. Clinically, this added mechanistic difference suggests a more cytotoxic response in patients treated with CC-92480/BTZ/DEX compared to POM/BTZ/DEX. Disclosures Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kang: BMS: Current equity holder in publicly-traded company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Hagner: BMS: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.
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- 2021
30. Large-Scale Mass Cytometry Reveals Significant Activation of Innate and Adaptive Immunity in Bone Marrow Tumor Microenvironment of Iberdomide-Treated Myeloma Patients
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William E. Pierceall, Geoffrey Kelly, Sarah Gooding, Samir Parekh, Oliver Van Oekelen, Udo Oppermann, Manman Guo, Sundar Jagannath, Alessandro Laganà, Seunghee Kim-Schulze, Bhaskar Upadhyaya, Michael Amatangelo, Anjan Thakurta, and Manishkumar Patel
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Tumor microenvironment ,medicine.anatomical_structure ,Scale (ratio) ,Immunology ,Cancer research ,medicine ,Mass cytometry ,Cell Biology ,Hematology ,Bone marrow ,Biology ,Acquired immune system ,Biochemistry - Abstract
Background: Iberdomide (IBER) is a potent cereblon E3 ligase modulator (CELMoD agent) with direct anti-tumor and immunomodulatory activities in multiple myeloma (MM). An ongoing phase 1b/2a multicenter, open-label, dose-escalation study has reported favorable efficacy and safety of IBER plus low-dose dexamethasone (DEX) in heavily pretreated patients with relapsed/refractory MM (RRMM). While previous studies focused on IMiD/CELMoD agent immune effects in peripheral blood (Amatangelo, ASH 2019), a large-scale characterization of pharmacodynamics in the bone marrow (BM) tumor microenvironment (TME) has not been reported. Here, we present data on BM aspirates (BMAs) of 99 individuals pre-/post-treatment with IBER in the largest study of TME immune dynamics of RRMM patients to date. Methods: A mass cytometry (CyTOF) panel was designed/validated to capture deep and comprehensive immunophenotyping of T, B, and NK cell subpopulations. In all, staining with 37 metal-tagged Abs characterized 110 supervised cell phenotypes. Paired longitudinal assessment was conducted for viably preserved BMAs of IBER±DEX treated patients in dose-escalation of the CC220-MM-001 Ph 1b/2a study pre (SCREEN) and post treatment (cycle 2 day 15, C2D15). For a subset of patients (n=12), samples were available at disease progression (PD) . A training set analysis (n=64, data reported here) collected in North America, was validated against an independent test set (n=35) recruited in Europe. An R-based computational workflow and manual hierarchical gating identified subpopulations of B, T, NK and myeloid cells. In total, more than 5M immune cells were captured (approx. 40K per specimen). Cell populations are expressed as percentage of non-tumor bone marrow mononuclear cells (BMMC, i.e. CD45+CD66b-). P values are by Mann-Whitney U test for unpaired and Wilcoxon test for paired analyses. Results: IBER treatment resulted in profound immune shifts in the TME. B cells decreased (median SCREEN 3.3% vs C2D15 0.7%, p=0.001), with significant reduction of naïve (p=0.001) and regulatory B cells (p NK cells increased (median 4.2% vs 8.7%, p=0.003), with increase of both CD56hi cytokine-producing (median 1.9% vs 4.5%, p Conclusion: Our analysis on a large cohort of RRMM patients provides unique insights into the heterogeneity of the immune TME of heavily pretreated MM patients and how it changes upon treatment with IBER±DEX. We found significant increases of effector T and NK cells in paired analysis, demonstrating innate and adaptive immune enhancement in the MM bone marrow niche as an important mechanism of action. Importantly, these changes were observed throughout dose escalation and in patients previously refractory to lenalidomide/pomalidomide. The presented platform of large-scale immune profiling demonstrates a strategy to design and study rational combinations with (other) immune-enhancing therapies in MM. Figure 1 Figure 1. Disclosures Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gooding: Bristol Myers Squibb: Research Funding. Jagannath: Legend Biotech: Consultancy; Bristol Myers Squibb: Consultancy; Karyopharm Therapeutics: Consultancy; Janssen Pharmaceuticals: Consultancy; Takeda: Consultancy; Sanofi: Consultancy. Pierceall: BMS: Current Employment, Current equity holder in publicly-traded company. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.
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- 2021
31. Repressed Chromatin Drives Leukaemogenesis in Mutant IDH2 Acute Myeloid Leukaemia Via Inhibition of Granulocyte Differentiation and Cell Cycle Progression
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Chun-Xiao Song, Douglas Ra Silveira, Anjan Thakurta, Marlen Metzner, Virginie Penard-Lacronique, Anna Corby, Prodromos Chatzikyriakou, B. L. Brown, Maroof Hasan, Alastair L. Smith, Roan Hulks, Lynn Quek, Sarah Mackie, Olena Yavorska, Paulina Siejka-Zielińska, Skirmantas Kriaucionis, Paresh Vyas, and Thomas A. Milne
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Immunology ,Cell cycle progression ,Mutant ,Cancer research ,Cell Biology ,Hematology ,Myeloid leukaemia ,Biology ,Biochemistry ,Granulocyte differentiation ,IDH2 ,Chromatin - Abstract
Differentiation arrest in acute myeloid leukaemia (AML) results in accumulation of leukaemic progenitors (L-Prog) and bone marrow failure. Mutant isocitrate dehydrogenase enzyme produces d-2-hydroxyglutarate (2HG), which inhibits α-ketoglutarate-dependent dioxygenases, including Jumonji histone demethylases (JKDM) and TET2, but how this causes AML is unclear. Inhibitors of mutant IDH enzyme (mIDHi) restore differentiation in IDH-mutant (mIDH) AML (Amatangelo et al., 2018). Here, we studied transcriptional networks involved using single-cell (SC) gene expression (GEX) and transcription factor (TF) motif accessibility in primary AML treated with the mIDH2 inhibitor enasidenib (ENA) and found that ENA activates cell cycle (CC) and pro-differentiation programmes through increased promoter accessibility of granulocyte-monocyte (GM)-TF targets. We treated patient L-Prog in vitro with ENA or vehicle, and performed SC RNA-seq (Chromium 10x) in 4 responsive (R), and one non-responsive (NR) patient samples in early, mid and late timepoints. GEX signatures were used to annotate cells according to function (undifferentiated [U], early and late GM [EGM and LGM]) and CC states. In R samples, ENA yielded more dividing late-GM at mid-late timepoints than DMSO (18% vs 6.5%), and more terminally differentiated neutrophils at late timepoints (46% vs 16%). Using SCENIC (Aibar et al., 2017) to assign highly differentially-expressed genes to TF motifs, we computed regulatory networks (regulons, 'R'). Expression of the SP1 R was strongly correlated with active proliferation and ENA conditions led to generation of more cells that co-expressed CEBPA R or CEBPE R with SP1 R, emphasising simultaneous engagement of CC and GM programmes. SP1 function is associated with CC and GM differentiation, and silencing of its binding to its targets contributes to AML pathogenesis (Maiques-Diaz et al., 2012). Control and NR samples failed to produce neutrophils, had reduced co-expression of CEBPE/SP1 R and yielded more poorly differentiated cells expressing GATA2 R. At the individual gene level, ENA stimulated downregulation of GATA2, GFI1B, IKZF1/2, and RUNX3 together with upregulation of immediate early genes which respond to cytokine and mitogenic stimuli (EGR1, IER2, AP-1) in early-mid phase. Later there is upregulation of CEBP TFs and effector genes FUT4, ELANE, AZU1 and PRTN3. Interestingly, expression of some GM-TFs (RUNX1, SPI1/PU.1, GFI1) was similar between ENA and DMSO, indicating that gene expression alone was insufficient for GM differentiation. Given the effects of 2-HG on JKDM, we assessed chromatin accessibility and TF binding using SC ATAC-seq. Overall, we had 25% of differentially accessible (DA) peaks, from which 75% were more accessible in ENA than in DMSO. ENA DA peaks were highly enriched in promoters. Using ArchR (Granja et al., 2021), we clustered cells and used ELANE expression levels to compute trajectories in parallel with SC RNA-seq data. ENA peaks were sequentially enriched for CBF/RUNX and GATA families, followed by AP-1 (JUN/FOS) and EGR/CEBP/KLF motifs. Footprinting analysis showed sequential decrease and increase of TF binding for GATA2 and CEBPA/E respectively during ENA-induced differentiation. Although it did not cause higher expression of SPI1/PU.1, ENA induced increased accessibility of its target binding sites at promoters, which included CEBPA/E and GM effectors (MPO, FUT4, PRTN3). This provides a novel mechanism by which ENA induces differentiation of L-prog. Regulatory network analysis around active, differentially expressed TFs at different phases of ENA-induced differentiation showed a switch from a repressive transcriptional landscape driven by stem-progenitor TFs, to one where AP-1 and GM-TFs activate expression of GM-effector genes. We postulate a model where MYC, E2F8 and EGR1 upregulate the CEBP family in early-mid differentiation. In addition to stimulation of promoter accessibility of TFBS, we find that ENA increases accessibility of cis-regulatory elements of CEBP TFs, adding another mechanism by which differentiation of L-Prog occurs. Our data on the mechanism of action of ENA suggest that differentiation arrest in IDHm AML involves suppression of CC and GM differentiation programs in a repressive chromatin landscape, likely via inhibition of KDM6A and demethylation of repressive H3K27me3 marks. Disclosures Silveira: Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Servier/Agios: Research Funding; BMS/Celgene: Research Funding. Hasan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Vyas: Gilead: Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding.
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- 2021
32. Loss of COP9 Signalosome Gene-Containing 2q Region Is Associated with Lenalidomide and Pomalidomide Resistance in Myeloma Patients
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Fadi Towfic, Kubra Karagoz, Naser Ansari-Pour, Erin Flynt, Sarah Gooding, William E. Pierceall, Evelyn Fitzsimons, Anjan Thakurta, Mohammad H Kazeroun, Kwee Yong, Mirian Angulo Salazar, Maria Ortiz Estevez, and Paresh Vyas
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business.industry ,Immunology ,Cancer research ,Medicine ,Cell Biology ,Hematology ,COP9 signalosome ,business ,Pomalidomide ,Biochemistry ,Gene ,medicine.drug ,Lenalidomide - Abstract
Introduction Identification of the causes of, and biomarkers for, drug resistance in myeloma is important for understanding treatment failures, and for future instigation of targeted therapeutics for myeloma. Using the largest set of whole genome sequencing (WGS) of advanced and drug resistant multiple myelomas to date, we reported that even heterozygous loss of the 3p region, which harbours immunomodulatory drug (IMiD) and CRBN E3 ligase modulator drug (CELMoD)-binding protein Cereblon (CRBN), undergoes strong therapeutic selection on lenalidomide (LEN) and/or pomalidomide (POM) treatment (Gooding et al 2021, PMC7893409). We hypothesized that copy loss of other genes required for IMiD activity may also have clinical relevance. Several groups have reported pharmacogenetic screens identifying genes essential for IMiD sensitivity in vitro, particularly genes required for the maintenance of the CUL4-DDB1-CRBN E3 Ubiquitin Ligase, such as members of the COP9 signalosome complex, function of which prevents CRBN protein degradation. However, loss of these genes has hitherto not been reported in myeloma. Methods and results We identified candidate genes whose loss may favor IMiD drug resistance from published pharmacogenetic screens (n=5), and shortlisted genes consistently identified as essential for LEN or POM function in ≥2 screens (n=23). In our WGS dataset of 455 patients (cohorts: newly diagnosed (ND) n = 198, LEN-refractory n = 203; and LEN-then-POM-refractory n = 54), the incidence of mutation of shortlisted LEN/POM-essential genes in drug-refractory cohorts was rare (10% copy loss at the LEN-then-POM-refractory state, plus incidence of copy loss that increased from ND to LEN-then-POM-refractory states by ≥1.5-fold. This delivered 3 copy loss regions for further investigation: a) 3p, which we had already reported; b) 17p, loss of which is known to be strongly selected in myeloma as the site of TP53; and c) 2q, previously unidentified as relevant in myeloma, but whose minimal common region contained two members of the COP9 signalosome (COPS7B, COPS8). Proportion of loss of this region increased between ND (5.5%), LEN-refractory (9.8%) and LEN-then-POM-refractory states (16.6%), p=0.009. Those patients who had lost a copy of these genes also demonstrated a significant reduction in COPS7B/COPS8 gene expression (p In a separate cohort of myeloma patients (n=24) with sequential sample WGS analysis before and after LEN and/or POM resistance acquisition, we traced acquisition of CNA-defined subclones. 5/24 (21%) patients had acquired either clonal or subclonal loss of the 2q region containing COPS7B and COPS8 at IMID resistance, which had been either absent or below limit of detection pre-IMiD exposure. No other CNA newly-emerged in such a high proportion during IMiD treatment. Relative decrease in even one COP9 signalosome gene has been shown to cause CRBN protein level to fall, and reduce LEN efficacy (Sievers et al 2018, PMC6148446). We are now analysing CRBN protein levels in sequential biopsies from these cases. Conclusion Copy number aberrations have not previously been shown to drive a therapy-specific clonal advantage in myeloma in the clinic. We have now identified a second novel CNA, 2q loss, which increases in incidence through LEN- and POM-refractory states to emerge as a marker of dominant clones in advanced, IMiD-resistant disease. Whether these CNAs will mark resistance to novel CELMoDs remains to be seen. The CRBN protein is key to the function of these drugs, and many novel proteolysis targeting chimeras (PROTACs) in development, but whether the kinetics of their CRBN binding are as sensitive to relative CRBN protein loss remains a key question. CNAs may be easily and cost-effectively detected in the clinic by targeted sequencing approaches, and may prove valuable in future therapeutic decision making. Disclosures Gooding: Bristol Myers Squibb: Research Funding. Ansari-Pour: Bristol Myers Squibb: Consultancy. Karagoz: h.: Research Funding. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Pierceall: BMS: Current Employment, Current equity holder in publicly-traded company. Yong: Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding. Vyas: Astellas: Consultancy, Honoraria; Takeda: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Gilead: Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.
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- 2021
33. Pre-Clinical and Clinical Immunomodulatory Effects of Pomalidomide or CC-92480 in Combination with Bortezomib in Multiple Myeloma
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Patrick Hagner, Tiziana Civardi, Jessica Katz, Nizar J. Bahlis, Jian Kang, Michael Pourdehnad, Hsiling Chiu, Paulo Maciag, Michael Amatangelo, Paul G. Richardson, Anjan Thakurta, and Chad C Bjorklund
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Oncology ,medicine.medical_specialty ,business.industry ,Bortezomib ,Immunology ,Cell Biology ,Hematology ,Pomalidomide ,medicine.disease ,Biochemistry ,Internal medicine ,medicine ,business ,Multiple myeloma ,medicine.drug - Abstract
Background: Pomalidomide (POM) is an established agent in relapsed/refractory (R/R) multiple myeloma (MM) with direct cytotoxicity against MM cells and immunostimulatory activities in multiple cell types including T cells and NK cells. CC-92480 is a novel Aiolos/Ikaros degrading cereblon E3 ligase modulator (CELMoD ®) agent is currently being investigated in combination with the proteasome inhibitor (PI) bortezomib (BTZ) and corticosteroid dexamethasone (DEX), or with DEX only in R/R MM (CC-92480-MM-002 and CC-92480-MM-001). Previous results indicate that triplet combination of POM/BTZ/DEX may enhance some T, B and NK cell subpopulations, overcoming immunosuppression when compared to BTZ/DEX-only treated patients (Rao et al, 2019). Mechanisms of action (MOA) of CC-92480- and POM-mediated substrate depletion occurs via ubiquitination and proteasome degradation, where BTZ has been speculated as potentially antagonistic as a PI. Here, we report pre-clinical and clinical observations of an immune MOA of CC-92480 or POM in combination with BTZ. Results: To mimic the clinical pharmacokinetics, BTZ was utilized as a high-dose pulse method alone and in combination with POM or CC-92480, followed by flow cytometric measurements of Aiolos and Ikaros protein abundance in healthy donor (HD) T cells. The addition of BTZ modestly delayed CRBN-dependent substrate depletion compared to single agent POM or CC-92480; however, this effect was only apparent at early time points (1-6 hr) where the effect was negligible by 24 hr. To understand the functional implications of BTZ combination, we conducted CD3-stimulated PBMC-mediated cytotoxicity assay against H929 MM target cells in a co-culture model. The efficiency of POM or CC-92480 induced PBMC-mediated killing in a dose dependent manner (~65% increase compared to DMSO) were similar at a 100-fold lower dose range of CC-92480 compared to POM, with the effect not being altered by co-treatment with BTZ. These data were replicated with a POM or CC-92480 treated supernatant stimulation of purified NK cells co-culture, which induced an 80% reduction in target cell viability with the BTZ combination having no negative effects on CELMoD-mediated activity. Cytokine analysis on PBMC supernatants treated with either POM or CC-92480 in the absence or presence of BTZ-pulse showed a dose-dependent increase in IL-2 (>2.4-fold) and Granzyme B (>3.1-fold), which were not impacted by BTZ co-treatment. As a secondary readout on activation status, we measured multiple signaling molecules and activation markers on the cell surface of T and NK cell subsets in CD3 stimulated HD PBMCs treated with dose-dependent POM or CC-92480 with or without co-treatment of BTZ. Compared to DMSO controls, elevated expression levels of CD25 (IL2RA), CD278 (ICOS), Granzyme B, CD134 (OX40R) and HLA-DR were observed with both POM and CC-92480 on CD4, CD8 and NK cells demonstrating a CELMoD-mediated increase in immune activation. These effects were not impacted by the co-treatment of BTZ. Examination of peripheral blood samples from MM patients enrolled in the CC-92480-MM-001/002 (NCT03374085/NCT03989414) clinical trials revealed that CC-92480 promoted potent immunomodulation when administered in combination with DEX and with BTZ/DEX. These data included increased numbers of activated and central memory T cells, as well as increased Ki67+ proliferating T and NK cell populations compared to samples collected during the screening period before any drugs had been administered, consistent with earlier observation of POM in combination with BTZ/DEX treated patients. Conclusions: Taken together, these data demonstrate that POM and CC-92480 are potent immunomodulatory agents with enhanced induction of PBMC and NK mediated cell killing of MM tumor cells and activation of T and NK cells, at 100-fold lower concentrations of CC-92480 compared to POM. Additionally, we showed that combination with BTZ in preclinical assays and in the clinical setting did not antagonistically affect the immunostimulatory ability of POM or CC-92480. Disclosures Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kang: BMS: Current equity holder in publicly-traded company. Civardi: Bristol Myers Squibb: Current Employment. Katz: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Maciag: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hagner: BMS: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Bahlis: Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Richardson: Oncopeptides: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; AbbVie: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.
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- 2021
34. Luspatercept Redistributes Body Iron to the Liver in Transfusion-Dependent-Thalassemia (TDT) during Erythropoietic Response
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Manuel Ugidos, Martin Schwickart, Jeevan K. Shetty, Anjan Thakurta, Maciej W Garbowski, John B. Porter, Sadanand Vodala, Rajasekhar N.V.S. Suragani, and Alberto Risueño
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Body iron ,medicine.medical_specialty ,Endocrinology ,business.industry ,Internal medicine ,Luspatercept ,Immunology ,medicine ,Transfusion dependent thalassemia ,Cell Biology ,Hematology ,business ,Biochemistry - Abstract
Introduction: Luspatercept inhibits select ligands of the TGF-β superfamily implicated in thalassemic erythropoiesis and promotes late-stage erythroid maturation (Suragani RN, et al. Nat Med 2014;20:408-414). This leads to greater red blood cell (RBC) output from thalassemic marrow and reduces transfusion dependence in TDT (Cappellini MD, et al. N Engl J Med 2020;382:1219-1231). The underlying mechanisms for this clinical outcome are not well understood in a syndrome involving significant iron overload and cyclical stimulation of erythropoiesis between transfusions. Here we report novel physiological and clinical insights from a BELIEVE trial (NCT02604433) biomarker analysis, which demonstrate that hepcidin and erythropoietic changes in TDT lead to iron redistribution from macrophages to hepatocytes on luspatercept. Methods: 336 TDT patients ≥ 18 years of age who took part in the BELIEVE study, a multicenter, randomized, double-blind, placebo-controlled phase 3 trial (Cappellini et al. 2020), were randomized 2:1 to receive luspatercept or placebo subcutaneously every 21 days for 48 weeks. This ethically approved study was conducted in accordance with the Declaration of Helsinki. Patients provided written informed consent. Transfusion iron loading rate (ILR) was calculated assuming 1 unit = 200 mg Fe. Liver iron content (LIC) was measured by R2 MRI and R2* MRI, and total body iron stores by Angelucci formula. Serum was assayed for ferritin (SF) nephelometrically (Covance lab), and hepcidin, erythroferrone (ERFE), growth differentiation factor (GDF)15, and transferrin receptor (sTfR1) by ELISA at Intertek (San Diego, CA). Median and interquartile ranges are shown; P value < 0.05 was deemed significant. Results: Within 48 weeks on luspatercept, transfusion iron loading fell by 1.6 g (8 RBC units, ILR difference −0.08 mg Fe/kg/d ± 0.07, range −0.4 to 0.2). Regardless of thalassemic genotype, SF fell by 269.3 ± 963.7 and earliest at 12 weeks by 103.6 ± 690.3 µg/L (both P < 0.0001), with no change on placebo, indicating reduced macrophage iron. However, despite unchanged chelation exposure, no reduction in LIC (5.7 to 6.7 mg/g dw) or calculated body iron stores (3.6 to 4.2 g, not significant) occurred on luspatercept, even though saved iron loading from transfusion was 44% (1.6 g/3.6 g) of baseline body iron stores. On luspatercept, but not placebo, hepcidin fell by 53%, while erythropoietin (EPO), ERFE, GDF15, sTfR1, and reticulocytes rose by 93%, 51%, 59%, 66%, and 112%, respectively (all P < 0.0001). 71% (120/169) patients with and 47% without (17/36) ILR reduction had negative SF trends (P < 0.0001). In patients with SF reduction on luspatercept, bilirubin and LDH rose 50% and 67% (P < 0.0001) indicating increased RBC hemolysis from residual (effective and ineffective) erythropoiesis. ILR change correlated with changes in EPO, hepcidin, sTfR1, and bilirubin, but not with changes in ERFE, GDF15, reticulocytes, or LDH (Figure A). Hepcidin reduction was related to LIC increase post splenectomy (Figure B), suggesting a role for the spleen in preventing hemolytic iron redistribution to the liver. Decrease in SF thus associates with erythroid iron incorporation (sTfR1), hence RBC production that reduces transfusion needs (falling ILR) but enhances hemolytic rerouting of iron to the liver. In a mixed-effect linear regression analysis, transfusion, LIC, baseline SF, time, and treatment predicted SF changes in a benchmark model. We found high baseline LIC lessens the SF outcome, and hepcidin, EPO, and bilirubin jointly explained 66% of the treatment effect of luspatercept on SF. Conclusions: Luspatercept-increased ERFE, likely as a result of increased production or higher frequency of late-stage erythroblasts, partially reduces hepcidin production. Lower hepcidin mobilizes iron stores to facilitate bulk hemoglobinization, erythropoietic response, and transfusion burden reduction. Luspatercept leads to SF reduction that marks hepcidin-dependent iron egress from macrophage compartment to plasma. This relocated iron, variably utilized by thalassemic erythron, refluxes back to plasma for hepatocyte and extrahepatic iron uptake, or as heme iron shunts into the liver through hemolytic pathways from intramarrow ineffective erythropoiesis or peripheral breakdown of newly made thalassemic RBC. Macrophage to hepatocyte iron redistribution in TDT appears to be a mechanism of luspatercept. Figure 1 Figure 1. Disclosures Garbowski: Imara: Consultancy; Vifor: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Ugidos: Bristol Myers Squibb: Current Employment. Risueño: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Suragani: Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Shetty: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Vodala: BMS: Current Employment, Other: stock options. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Schwickart: BMS (Celgene): Current equity holder in publicly-traded company, Ended employment in the past 24 months, Other, Patents & Royalties; Exelixis: Current Employment, Current equity holder in publicly-traded company. Porter: Celgene (BMS): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla Pharmaceuticals: Honoraria; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
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- 2021
35. 16p Deletion Involving BCMA Locus Is Frequent and Predominantly Observed with del17p
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Romain Lannes, Hervé Avet-Loiseau, Anjan Thakurta, Mehmet Kemal Samur, Jill Corre, Kenneth C. Anderson, Anil Aktas-Samur, and Nikhil C. Munshi
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Genetics ,Immunology ,Locus (genetics) ,Cell Biology ,Hematology ,Biology ,Biochemistry - Abstract
New generation immunotherapies in Multiple Myeloma (MM) targeting BCMA, have shown remarkable clinical benefits. However relapse still occurs due to tumor intrinsic and extrisic resistance mechanisms including antigen loss related to mutation, deletion and splicing pattern changes. Two recent case reports including ours highlighted biallelic loss of BCMA as a cause for resistance to anti-BCMA targeting therapy. In both studies BCMA locus at 16p was deleted bringing in focus importance of del16p. Here, we have evaluated 2883 MM patients at diagnosis and relapse to understand frequency characteristics of somatic events targeting BCMA. We first evaluated the frequency of deletion involving the BCMA locus (16p13.13) in MM patients from multiple studies using WGS sequencing data as well as using Affymetrix Cytoscan HD and SNP 6.0 arrays. We observed del16p in 8.58 % (7.6% to 14.6% in individual studies) of newly-diagnosed patients (n=2458). Similar frequency was observed in relapsed MM patients not previously exposed to BCMA targeting therapy. Next, we evaluated genome wide copy number alterations (CNAs) in all patients with loss of BCMA locus and observed similar frequency of loss in both hyperdiploid MM (HMM) and non-HMM suggesting its independence from cytogentic subtypes of MM. Overall copy number loss was significantly higher in patients with BCMA loss compared to rest of the MM patients. Patients with loss of BCMA locus have increased mutational load (8202 with 95% HDI 6921 and 9535) compared to those without BCMA locus loss (6975 with 95% HDI 6626 - 7343); probability of difference greater than 0 was 96.8% and difference of the means were 1222 [95% CI -112 - 2589] We next evaluated co-occurrence of BCMA loss with other high risk events and observed del1p and del17p as being significantly associated with loss of BCMA locus [Odds ratio 19.37 (13.13-25.80), FDR = 1.57e-65; and 8.8 (6.39-12.15), FDR = 5.57E-39, respectively)]. Furthermore, we observed that when both BCMA and TP53 loss are present, they have same log ratio (sequencing) or smoothed copy numbers (SNP array). Similarly, we used CDKN2C as a proxy to chromosome 1p loss and observed that when both BCMA and CKDN2C loss are present in the same patient they tend to show similar copy number values. These data suggested a possibility of co-occurrence of these events in the same cell. To further investigate this observation, we used single cell DNA sequencing data from patients with sub clonal and clonal BCMA locus loss. scDNA sequencing showed that almost all cells with BCMA deletion also had TP53 deletion (95%). Interestingly, almost all cells with BCMA loss also had p53 loss, while not all cells with p53 loss had BCMA loss suggesting that the chronology of this copy number alternation may suggest first p53 loss followed by BCMA loss. We further investigated whether a bi-allelic BCMA loss was observed after anti-BCMA targeted CAR-T cell therapy by imputing the copy number alterations using single cell RNA sequencing data. Our data from this case also indicated that BCMA loss tend to co-occur with TP53 deletions (OR=5.67 [95% CI 4.12-7.84], p value < 0.0001). Moreover, TP53 mutations were also more frequent in patients with del16p and del17p, compared to patients who only had del16p or del17p. In summary, our data from large scale copy number profiles at the diagnosis and relapse showed that monoallelic BCMA deletions are frequent events, patients with these events show increased aneuploidy, mostly deletions, potentially making these cells vulnerable for biallelic loss of genes, especially under the pressure of targeted therapy. Our results also highlight that BCMA expressions in bulk sample may not detect the presence or absence of cells with target loss and therefore combining strategies at bulk and single cell level are necessary to understand the disease status. These results suggest the need to study del16p in patients being targeted for BCMA-directed therapy and its association with other risk factors in MM. Disclosures Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy.
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- 2021
36. CC-486 Mechanism Imparted By Extended Exposure of Azacitidine Upregulates Myeloid Differentiation Markers and Induces Cell Death in AML Cells
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Patrick Hagner, Kyle J. MacBeth, C.L. Beach, Dai Yumin, Anjan Thakurta, Wendy L. See, Danny V Jeyaraju, Diana Ronai Dunshee, Jessica C. Jang, Ignazia La Torre, Daniel Menezes, Xiaomin Wang, Keshava Kumar, Barry S. Skikne, and Alberto Risueño
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Myeloid ,Immune response gene ,Cell cycle checkpoint ,medicine.diagnostic_test ,business.industry ,Cellular differentiation ,Immunology ,Azacitidine ,Cell Biology ,Hematology ,Pharmacology ,Biochemistry ,Flow cytometry ,medicine.anatomical_structure ,Hypomethylating agent ,Apoptosis ,medicine ,business ,medicine.drug - Abstract
BACKGROUND: CC-486, a DNA hypomethylating agent and epigenetic modifier, is an oral formulation of azacitidine (AZA) that is administered at lower exposures for extended durations (300 mg/day [d] for 14 or 21d/28d cycle) compared with the injectable formulation of AZA, which is given in a high exposure, limited duration regimen of 75mg/m2 for 7d/28d cycle. AZA induces DNA damage and cytotoxicity, and promotes changes in gene expression leading to cellular differentiation. As DNA incorporation of AZA is S-phase-dependent, it has been hypothesized that extended dosing with CC-486 prolongs drug exposure and DNA incorporation to enhance epigenetic activity. The mechanism of action imparted by extended dosing schedules of CC-486 is not fully understood. In patients with myeloid malignancies, DNA hypomethylation in blood is sustained throughout the 28d Tx cycle with extended CC-486 dosing regimens (Laille, 2015; Garcia-Manero, 2016). To better understand the mechanism of CC-486, we assessed the kinetics of expression of myeloid markers of cellular differentiation and cytotoxicity with various AZA dosing schedules in in vitro and in vivo models of AML. METHODS: AML cell lines (AML-193, KG1a, and MV4-11) were treated in vitro with AZA (0.05 - 5 µM daily for 5d or 15d), and at cumulative concentrations of 1 or 3 µM administered once or fractionated over 2-5d to experimentally model CC-486 extended exposures: 1 µM cumulative dose (1 µM × 1d, 0.5 µM × 2d, 0.33 µM × 3d, 0.25 µM × 4d, or 0.2 µM × 5d); 3 µM cumulative dose (3 µM × 1d, 1.5 µM × 2d, 1 µM × 3d, 0.75 µM × 4d, or 0.6 µM × 5d). AZA- or vehicle-treated cells were analyzed by flow cytometry, DNA methylation (Illumina Infinium EPIC assay), and RNA-Seq. Temporal expression of CD11b was assessed as a surface marker of myeloid differentiation, and Annexin-V staining was used to determine the extent of apoptosis and cell death. In efficacy studies, mouse models of AML (syngeneic, cell line-derived xenografts) were treated intraperitoneally with AZA regimens at 1 mg/kg/d × 15d (extended) or 3 mg/kg/d x 5d. RESULTS: Tx of AML-193 cells with 0.05 - 5 µM daily AZA led to upregulation of markers of myeloid differentiation (including CD11b) at lower doses, and a dose-dependent increase in apoptosis up to 7d after Tx initiation. Following Tx with 1 µM AZA for 1d, maximal cellular differentiation (ie, CD11b expression) occurred at d3 in 30% of AML-193 cells; conversely, cells treated with 0.2 µM/d AZA for 5d showed greater differentiation (40%) peaking on d7 (Fig A). CD11b expression was increased upon each subsequent cell division; after 5 cell divisions, CD11b upregulation was 4-fold higher in cells treated with multiple, lower AZA doses than with 1 µM AZA administered for 1d (Fig B). CD11b upregulation was not observed in the absence of cell division under serum starvation conditions for 3d (to induce cell cycle arrest), further suggesting that cell division is a requirement for AZA-induced CD11b changes (Fig C). Similarly, AML-193 cells treated with a 3 µM cumulative AZA dose over 1, 2, 3, 4, or 5d showed greater changes in myeloid differentiation marker expression, with peak apoptosis at d7 with extended dosing regimens (Fig D). In KG1a and MV4-11 cells, Tx with 1 µM AZA QD for 5d led to induction of myeloid differentiation by d7, and cell death (followed by recovery of undifferentiated cells) by d28. In contrast, daily Tx with 0.3 µM AZA for 15d led to slower, more robust upregulation of differentiation markers, peaking at d21 and accompanied by a gradual loss of cell viability. Extended AZA exposure to cells led to pronounced changes in gene expression (Fig E) and DNA methylation (Fig F) at both d7 (immune response gene signature) and d28 (cell adhesion gene signature) compared with limited duration exposure AZA. In mice, low exposure, extended regimens of AZA exhibited higher DNA and RNA incorporation into peripheral blood mononuclear cells (PBMCs) and bone marrow cells when compared with higher exposure, limited duration regimens. Extended AZA dosing led to significant efficacy in murine AML models. CONCLUSIONS: In AML cell lines, low exposure, extended duration AZA schedules modeling CC-486 induced robust changes in differentiation. These results suggest that CC-486-mediated effects using extended exposure regimens preferentially promote a differentiation effect and cell death of AML tumor cells. These mechanistic insights may help inform rational CC-486 combination Tx strategies. Disclosures Dunshee: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Genentech Inc.: Current Employment, Current equity holder in publicly-traded company. Dai:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jang:Bristol Myers Squibb: Ended employment in the past 24 months. Risueño:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: Named in BMS (before Celgene) patent filings related to predictive patient response biomarkers in hematological malignancies. Jeyaraju:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hagner:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. See:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Wang:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. La Torre:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Skikne:Bristol Myers Squibb: Current Employment. Beach:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kumar:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopes de Menezes:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.
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- 2020
37. High-Dose Melphalan Significantly Increases Mutational Burden in Multiple Myeloma Cells at Relapse: Results from a Randomized Study in Multiple Myeloma
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Giovanni Parmigiani, Stephane Minvielle, Mehmet Kemal Samur, Raphael Szalat, Hervé Avet-Loiseau, Abdul Hamid Bazarbachi, Anjan Thakurta, Paul G. Richardson, Kenneth C. Anderson, Florence Magrangeas, Mariateresa Fulciniti, Adam S. Sperling, Masood A. Shammas, Philippe Moreau, Aurore Perrot, Anil Aktas-Samur, Nikhil C. Munshi, Marco Roncador, and Jill Corre
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,High dose melphalan ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,law.invention ,Randomized controlled trial ,law ,Internal medicine ,medicine ,business ,Multiple myeloma - Abstract
We recently shown that high-dose melphalan (HDM) followed by autologous stem cell transplant (ASCT) as first line therapy in young ( A significant change in frequency of driver mutations including RAS/RAF, FAM46C, TP53, and DIS3 was not observed at the time of relapse. Clonality level was increased only for KRAS (p=0.054), while all other specific driver genes had similar clonality level at diagnosis and relapse. Interestingly, a significant increase in mutations involving MYO16 and SLC7A8 genes was observed at relapse in both arms, implicating components of the induction regimen (RVD). Investigating the mutational signature utilization in only newly acquired mutations identified 4 signatures: APOBEC, HR Double Strand Repair, clock-like signature, and unknown. k-means clustering analysis of samples based on signature utilization showed four distinct clusters. All patients clustering with high DNA repair signature utilization were in the HDM arm (65% HDM patients), the majority of whom achieved CR or sCR (74%); these patients acquired 8308 (range 3302-19107) new mutations between diagnosis and relapse. None of the RVD only treated patients were in this cluster. The remaining 35% HDM group patients were clustered with RVD samples and showed unknown signature utilization. Furthermore, motif enrichment analysis identified CYWR and ATGAGATV (p < 1e-130) as enriched motifs around the new mutations in HDM compared to RVD cohort. Importantly and as expected, DNA damage repair pathway genes were frequently targeted in the HDM group: 72% HDM samples accumulated DDR gene mutations vs. only 17% in the RVD alone arm (p < 0.001). At the time of relapse, 100% HDM arm patients had at least one DDR gene mutation and 80% had two or more, while only 37% RVD only group had one or more such mutation. Finally, we have reconstructed phylogenetic and evolutionary trajectories based on mutation and copy-number data from samples at diagnosis and relapse. The clonal composition in both arms was similar at diagnosis; however, HDM caused a significant shift to more subclonal mutations at relapse. chromothripsis and chromoplexy events were detected in 30% patients at diagnosis, which remained constant at relapse regardless of treatment. In summary, we describe significant accumulation of mutations following high dose melphalan. This fundamental molecular change in the disease at relapse, suggests the need for reappraisal of the optimal use and sequencing of high dose melphalan in the era of novel agents. Disclosures Fulciniti: NIH: Research Funding. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Perrot:Amgen, BMS/Celgene, Janssen, Sanofi, Takeda: Consultancy, Honoraria, Research Funding. Moreau:Sanofi: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Honoraria; Novartis: Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Parmigiani:Phaeno Biotehnologies: Current equity holder in publicly-traded company; CRA Health: Current equity holder in publicly-traded company; Foundation Medicine Institute: Consultancy; Delphi Diagnostics: Consultancy; BayesMendel Laboratory: Other: Co-lead. Munshi:Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Legend: Consultancy.
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- 2020
38. Preclinical and Translational Data Support Development of Iberdomide in Combination with CD38- and SLAMF7-Directed Monoclonal Antibodies: Evidence for Rational Combinations
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Pieter Sonneveld, Anjan Thakurta, Jeffrey A. Zonder, Peilin Ma, Paula Rodriguez-Otero, Ashraf Z. Badros, April Sorrell, Michael Amatangelo, Sagar Lonial, Sundar Jagannath, Niels W.C.J. van de Donk, Monique C. Minnema, Krista Wollerman, Rakesh Popat, Chad C Bjorklund, Jeremy T. Larsen, William E. Pierceall, Tuong Vi Nguyen, Albert Oriol, and Kevin Hong
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medicine.drug_class ,business.industry ,SLAMF7 ,Immunology ,medicine ,Cancer research ,Cell Biology ,Hematology ,CD38 ,Monoclonal antibody ,business ,Biochemistry ,health care economics and organizations - Abstract
Introduction: Iberdomide (IBER; CC-220) is a potent, novel cereblon (CRBN) E3 ligase modulator (CELMoD) agent under investigation in a phase 1/2 dose-escalation study in relapsed/refractory multiple myeloma (MM) (CC-220-MM-001; NCT02773030). IBER modulates CRBN to induce ubiquitination and proteasome-dependent degradation of Ikaros/Aiolos, resulting in anti-myeloma and immunomodulatory activity. Compared with immunomodulatory drugs (IMiDs), IBER binds to CRBN with 20-fold higher affinity and is more efficient at degrading Ikaros/Aiolos. Additionally, IBER can overcome IMiD drug resistance. Here, we evaluate the preclinical activity of IBER in combination with monoclonal antibodies (mAbs) and assess immune pharmacodynamic changes of IBER + daratumumab (DARA) among patients in the CC-220-MM-001 study. Methods: Preclinical analyses were performed on MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy volunteers. In co-culture assays, cell lines and immune cells were treated with clinically relevant concentrations of lenalidomide (LEN; 1 μM), pomalidomide (POM; 300 nM), and IBER (20 nM) alone, and in combination with DARA or elotuzumab (ELO). Clinical biomarker data were obtained from blood samples of patients enrolled in IBER + dexamethasone (DEX) and IBER + DARA + DEX cohorts of the CC-220-MM-001 study. Immune profiling was evaluated by flow cytometry on Cycle (C) 1 Day (D) 1, C2D15, C4D1, and C4D15. Results: Treatment of CD3-stimulated PBMCs with IBER increased cytokine secretion more potently than with IMiD drugs, promoting natural killer (NK) cell proliferation and increasing NK cell numbers by > 30% in cultures. Treatment of CD3-stimulated PBMCs with IBER also promoted immune-mediated killing of MM cell lines, and pretreatment of MM cell lines for 48 h followed by washout sensitized cells to immune-mediated clearance. IBER treatment of MM cells increased protein expression of CD38, but had no discernable effect on SLAMF7 expression. IBER in combination with DARA or ELO enhanced the immune-mediated killing of MM cell lines and resulted in deeper cell killing compared with LEN or POM when combined with the same mAbs. Analogous to preclinical data, immune profiling of patients treated with IBER + DEX demonstrated an immune-stimulatory effect, including increased proliferating NK and T cells. Notably, patients coming on trial directly after a DARA-containing regimen showed depleted NK cell counts, which rebounded by nearly 2-fold after 1 cycle of IBER + DEX. Additionally, the effects of IBER on T and NK cell proliferation were similar in patients treated with IBER + DEX + DARA and IBER + DEX, except for overall reduction in CD38-expressing NK and T cells in patients receiving DARA. Conclusions: IBER induced immune-stimulatory activity more potently than LEN or POM, enhancing cytokine release and NK cell proliferation. MM cells treated with IBER were also sensitized to immune-mediated clearance. These data suggest that IBER may enhance the activity of biologics that exert anti-malignant activity through antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Accordingly, IBER enhanced the activity of DARA and ELO in co-culture. IBER also induced an immune-stimulatory effect on NK and T cells in vivo, confirming immune-stimulatory effects of IBER in patients with MM. Notably, the effect of IBER on NK cells was more pronounced in patients whose last treatment regimen contained DARA, suggesting IBER treatment may contribute to NK cell recovery after DARA treatment. Furthermore, immune changes observed by the addition of DARA to IBER + DEX were similar, suggesting IBER was the primary agent promoting immunomodulation in this triplet therapy. Together, these data support continued clinical development of IBER in combination with CD38- and SLAMF7-directed mAbs as well as other immune-directed therapies for the treatment of MM. Disclosures Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ma:Bristol Myers Squibb: Current Employment. Wollerman:Bristol Myers Squibb: Current Employment. Pierceall:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lonial:JUNO Therapeutics: Consultancy; Merck: Consultancy, Honoraria, Other: Personal fees; Abbvie: Consultancy; GSK: Consultancy, Honoraria, Other: Personal fees; BMS: Consultancy, Honoraria, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; Takeda: Consultancy, Other: Personal fees, Research Funding; Genentech: Consultancy; Karyopharm: Consultancy; Sanofi: Consultancy; Amgen: Consultancy, Honoraria, Other: Personal fees; Janssen: Consultancy, Honoraria, Other: Personal fees, Research Funding; Onyx: Honoraria; Millennium: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. van de Donk:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ferrer: Membership on an entity's Board of Directors or advisory committees. Popat:Celgene: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding; AbbVie: Consultancy, Honoraria; GSK: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Jagannath:BMS, Janssen, Karyopharm, Legend Biotech, Sanofi, Takeda: Consultancy. Zonder:Intellia: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; BMS: Consultancy, Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Celgene: Research Funding; Prothena: Consultancy; Janssen: Consultancy, Other: Personal fees; Oncopeptide: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Caelum: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees. Minnema:Kite, a Gilead Company: Speakers Bureau; Servier: Consultancy; Amgen: Consultancy; Celgene: Other: travel support, Research Funding. Oriol:Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Larsen:Janssen Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Badros:Amgen: Consultancy; University of Maryland: Current Employment. Rodriguez-Otero:Sanofi: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); GlaxoSmithKline: Consultancy, Current Employment, Current equity holder in publicly-traded company, Honoraria; Medscape: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Kite: Consultancy, Honoraria. Sonneveld:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Skyline Dx: Honoraria, Research Funding; Sanofi: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Nguyen:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hong:Bristol Myers Squibb: Current Employment. Sorrell:Bristol Myers Squibb: Current Employment; Children's Oncology Group: Other: Non-member; Previous Study Chair AAML08B1. Thakurta:Bristol Myers Squibb: Current Employment; Oxford University: Other: Visiting professor.
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- 2020
39. Preclinical and Translational Support for Clinical Development of Iberdomide in Combination with Proteasome Inhibitors: Mechanism of Synergy in Clinical Trial CC-220-MM-001
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Pieter Sonneveld, Paula Rodriguez-Otero, Sundar Jagannath, Jian Kang, Albert Oriol, Anjan Thakurta, William E. Pierceall, Sagar Lonial, Tuong Vi Nguyen, Ashraf Z. Badros, Jeffrey A. Zonder, Rakesh Popat, María Dolores Jiménez Nuñez, Chad C Bjorklund, Jeremy T. Larsen, Lilly Wong, Monique C. Minnema, Michael Amatangelo, April Sorrell, Kevin Hong, Archana Mukhopadhyay, and Niels W.C.J. van de Donk
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Clinical trial ,Proteasome ,business.industry ,Mechanism (biology) ,Immunology ,Cancer research ,Medicine ,Cell Biology ,Hematology ,business ,Biochemistry - Abstract
Introduction: Iberdomide (IBER; CC-220) is a potent, novel cereblon (CRBN) E3 ligase modulator (CELMoD) agent under investigation in a phase 1/2 dose-escalation study for relapsed/refractory multiple myeloma (MM) (CC-220-MM-001; NCT02773030). IBER modulates CRBN to induce ubiquitination and proteasome-dependent degradation of Ikaros/Aiolos, resulting in antimyeloma and immunomodulatory activity. Compared with immunomodulatory drugs (IMiDs); IBER binds to CRBN with 20-fold higher affinity and is more efficient at degrading Ikaros/Aiolos. Additionally, IBER can overcome IMiD drug resistance. Here, we compare the preclinical activity of IBER versus IMiD drugs + proteasome inhibitors (PIs), and assess the pharmacodynamic (PD) activity of IBER in patients treated with IBER + dexamethasone (DEX), IBER + DEX + bortezomib (BORT), and IBER + DEX + carfilzomib (CFZ). Methods: MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were analyzed. Cell lines treated with PIs (BORT and CFZ) were pulsed with drug for 1 h, and then treated with clinically relevant concentrations of lenalidomide (LEN; 1 μM), pomalidomide (POM; 300 nM), or IBER (20 nM). Biomarker data for PD analyses were obtained from blood samples of patients enrolled in the IBER + DEX, IBER + DEX + BORT, and IBER + DEX + CFZ cohorts of the CC-220-MM-001 study. Degradation of Ikaros/Aiolos in peripheral blood was analyzed on Cycle (C) 1 Day (D) 1, pre-dose, and post IBER/IBER + PI dose by flow cytometry. Immune profiling was evaluated by flow cytometry from peripheral blood at C1D1, C2D15, C4D1, and C4D15. Results : Proteasome inhibition in cell lines after 1 h pulse of PI was maintained after washout. However, substrate degradation by IBER was affected only minimally by combination with PIs, and was more potent than by LEN or POM. These results correlate with synergistic antiproliferative activity and more potent tumoricidal activity in cell lines treated with IBER + PI than IMiD + PI combinations. Furthermore, ex vivo treatment of PBMCs with IBER + PIs minimally affected cytokine induction by IBER. Analysis of patient samples from the CC-220-MM-001 study confirmed preclinical observations showing minimal inhibition of IBER PD with the addition of PIs to the treatment regimen. In patient T cells, Ikaros and Aiolos protein levels decreased by > 50% and > 70%, respectively, with IBER + DEX, and by > 30% and > 45% (at lower IBER doses) with IBER + DEX + BORT or IBER + DEX + CFX. In all cohorts, the nadir of substrate expression was determined to be 6 h. After repeated dosing (mid-C1) patients treated with IBER + PIs showed > 70% decreases in both Ikaros and Aiolos expression. Furthermore, immune profiling of patients showed that the addition of PIs to the IBER treatment regimen did not inhibit immune-stimulatory activity of IBER, including induction of NK and T cell proliferation. Conclusions: Preclinically, IBER induced substrate degradation in the presence of PIs, and treatment with IBER + PIs resulted in substantially increased tumoricidal activity. Deeper substrate degradation and increased apoptosis were also observed when PIs were combined with IBER versus IMiD drugs. Clinically, IBER led to rapid decreases in substrate levels, even in the presence of PIs, and levels were reduced further with repeated dosing, suggesting that concurrent administration of PIs minimally affects proteasomal degradation of substrates mediated by IBER. Furthermore, increases in NK and T cell proliferation in IBER-treated patients were consistent whether or not they also received a PI, further confirming that IBER has immune-stimulatory activity in combination with PIs. These data support continued clinical development of IBER in combination with PIs for the treatment of MM. Disclosures Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kang:Bristol Myers Squibb: Current Employment. Mukhopadhyay:Bristol Myers Squibb: Other: Contract scientist, Research & Early Development. Jiménez Nuñez:CITRE, a Bristol-Myers Squibb Company: Current Employment. Wong:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierceall:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lonial:Merck: Consultancy, Honoraria, Other: Personal fees; Genentech: Consultancy; Karyopharm: Consultancy; Sanofi: Consultancy; BMS: Consultancy, Honoraria, Other: Personal fees, Research Funding; Janssen: Consultancy, Honoraria, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; Takeda: Consultancy, Other: Personal fees, Research Funding; Amgen: Consultancy, Honoraria, Other: Personal fees; GSK: Consultancy, Honoraria, Other: Personal fees; Abbvie: Consultancy; Millennium: Consultancy, Honoraria; JUNO Therapeutics: Consultancy; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria. van de Donk:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Popat:GSK: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Jagannath:BMS, Janssen, Karyopharm, Legend Biotech, Sanofi, Takeda: Consultancy. Zonder:Caelum: Consultancy; Intellia: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Alnylam: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Oncopeptide: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Celgene: Research Funding; Janssen: Consultancy, Other: Personal fees; Prothena: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; BMS: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees. Minnema:Celgene: Other: travel support, Research Funding; Servier: Consultancy; Amgen: Consultancy; Kite, a Gilead Company: Speakers Bureau. Oriol:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Amgen: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Larsen:Janssen Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Badros:Amgen: Consultancy; University of Maryland: Current Employment. Rodriguez-Otero:Sanofi: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Current Employment, Current equity holder in publicly-traded company, Honoraria; Amgen: Consultancy, Honoraria; Medscape: Membership on an entity's Board of Directors or advisory committees; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Oncopeptides: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Sonneveld:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Skyline Dx: Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding. Nguyen:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hong:Bristol Myers Squibb: Current Employment. Sorrell:Children's Oncology Group: Other: Non-member; Previous Study Chair AAML08B1; Bristol Myers Squibb: Current Employment. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.
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- 2020
40. Immune Cell Profiles in Patients Treated with Lenalidomide and Alternate Day Prednisolone Maintenance Post Upfront ASCT for Multiple Myeloma (LEOPARD Trial)
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Tiffany Khong, Samuel E Norton, John V. Reynolds, William E. Pierceall, Malarmathy Ramachandran, Mary H. Young, Udo Oppermann, Andrew Spencer, Manman Guo, Anjan Thakurta, and Anna Kalff
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Oncology ,education.field_of_study ,medicine.medical_specialty ,Myeloid ,business.industry ,Immunology ,Population ,Cell Biology ,Hematology ,medicine.disease ,NKG2D ,Biochemistry ,medicine.anatomical_structure ,Internal medicine ,medicine ,Prednisolone ,IL-2 receptor ,CD5 ,education ,business ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
The LEOPARD trial evaluated lenalidomide and alternate day prednisolone (RAP) as post ASCT maintenance in newly diagnosed transplant eligible MM patients (TE NDMM). 60 patients were recruited. Estimated median potential follow-up was 44 months (IQR 26m - 52m). Median PFS from time of commencing RAP was 38.3m (95% CI, 25.8 to 54.8); median OS was not reached (71.4% of patients were alive at 36 months). Here we present the findings from correlative immune studies of this trial. Aims: To undertake mass cytometry (CyTOF) based immune profiling in patients with TE NDMM treated with RAP maintenance post ASCT. Methods: The LEOPARD trial was a phase II, multi centre, open label, single arm study of RAP maintenance after a single melphalan conditioned (200mg/m2) ASCT as part of up-front therapy. Patients were restaged at D+42 ASCT, and if eligible, were commenced on RAP maintenance (LEN 10mg daily increasing to 15mg daily after 8 weeks and alternate day prednisolone 50mg) within 8 weeks of D+0 of the ASCT. Therapy continued until toxicity/progression. CyTOF was performed in sequential samples in two selected groups of patients: long runners (LR, n=7), defined as those with PFS > 36 months (median) and early relapsers (ER, n=8), defined as those who progressed/died before reaching the lower quartile of PFS. [All patients had peripheral blood collected at baseline (pre-ASCT), 6w post-ASCT and weeks 4, 8, 12, 20, 28 and 40 of RAP]. Cells were barcoded using the Cell-ID 20-Plex Pd barcoding kit (Fluidigm) followed by staining with sub-set/function defining antibodies (targeting myeloid, B, T and NK cells: CD45, CD3, CD19, CD5, CD1c, CD226. CD8, CD11c, CD16, CD127, CD138, CD123, NKG2A, TIGIT, TIM3, CD45RA. CD274, CD27, CD197, CD28, Ki67, CD66b, CD183, KLRG1, CD43, NKG2D, CD38, CD278/ICOS, CD25, HLA-DR, CD4, CD57, GramB, PD-1, CD14, CD56, CD11b, Tbet, CD33). Samples were acquired on the Helios instrument. Supervised analysis was performed to determine differences in canonical immune cell populations. Unsupervised analysis was then performed: data were clustered in the VORTEX package. Significant differences in cluster frequency were assessed by Mann-Whitney test for statistical significance. Cluster phenotypes were determined and validated via multiple visualisation approaches. Results: Median age was 56yrs for LR versus 63yrs for ER. Median PFS for LR was 46.3m (38.4 - 51.5m) versus 10.2 m (2.1 - 21.3m) for ER. Supervised analysis was performed on all samples, dichotomized into baseline and last time point sampled for each patient. At baseline, Ki67+CD8+ T cells, ICOS+CD8+ T cells, HLA-DR+CD4+ T cells and CD11c+ myeloid cells were enriched in LR compared to ER. At the last timepoint sampled, Ki67+CD8+ T cells and ICOS+CD8+ T cells were again enriched in LR compared to ER. Conversely, B-reg-like cells (CD19+CD5+CD43-) were enriched in ER compared to LR at the last timepoint sampled. Unsupervised analysis was performed on all samples (all timepoints were pooled). Five clusters were significantly enriched in LR compared to ER. Four of these clusters represented activated/cytotoxic NK cells: CD56 dim, CD16-, NKG2A(CD159a)+, NKG2D(CD314)+, Granzyme B+ and CD38+, and additional expression of CD57 on one cluster; one cluster represented a mature myeloid population, with high expression of HLA-DR, CD11b and CD11c and low expression of CD33. One cluster was significantly enriched in ER compared to LR, representing activated neutrophils, with high expression of CD66b, CD11b and CD16. The clusters that were enriched were then assessed longitudinally over all time points. There was no difference in the kinetics of these populations between groups. Conclusions Significant differences in both T-cell and NK cell populations were demonstrable at baseline in LR versus ER patients. Subsequently, durable responses to post-ASCT lenalidomide maintenance were associated with a cytotoxic, controlled immune response whereas early relapse was characterised by a more uncontrolled inflammatory response and the emergence of B-reg-like cells prior to relapse. We conclude that immune profiling at baseline and after initiation of therapy may help to predict a more sustained response to lenalidomide maintenance enabling pre-emptive tailored treatment decisions. Disclosures Kalff: Roche: Honoraria; Janssen: Honoraria; Amgen: Honoraria; CSL: Honoraria; Celgene: Honoraria. Young:Bristol Meyers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierceall:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company; Oxford University: Other: visiting professor. Oppermann:Bristol Meyers Squibb: Research Funding. Guo:Bristol Meyers Squibb: Research Funding. Reynolds:Novartis AG: Current equity holder in publicly-traded company. Spencer:AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; Celgene, Janssen and Takeda: Speakers Bureau.
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- 2020
41. Recurrent somatic Alterations in the Non-Coding Genome Alter Gene Expression Levels and Correlate With Clinical Outcome
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Hervé Avet-Loiseau, Anjan Thakurta, Philippe Moreau, Mehmet Kemal Samur, Masood A. Shammas, Anil Aktas Samur, Thierry Facon, Kenneth C. Anderson, Paul G. Richardson, Nikhil C. Munshi, Michel Attal, Florence Magrangeas, Mariateresa Fulciniti, Raphael Szalat, Giovanni Parmigiani, and Stephane Minvielle
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Genetics ,Cancer Research ,Oncology ,Somatic cell ,business.industry ,Gene expression ,Medicine ,Hematology ,business ,Outcome (game theory) ,Genome ,Coding (social sciences) - Published
- 2019
42. Mass cytometry reveals increase in marrow resident monocytes associated with response to daratumumab and pomalidomide
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Paola Neri, Nizar J. Bahlis, Adeeb Rahman, Mary Young, Anjan Thakurta, William E. Pierceall, Samir Parekh, Oliver Van Oekelen, and Alessandro Laganà
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Cancer Research ,Oncology ,business.industry ,Immunology ,Medicine ,Daratumumab ,Mass cytometry ,Hematology ,business ,Pomalidomide ,medicine.drug - Published
- 2019
43. Enrichment for copy number alterations and a unique pattern of gene mutations characterize multiple myeloma in elderly patients
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Michael A Bauer, Faith E. Davies, Brian A Walker, Louis Williams, Anjan Thakurta, Gareth J. Morgan, Christopher P. Wardell, Eileen M Boyle, Cody Ashby, and Erin Flynt
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Cancer Research ,Oncology ,business.industry ,Cancer research ,Medicine ,Hematology ,Gene mutation ,business ,medicine.disease ,Multiple myeloma - Published
- 2019
44. High subclonal fraction of 17p deletion is associated with poor prognosis in multiple myeloma
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Zhinuan Yu, Pieter Sonneveld, Hervé Avet-Loiseau, Erin Flynt, Pedro Blecua, Anjan Thakurta, Natalya Serbina, Maria Ortiz, Fadi Towfic, Hartmut Goldschmidt, Meletios A. Dimopoulos, Zhihong Yang, Norma C. Gutiérrez, Jill Corre, Antonio Palumbo, Celgene, and Hematology
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Oncology ,medicine.medical_specialty ,Poor prognosis ,17p deletion ,Immunology ,Biochemistry ,Somatic evolution in cancer ,Fluorescent in situ hybridization ,Clonal Evolution ,Cytogenetics ,Text mining ,Multiple myeloma ,Internal medicine ,medicine ,Chromosome 17p deletion ,Biomarkers, Tumor ,Datasets ,Humans ,Survival rate ,Cancer ,Lymphoid Neoplasia ,medicine.diagnostic_test ,business.industry ,High-Throughput Nucleotide Sequencing ,Cell Biology ,Hematology ,medicine.disease ,tp53 gene ,Prognosis ,Survival Rate ,Mutation ,Chromosome Deletion ,Tumor Suppressor Protein p53 ,business ,Multiple Myeloma ,Fluorescence in situ hybridization ,Chromosomes, Human, Pair 17 - Abstract
Deletions of chromosome 17p (del17p) that span the TP53 gene are associated with poor outcome in multiple myeloma (MM), but the prognostic value of del17p cancer clonal fraction (CCF) remains unclear. We applied uniform cytogenetic assessments in a large cohort of newly diagnosed MM (NDMM) patients carrying varying levels of del17p. Incremental CCF change was associated with shorter survival, and a robust CCF threshold of 0.55 was established in discovery and replication data sets. After stratification on the 0.55-CCF threshold, high-risk patients had statistically significantly poorer outcomes compared with low-risk patients (median progression-free survival [PFS] and overall survival [OS], 14 and 32 vs 23.1 and 76.2 months, respectively). Analyses of a third data set comprising whole-exome sequencing data from NDMM patients identified presence of TP53 deletions/mutations as a necessary requirement for high-risk stratification in addition to exceeding the del17p CCF threshold. Meta-analysis conducted across 3 data sets confirmed the robustness of the CCF threshold for PFS and OS. Our analyses demonstrate the feasibility of fluorescence in situ hybridization– and sequencing-based methods to identify TP53 deletions, estimate CCF, and establish that both CCF threshold of 0.55 and presence of TP53 deletion are necessary to identify del17p-carrying NDMM patients with poor prognosis., This work was supported by the Myeloma Disease Team in Celgene (A.T.), by the Association Recherche Cancer (IUC-Oncopole Laboratory), and by Celgene, which provided data processing and storage.
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- 2018
45. Prospective Study Reveals Increased Platelet Function Associated with Multiple Myeloma and Its Treatment
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Anjan Thakurta, Susie Shapiro, Dalia Khan, Rekha Rana, Michael Laffan, Neline Kriek, Joanne L. Mitchell, Amanda J. Unsworth, Jonathan M. Gibbins, Tanya Sage, and Karthik Ramasamy
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Oncology ,medicine.medical_specialty ,business.operation ,business.industry ,Immunology ,Fibrinogen binding ,Cell Biology ,Hematology ,Octapharma ,medicine.disease ,Biochemistry ,Thalidomide ,Internal medicine ,medicine ,Proteasome inhibitor ,Platelet ,Platelet activation ,business ,health care economics and organizations ,Multiple myeloma ,medicine.drug ,Lenalidomide - Abstract
Background: Multiple Myeloma (MM) is a rare incurable bone marrow cancer characterised by a malignant proliferation of plasma cells. MM is usually preceded by a premalignant and benign Monoclonal Gammopathy of Undetermined Significance (MGUS). The incidence of arterial and venous thrombosis in MM is substantially higher than in the normal population, however the cause of this increased thrombosis risk and the impact of MM on platelet function is unclear. Treatments for both newly diagnosed and relapsed/refractory patients with MM include Immunomodulatory drugs (IMiDs) such as thalidomide/lenalidomide-based combinations. These treatments improve considerably patient outcomes, however iMiD treatment also increases the risk of thrombotic complications in these patients. Aims: In this prospective study we explored the impact of MM and its treatment on platelet function. Methods: High throughput functional analysis was performed using platelets from normal healthy controls (n=31) and patients with MGUS (n=18), smouldering multiple myeloma (SMM, n= 20), and MM (26). The MM group was further divided into 3 treatment cohorts; (1) no treatment, (2) treatment with proteasome inhibitor (PI) and dexamethasone (Dex), and (3) treatment with PI, Dex, immunomodulatory drug (iMiD) and direct oral anticoagulant. Platelet aggregation and activation (fibrinogen binding and P-selectin exposure) were measured in response to a concentration range of agonists including ADP, the thrombin receptor agonist TRAP-6, collagen, collagen-related peptide (CRP), a thromboxane receptor agonist U46619 and epinephrine. Cereblon protein was detected in platelet protein extracts by immunoblot analysis. Results: Consistent with previous reports, modestly increased VWF and factor VIII levels were detected in MM patients, but no additional differences in coagulation parameters were detected in patient groups compared to normal healthy controls (other than expected due to anticoagulant usage). Platelet aggregation in response to each agonist was increased significantly in the MM patient group compared to the normal healthy controls, suggesting that platelet reactivity is elevated in MM patients through a common mechanism that is shared by different activation pathways or the involvement of multiple mechanisms. P-selectin exposure on platelets from MM patients was not significantly different from normal healthy donors, indicating that enhanced platelet reactivity in MM is specifically through modulation of integrin αIIbβ3 activation, fibrinogen binding and therefore enhanced aggregation. The effects of treatment on platelet function in patients on iMiD vs. non iMiD treatment were assessed. In the iMiD treatment group, patient platelets aggregated in response to lower concentrations of ADP, collagen, epinephrine and CRP in samples taken post-treatment compared to those taken before and during treatment. This demonstrates an increased sensitivity to platelet activation in these patients induced by treatment. Immunoblot analysis revealed that platelets contain cereblon, a therapeutic target of lenalidomide. The potential direct effects of iMiDs on platelets in vitro was therefore explored. Lenalidomide treatment (10mM) increased the ability of platelets to aggregate in response to low concentrations of each agonist tested when compared to normal controls. Conclusions: Platelet reactivity is increased in multiple myeloma and increased further upon iMiD treatment. The presence of the key therapeutic target for iMiDs in platelets and the ability of lenalidomide to modulate platelet function directly, reveals new avenues for investigation to determine the underlying mechanism of action. Disclosures Laffan: CSL: Consultancy; Pfizer: Consultancy; Sobi: Consultancy; Roche: Consultancy; LFB: Consultancy; Shire: Consultancy; Octapharma: Consultancy; Bayer: Speakers Bureau; Roche-Chugai: Speakers Bureau; Takeda: Speakers Bureau; Leo-Pharma: Speakers Bureau; Pfizer: Speakers Bureau. Shapiro:Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Chugai/Roche: Consultancy, Speakers Bureau; Shire/Takeda: Consultancy, Speakers Bureau. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy:Takeda: Research Funding; Janssen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Amgen: Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Oncopeptides: Honoraria; Takeda: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Bristol Myers Squibb: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers squibb: Membership on an entity's Board of Directors or advisory committees. Gibbins:Bristol Myers Squibb: Research Funding; Arena Pharmaceuticals: Research Funding.
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- 2020
46. Pomalidomide in combination with dexamethasone results in synergistic anti-tumour responses in pre-clinical models of lenalidomide-resistant multiple myeloma
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Brian E. Cathers, Thomas O. Daniel, Emily Rychak, Rama K. Narla, Heather Raymon, Ling Lu, Rajesh Chopra, Yuhong Ning, Anita Gandhi, Antonia Lopez-Girona, Chad C. Bjorklund, Karen Miller, Tao Shi, Jim Leisten, Derek Mendy, Anjan Thakurta, and Robert Z. Orlowski
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0301 basic medicine ,Apoptosis ,Mice, SCID ,Pharmacology ,Biology ,Dexamethasone ,Immunomodulation ,03 medical and health sciences ,0302 clinical medicine ,Antineoplastic Combined Chemotherapy Protocols ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Lenalidomide ,Multiple myeloma ,Cell Proliferation ,Bortezomib ,Cell growth ,Drug Synergism ,Hematology ,Pomalidomide ,medicine.disease ,Xenograft Model Antitumor Assays ,IKZF3 ,Thalidomide ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Female ,Multiple Myeloma ,medicine.drug - Abstract
Pomalidomide is an IMiD(®) immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti-proliferative and pro-apoptotic activity in both lenalidomide-sensitive and lenalidomide-resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single-agent treatment in xenografts of lenalidomide-resistant H929 R10-1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide-sensitive H929 MM cell lines, were also observed in PomDex-treated lenalidomide-resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide-sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex-treated samples, highlighted by the modulation of pro-apoptotic pathways in lenalidomide-resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide-resistant setting.
- Published
- 2016
47. Cereblon loss and up-regulation of c-Myc are associated with lenalidomide resistance in multiple myeloma patients
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Mark-David Levin, Laurens E. Franssen, Laura M. Faber, Anjan Thakurta, Gerard M. J. Bos, Harry R. Koene, Suzana Couto, Henk M. Lokhorst, Sonja Zweegman, Ellen van der Spek, Niels W.C.J. van de Donk, Saskia K. Klein, Reinier Raymakers, Inger S. Nijhof, Pieter Sonneveld, Roos J. Leguit, Andries C. Bloem, Xiaozhong Qian, Maria Wang, Yan Ren, Tuna Mutis, Annemiek Broijl, Aart Beeker, Internal medicine, Hematology laboratory, AII - Inflammatory diseases, Hematology, CCA - Cancer biology and immunology, RS: GROW - R3 - Innovative Cancer Diagnostics & Therapy, Interne Geneeskunde, and MUMC+: MA Hematologie (9)
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Adult ,Male ,0301 basic medicine ,EXPRESSION ,Ubiquitin-Protein Ligases ,Drug Resistance ,Drug resistance ,Proto-Oncogene Proteins c-myc ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Refractory ,Humans ,Medicine ,Online Only Articles ,Lenalidomide ,Multiple myeloma ,Adaptor Proteins, Signal Transducing ,Aged ,Aged, 80 and over ,medicine.diagnostic_test ,business.industry ,Cereblon ,POMALIDOMIDE ,Bone Marrow Examination ,Hematology ,Middle Aged ,Pomalidomide ,medicine.disease ,Up-Regulation ,Bone marrow examination ,030104 developmental biology ,030220 oncology & carcinogenesis ,CELLS ,Cancer research ,Female ,Multiple Myeloma ,business ,Peptide Hydrolases ,medicine.drug - Abstract
Multiple myeloma (MM) patients who become refractory to anti-MM drugs have a very poor prognosis. Therefore, it is important to gain insight into the mechanisms of resistance to these drugs. Immunomodulatory drugs (IMiDs) have immune-stimulatory and anti-angiogenic properties as well as direct anti
- Published
- 2018
48. Large-Scale Analysis of Relapsed/Refractory Multiple Myeloma Genome Reveals Increased Prevalence of High-Risk Molecular Features and Oncogenic Drivers
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Sneh Lata, Naser Ansari-Pour, Fadi Towfic, Nicholas Stong, Sitharthan Kamalakaran, Maria Ortiz, Sarah Gooding, Victoria Zadorozhny, Erin Flynt, Anjan Thakurta, Kao-Tai Tsai, Konstantinos Mavrommatis, and Dan Rozelle
- Subjects
Oncology ,medicine.medical_specialty ,Immunology ,Treatment outcome ,Cancer ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Genome ,Leukemia ,Internal medicine ,Relapsed refractory ,medicine ,Multiple myeloma ,Protein p53 - Abstract
Introduction Multiple myeloma (MM) is a heterogeneous disease defined by genetic lesions including translocations, chromosomal copy number aberrations (CNA), and mutations. Large-scale analyses of genomic data from newly-diagnosed patients (ndMM) have defined key oncogenic drivers beyond previously known primary events. In relapsed/refractory MM (rrMM), analyses have been completed on small numbers of patient samples for mutational profiles; however, a large-scale analysis of genomic and transcriptomic data has not been performed. In this initial analysis, we describe the rrMM genomic landscape including prevalence of key translocations, oncogenic/tumor suppressor mutational drivers, and chromosomal CNA. Methods We generated whole genome sequencing (WGS) and whole transcriptome expression (RNA-seq) from 485 rrMM patient samples derived from clinical trials: NCT01712789/CC-4047-MM-010 (N=236), NCT02045017/CC-4047-MM-013 (N=17), NCT02773030/CC-220-MM-001 (N=45) and NCT01421524/CC-122-ST-001MM2 (N=10). Somatic single nucleotide variants (SNVs) and indels were derived from WGS from 308 samples using GATK/MuTect2 and annotated using ANNOVAR. Clonal and subclonal CNA were identified using Sclust. Translocations were determined using Manta. Oncogenic/tumor suppressor drivers were identified using cDriver, which utilizes recurrence and functional consequence and cancer cell fraction (CCF) of each mutation. Results Key translocations included: t(11;14) [76/308 (25%)], t(8;14) [79/308 (25%)], t(4;14) [44/308 (14%)], t(14;16) [24/308 (8%)], t(6;14) CCND3 [18/308 (6%)], t(14;20) [17/308 (5.5%)], and t(6;14) [IRF4 17/308 (5.5%)]. Hyperdiploidy was detected in 140/308 (46%) patients. Key chromosomal CNA included del14q 134/308 (44%), del13q 131/308 (43%), del8p 113/308 (37%), del17p 53/308 (17%), amplification of 1q (≥4 copies) 45/308 (15%), and del1q 31/308 (10%). Compared to samples from ndMM patients, we saw an increase of del17p (17% vs. 8%) and t(11;14) (25% vs. 15%) in rrMM. Further, out of the 53 del17p patients, 45 (85%) were high CCF (>0.55) versus 63/107 (59%) reported in ndMM (Thakurta et al, Blood. 2019) Double Hit patients (biallelic inactivation of TP53 or amplification of 1q on a background of ISS3) was detected in 12% patients which is significantly higher than ndMM (6%, p < 0.05) (Walker et al, Leukemia. 2018) We identified a total of 107 driver genes (FDR Conclusions We have established and analyzed the largest molecular rrMM dataset with associated clinical outcome data. These analyses are revealing the evolution of genetic drivers of resistance to therapy and will assist in identification of subsets of poor prognostic groups (eg, Double Hit) and new molecular subsets of rrMM where novel targeted therapies could be developed. Disclosures Towfic: Celgene Corporation: Employment, Equity Ownership. Ansari-Pour:Celgene Corporation: Consultancy. Ortiz:Celgene Corporation: Employment, Equity Ownership. Gooding:Celgene Corporation: Research Funding. Rozelle:Celgene Corporation: Other: Contractor for Celgene. Zadorozhny:Celgene Corporation: Other: Contractor for Celgene. Mavrommatis:Celgene Corporation: Employment, Equity Ownership. Lata:Celgene Corporation: Employment, Equity Ownership. Stong:Celgene Corporation: Employment, Equity Ownership. Kamalakaran:Celgene Corporation: Employment, Equity Ownership. Tsai:Celgene Corporation: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership.
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- 2019
49. Higher Expression of Nuclear Cereblon in Bone Marrow Biopsies of Patients with Multiple Myeloma Treated with Imids in the HOVON-87/Nmsg-18 Trial Is Associated with Longer PFS and OS
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Yan Ren, Heleen Visser-Wisselaar, Mark van Duin, B Beverloo, Ruth Wester, Anjan Thakurta, Suzana Couto, Annemiek Broyl, Maria Wang, Bronno van der Holt, Pieter Sonneveld, Sonja Zweegman, Alex L. Nigg, and King H. Lam
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Oncology ,Melphalan ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,Cereblon ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Thalidomide ,medicine.anatomical_structure ,Prednisone ,Internal medicine ,Biopsy ,medicine ,Bone marrow ,business ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
Introduction Response to treatment in patients with multiple myeloma (MM) is variable. With increasing possibilities of treatment regimens, predictive factors for response are important. Immune modulating agents (IMiDs) require Cereblon (CRBN) for activity. Therefore, the aim of this study was to identify the genes involved in the CRBN pathway which predict the response to therapy with IMiDs. Methods Paraffin embedded bone marrow (BM) biopsies were used from newly diagnosed patients included in HOVON-87/NMSG-18 trial obtained at inclusion. In this trial, elderly patients with MM were randomized between treatment with Melphalan-Prednisone (MP)-Thalidomide (MPT) followed by thalidomide maintenance versus MP-Lenalidomide (MPR) followed by lenalidomide maintenance (Zweegman et al. Blood 2016;127:1109-1116). BM biopsies were stained with a fully automated dual color, bright-field immunohistochemical assay for CRBN, its neosubstrates Ikaros and Aiolos and the downstream targets IRF4 and c-MYC. CD138 was used to identify MM plasma cells in the BM samples. For CRBN, both nuclear and cytoplasmic staining was evaluated. The distribution and intensity of the immunostaining was assessed using the H-score. The H-scores were calculated using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)] and range from 0-300 (0-600 for combined cytoplasmic-nuclear CRBN H-score). For the Cox regression analysis H-scores were corrected by dividing these by a factor 100: hazard rates were considered per 100 points increase of the H-score. Protein levels of the CRBN pathway were compared between patients with complete response (CR) or very good partial response (VGPR) vs partial response (PR) and no change/progressive disease (NC/PD). High-risk cytogenetic aberrations (FISH) were defined as having deletion of 17p, and/or translocation t(4;14) and/or t(14;16). Statistical analysis was done using univariate and multivariate Cox regression analysis for progression free survival (PFS) and overall survival (OS), and the Mann-Whitney test for comparing response groups. Kaplan-Meier survival curves were generated to illustrate survival. Results BM samples obtained at diagnosis from 149 patients were evaluated. Seventy-one patients were treated in the thalidomide arm vs 78 patients in the lenalidomide arm. Median age was 73 years [range 60-90]. Revised ISS stages I/II/III were 12%/80%/8% respectively. At the time of analysis, median follow up of the 45 patients still alive was 83 months [range 23 - 114 months]. Best response on protocol treatment was sCR/CR in 22%, VGPR in 30%, PR in 36% and NC/PD in 12%. Protein expression across the response groups showed higher nuclear CRBN in patients who responded better (sCR/CR/VGPR; median H-score: 178 (49-273)) compared to patients with a worse response (PR/NC/PD; median H-score: 157 (67-251)), albeit not statistically significant (Mann-Whitney p-value=0.06). Higher H-score of nuclear staining of CRBN was associated with a longer PFS and OS, with a hazard ratio (HR) of 0.52 for PFS (95% confidence interval (CI)=0.37-0.86, p In a multivariate analysis (which included study arm (MPT vs MPR), nuclear CRBN, high-risk cytogenetic aberrations and R-ISS), nuclear CRBN remained independently associated with OS as well as R-ISS and study arm. For PFS, only nuclear CRBN remained statistically significant after backward selection. Despite treatment arm being a statistically significant term in the multivariate Cox model for OS, no relation was found for treatment arm and nuclear CRBN, in terms of OS. Conclusions In this study we demonstrate that higher expression of nuclear CRBN in myeloma cells in BM of patients with MM was associated with a superior PFS and OS. Disclosures Couto: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Ren:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership. Zweegman:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Broyl:Celgene, amgen, Janssen,Takeda: Honoraria. Sonneveld:Amgen: Honoraria, Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; SkylineDx: Research Funding.
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- 2019
50. Chromoplexy and Chromothripsis Are Important Prognostically in Myeloma and Deregulate Gene Function By a Range of Mechanisms
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Judith A. Dent, Cody Ashby, Gareth J. Morgan, Faith E. Davies, Eileen M Boyle, Brian A Walker, Katie R. Ryan, Anjan Thakurta, Michael A Bauer, and Erin Flynt
- Subjects
Chromothripsis ,business.industry ,Immunology ,Alpha interferon ,Cell Biology ,Hematology ,Chromoplexy ,medicine.disease ,Biochemistry ,Cancer research ,medicine ,business ,Gene ,Protein p53 ,Multiple myeloma ,Function (biology) - Abstract
Background: Structural variants are key recurrent molecular features of myeloma (MM) with two types of complex rearrangement, chromoplexy and chromothripsis, having been described recently. The contribution of these to MM prognosis, rapid changes in clinical behavior and punctuated evolution is currently unknown as is the mechanism by which they deregulate gene function. Methods: We analyzed two sets of newly diagnosed MM data: 85 cases with phased whole genome sequencing; and 812 cases from CoMMpass where long-insert whole-genome sequencing was available. Patient derived xenografts from five MM cases were used to generate epigenetic maps for the histone marks, BRD4, MED1, H3K27Ac, H3K4me1, H3K4me3, H3K9me3, H3K36me3 and H3K27me3. Results: In the 10X data the median number of structural events per case was 25 (range 1 - 182); with a median of 14 intra-chromosomal events (range 1 - 179; P Using an elbow test defined cutoff, we identified cases with high structural variant load in 10% of cases. Chromoplexy called by "Chainfinder" was seen in 18% of cases. Chromothripsis called by "Shatterseek" was seen in 9% of cases. Cases with a high structural load alone were not associated with an adverse outcome whereas cases with chromoplexy or chromothripsis were associated with adverse PFS and OS, p=0.001. A new high-risk subgroup comprising approximately 5% of cases was identified with chromoplexy, chromothripsis and a high structural load. Gene set enrichment analysis of cases with chromoplexy and chromothripsis showed an excess of MYC, E2F and G2M targets, and a reduction in RAS signaling. Interferon a and g responses, an excess of TP53 and reduction in TRAF3 mutations was associated predominantly with chromothripsis. How chromoplexy and chromothripsis are tolerated by the cell is unknown and the association with the cGAS/STING response is further being explored. To determine how chromoplexy may deregulate multiple genes we identified the full spectrum of structural variants to the immunoglobulin (Ig) and non-Ig loci. A range of genes are deregulated by Ig loci including MAP3K14 at a frequency of 2% confirming the importance of non-canonical NFkB signaling. A novel intra-chromosomal rearrangement to ZFP36L1 was upregulated in 10% of cases but was not prognostic. Gene upregulation by non-Ig super enhancers is frequent and targets include PAX5, GLI3, CD40, NFKB1, MAP3K14, LRRC37A, LIPG, PHLDA3, ZNF267, CENPF, SLC44A2, MIER1, SOX30, TMEM258, PPIL1, and BUB3. The topologically associating domain (TADs) containing super enhancers bringing about gene deregulation include TXNDC5, FOXO3, FCHSD2, SP2, FAM46C, CACNA1C, TLCD2 and PIK3C2G. These super enhancers frequently contain important MM genes, the coding sequence of which are disrupted by the rearrangement and could contribute to the clinical phenotype. Accurately reconstructing the structure of the complex rearrangements will allow us to identify the mechanism of gene deregulation and to distinguish between either gene stacking, receptor stacking or both. Conclusions: Upregulation of gene expression by super enhancer rearrangement is a major mechanism of gene deregulation in MM and complex structural events contribute significantly to adverse prognosis by a range of mechanisms as well as simple gene overexpression. Disclosures Boyle: Amgen, Abbvie, Janssen, Takeda, Celgene Corporation: Honoraria; Amgen, Janssen, Takeda, Celgene Corporation: Other: Travel expenses. Walker:Celgene: Research Funding. Thakurta:Celgene: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Davies:Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor; Janssen, Celgene: Other: Research Grant, Research Funding. Morgan:Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding.
- Published
- 2019
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