31 results on '"Shruti Bhatt"'
Search Results
2. PPM1D-truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells
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Marie McConkey, Rob S. Sellar, Benjamin L. Ebert, John G. Doench, Siddhartha Jaiswal, Josephine Kahn, Karsten Krug, Shaunt Fereshetian, Dylan N. Adams, Shruti Bhatt, Peter Miller, Brenton G. Mar, Haoling Zhu, Christopher J. Gibson, Steven A. Carr, Alexander J. Silver, Anthony Letai, and Philipp Mertins
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0301 basic medicine ,Myeloid ,DNA damage ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Chemotherapy regimen ,Phenotype ,03 medical and health sciences ,Haematopoiesis ,Exon ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Apoptosis ,Cell culture ,030220 oncology & carcinogenesis ,Cancer research ,medicine - Abstract
Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.
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- 2018
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3. Maturity State and MCL-1 Dependence Predetermines Response to NOTCH1 Inhibition in T-ALL
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Anna Montanaro, David M. Weinstock, Shruti Bhatt, Noori Sotudeh, Praveen Anand, Antonis Kokkalis, Jake A. Kloeber, Johannes M. Waldschmidt, Jens G. Lohr, Alexandria Van Scoyk, Anthony Letai, Huiyoung Yun, Sayalee Potdar, Julia Frede, Birgit Knoechel, and Valeriya Dimitrova
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Maturity (geology) ,Animal science ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry - Abstract
Introduction: Acute T cell lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and young adults that frequently becomes treatment-refractory and relapses. The Notch1 pathway is a key oncogenic driver in T-ALL and is aberrantly activated in more than 50% of the cases. Despite promising pre-clinical data using gamma secretase inhibitors such as DBZ to target NOTCH1, resistance is rapidly occurring in vivo. As molecular heterogeneity has been linked to treatment escape, we focused our study on defining transcriptional cell states driving resistance to NOTCH inhibition and understanding their relation to mitochondrial priming. Methods: 5 primary T-ALLs harboring NOTCH activating mutations were engrafted in NSG (NOD-scidIL2Rgnull) mice. Upon reaching ~ 10% of human CD45+ positive leukemic blasts in the peripheral blood, randomized groups of 8 mice per primary T-ALL were treated with DBZ (Dipenzazepine; 10 μM/kg every other day through tail vein) or vehicle (VEH). 3 mice per group were sacrificed after one week of treatment to assess short-term effect of DBZ, while the remaining 5 mice were weekly monitored for disease progression, leukemic blasts were collected from lymphoid organs and overall survival was determined. Full-length transcriptome analysis of 3188 blasts present in the blood of 20 sensitive and 22 refractory mice was performed by Smart-Seq2. Based on scRNA features, 'scVelo' and 'CytoTRACE' were used to identify developmental potential and differentiation trajectories. Cell fate and transcriptional regulatory networks were defined and reconstructed using 'SCENIC'. Assessment of mitochondrial priming as measured by BH3 profiling was used to identify anti-apoptotic vulnerabilities present in these PDX models. Results: Upon DBZ, short or long-term disease control was observed in two strains, while rapid resistance occurred in three strains, thus establishing two sensitive and three refractories to NOTCH inhibition PDX models. Immunohistochemical analysis showed decreased expression of active NOTCH1 in spleen biopsies of all strains, validating the efficacy of DBZ and suggesting a mechanism of resistance independent of ICN1. Single cell transcriptional profiling showed enrichment of immature hematopoietic signatures and co-expression of lymphoid and myeloid progenitor programs in refractory models. Interestingly, pre-existing cells harboring refractory-like transcriptional circuits within the untreated sensitive population were identified. Upon treatment, despite increased differentiation in all models, lineage promiscuity was maintained in refractory strains, suggesting that cellular plasticity mediates treatment escape. Next, we characterized cell states driving treatment refraction. RNA velocity projections identified two distinct immature states differing in cell cycle and oncogenic signaling. Clustering of untreated, sensitive leukemic cells in immature state imply that aberrant lineage commitment can predict response to NOTCH inhibition in vivo. These observations were further confirmed by differentiation state analysis, where prior to treatment, high developmental potential was correlated to treatment escape. Surprisingly, in addition to early lineage differentiation drivers such as BCL11A, state-specific regulons analysis associated immature states with BCLAF1 a transcriptional regulator of apoptosis. We postulated that these transcriptional circuits lead to differential apoptotic priming, therefore the dependence on individual anti-apoptotic proteins was evaluated. Mitochondrial priming at baseline revealed BCL-2 dependence in sensitive strains whereas MCL1-dependence was observed in refractory ones. Upon DBZ treatment, while dependency profiles in refractory strains remained unchanged, a functional switch from BCL-2 to MCL1-dependency occurred in sensitive models. Conclusion: Our results suggest that response to NOTCH inhibition is predetermined by cell maturity states and their associated transcriptional circuits responsible for differential sensitivity to apoptotic priming via BCL2 and MCL1. These data suggest that combining BH3 and lineage commitment profiling may predict drug responses in vivo. Moreover, our findings highlight the importance of targeting co-existing cell states to overcome transcriptional heterogeneity as a driver of treatment escape. Disclosures Letai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Weinstock: Daiichi Sankyo: Consultancy, Research Funding; Verastem: Research Funding; Abcuro: Research Funding; Bantam: Consultancy; ASELL: Consultancy; SecuraBio: Consultancy; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.
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- 2021
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4. Identification of Novel Targets Based on Splicing Alterations for Undruggable RAS/CDK Signaling Cascade in Multiple Myeloma
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Yu-Tzu Tai, Shruti Bhatt, Sophia Adamia, Kenneth Wen, Kenneth C. Anderson, Teru Hideshima, David M. Dorfman, Anthony Letai, Zuzana Chyra, Ivane Abiatari, Morgan O'Keefe, and Geoffrey Fell
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biology ,Immunology ,Cell Biology ,Hematology ,Computational biology ,medicine.disease ,Biochemistry ,Cascade ,Cyclin-dependent kinase ,RNA splicing ,medicine ,biology.protein ,Identification (biology) ,Multiple myeloma - Abstract
Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets. Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. Disclosures Letai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Anderson: Bristol Myers Squibb: 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; 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; 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
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5. Anti-CD20-interleukin-21 fusokine targets malignant B cells via direct apoptosis and NK-cell–dependent cytotoxicity
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Joseph D. Rosenblatt, Salma Parvin, Shruti Bhatt, John M. Timmerman, Francisco Vega, Kranthi Kunkalla, Yu Zhang, Izidore S. Lossos, Hyun Mi Cho, and Seung Uon Shin
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Cytotoxicity, Immunologic ,0301 basic medicine ,Recombinant Fusion Proteins ,Immunology ,Gene Expression ,Antineoplastic Agents ,Apoptosis ,Mice, Transgenic ,Biochemistry ,Antibodies, Monoclonal, Murine-Derived ,Interferon-gamma ,Mice ,03 medical and health sciences ,Interleukin 21 ,0302 clinical medicine ,Antigen ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Molecular Targeted Therapy ,Functional ability ,Cytotoxicity ,B-Lymphocytes ,Mice, Inbred BALB C ,biology ,Interleukins ,Lymphoma, Non-Hodgkin ,Cell Biology ,Hematology ,Antigens, CD20 ,medicine.disease ,Lymphoma ,Killer Cells, Natural ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,Receptors, Interleukin-21 ,Rituximab ,Antibody ,Half-Life ,Signal Transduction ,medicine.drug - Abstract
In spite of newly emerging therapies and the improved survival of patients with non-Hodgkin lymphoma (NHL), relapses or primary refractory disease are commonly observed and associated with dismal prognosis. Although discovery of the anti-CD20 antibody rituximab has markedly improved outcomes in B-cell NHL, rituximab resistance remains an important obstacle to successful treatment of these tumors. To improve the efficacy of CD20-targeted therapy, we fused interleukin 21 (IL-21), which induces direct lymphoma cytotoxicity and activates immune effector cells, to the anti-CD20 antibody (αCD20-IL-21 fusokine). We observed substantially enhanced IL-21R-mediated signaling by the fusokine compared with native IL-21 at equimolar concentrations. Fusokine treatment led to direct apoptosis of lymphoma cell lines and primary tumors that otherwise were resistant to native IL-21 treatment. In addition to direct cytotoxicity, the fusokine enhanced NK cell activation, effector functions, and interferon γ production, resulting in greater antibody-dependent cell-mediated cytotoxicity compared with IL-21 and/or anti-CD20 antibody treatments. Further, the αCD20-IL-21 fusokine stabilizes IL-21 and prolongs its half-life. In vivo αCD20-IL-21 therapy resulted in a significant tumor control in the rituximab-resistant A20-huCD20 tumors. Collectively, the dual functional ability of the αCD20-IL-21 fusokine to induce direct apoptosis and activate immune effector cells may provide benefit over existing treatments for NHL.
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- 2017
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6. Pre-Clinical Validation of a Novel Erk1/2 and CDK4/6 Inhibitor Combination in Multiple Myeloma (MM)
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Shruti Bhatt, Ivane Abiatari, Sophia Adamia, Yu-Tzu Tai, Catherine A Nicholas, Anthony Letai, Kenneth C. Anderson, Kenneth Wen, and Marissa S. Pioso
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MAPK/ERK pathway ,Cell cycle checkpoint ,biology ,Cell growth ,business.industry ,Immunology ,Cell Biology ,Hematology ,Cell cycle ,Biochemistry ,Downregulation and upregulation ,Cancer research ,biology.protein ,Medicine ,Cyclin-dependent kinase 6 ,Signal transduction ,business ,G1 phase - Abstract
Whole-genome sequencing analysis of newly diagnosed and relapsed multiple myeloma (MM) samples identified recurrent mutations in genes involved in the MAPK pathway, highlighting the potential of RAS/RAF/MEK/ERK signaling as a therapeutic target. Genomic studies identified translocations that involve IGH and set of partner genes MMSET, FGFR3, and CCND1 as primary events in MM. CDK4/CDK6 is overexpressed in MM, and CDK6 overexpression correlates with poor OS, suggesting that CDK4/6 are promising targets for MM therapy. Recent studies demonstrated synergistic activity of combined novel ERK1/2i inhibitor LY3214996 and CDK4/6i LY2835219 in solid tumors, but analogous studies have not been done in MM. Here we used preclinical models of MM to investigate inhibiting Erk1/2, CDK4/6, or both using ERK1/2i, CDK4/6i, or combination therapy. MM cell lines, RAS mutated or wild type (WT), were sensitive to ERK1/2i at IC50 To address the underlying mechanism of the synergism between Erk1/2i and CDK4/6i, we evaluated their cellular and transcriptional activity in MM cells. Gene expression profiling showed significant downregulation of RAS and CDK4/6 signaling pathway genes in MM cells as a result of ERK1/2i and CDK4/6i treatment at specific concentration ratios (3:1/1:3). Further evaluation of functional effects of ERK1/2i and CDK4/6i, alone or in combination, demonstrated that the synergistic effect of these inhibitors in MM cells is achieved through inhibition of p-S6, downregulation of c-myc, and correlate with ERK1/2i+CDK4/6i induced cell arrest in the G1 cell cycle phase. We noted increased ERK1/2 phosphorylation, which generally results in compensatory activation of parallel signaling pathways or in the loss of negative feedback. Regardless, ERK1/2i+CDK4/6i retained the inhibitory activity of the downstream signaling network, as demonstrated by the inhibition of cytoplasmic (p-RSK1) and nuclear (c-myc) targets of ERK at protein and mRNA levels. Treatment with ERK1/2i+CDK4/6i significantly decreased the levels of p-Rb and E2F1, downstream targets of CDK4/6. Recent studies shown that, in addition to cell cycle regulation, CDK4 and CDK6 induce tumorigenesis through regulation of inflammatory cytokines that are induced via NFκB pathway activation. CDK4/6i functional effects on MM cells cannot be limited to cell cycle arrest, CDK4/6i might also inhibit cytokines, which are produced in MM cells by NFκB activation. Overall, we shown that ERK1/2i+CDK4/6i induced cell proliferation and led to the key target molecule (p-c-myc, p-RSK, p-S6, p-RB, and E2F1) downregulations suggesting on-target activity of these inhibitors in MM cells. Importantly, our studies demonstrate strong synergistic anti-MM activity with ERK1/2+CDK4/6 therapy, providing a preclinical framework for clinical trials to improve patient outcome in MM. Disclosures Letai: Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees; Chugai: Other: Lecture Fees. Anderson: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; 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; 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; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics..
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- 2020
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7. Patterns of substrate affinity, competition, and degradation kinetics underlie biological activity of thalidomide analogs
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Mikolaj Slabicki, Eric Kuhn, Quinlan L. Sievers, Namrata D. Udeshi, Jessica A. Gasser, Adam S. Sperling, Rob S. Sellar, Hasmik Keshishian, Michael Burgess, Elyse A. Olesinski, Benjamin L. Ebert, Steven A. Carr, Rohan Sharma, Anthony Letai, Peter Miller, Shruti Bhatt, Max Jan, Brian J. Liddicoat, Mariateresa Fulciniti, Emma C. Fink, Dylan N. Adams, and Nikhil C. Munshi
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biology ,Chemistry ,Ubiquitin-Protein Ligases ,Immunology ,Biological activity ,Cell Biology ,Hematology ,Drug resistance ,Protein degradation ,Biochemistry ,In vitro ,Ubiquitin ligase ,Substrate Specificity ,Thalidomide ,Targeted mass spectrometry ,In vivo ,Hematologic Neoplasms ,Proteolysis ,biology.protein ,medicine ,Humans ,medicine.drug - Abstract
Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.
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- 2018
8. Individualized Mitochondrial Functional Approach to Combination of BCL-2 and MCL-1 Antagonism in Acute Myeloid Leukemia
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Ensar Halilovic, Elyse A. Olesinski, David M. Weinstock, Holly Zhu, Sophia Adamia, Leutz Buon, Jeremy Ryan, Shruti Bhatt, Morris Erick, Marissa S. Pioso, Binyam Yilma, Anthony Letai, Youzhen Wang, and Jacqueline S. Garcia
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Venetoclax ,business.industry ,Immunology ,Azacitidine ,Myeloid leukemia ,Cell Biology ,Hematology ,Mitochondrion ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,Leukemia ,chemistry ,Apoptosis ,medicine ,Cytarabine ,Cancer research ,Antagonism ,business ,medicine.drug - Abstract
BCL-2 antagonist venetoclax combined with hypomethylating agents or low-dose cytarabine is a new standard of care for treatment-naive elderly or unfit AML patients. Despite the striking overall response of 70%, only 30% achieved MRD negative status with a short remission duration of 11.3 months. This highlights the need for new agents that can overcome venetoclax resistance. We exploited mitochondrial functional assay called BH3 profiling to identify predictors of clinical responses and to discover combination partners of venetoclax. BH3 profiling measures apoptotic priming of a cell (proximity to the apoptotic threshold) by measuring mitochondrial response to a standardized panel of pro-apoptotic BH3 oligopeptides. By using pretreatment myeloblasts of patients (N=16) from clinical trial of venetoclax and azacitidine, we found that response to HRK + MS1 peptides (infers BCL-XL and MCL-1 dependency, respectively) inversely correlates with the achievement of remission. The serial sampling on treatment revealed the emergence of differential patterns of anti-apoptotic dependency that correlated with patient response. Of note, mitochondrial sensitivity to the MS-1 peptide, an indication of MCL-1 dependence, increased from 0% at diagnosis to 40% at relapse, suggesting an opportunity for MCL-1 antagonism in the relapsed setting. First, we exposed 6 AML cell lines with a BCL-2 (venetoclax) and MCL-1 antagonist (S63845). As previously reported, combination treatment resulted in synergistic cell line killing. Venetoclax treatment (within an hour) led to a dynamic increase in mitochondrial sensitivity to the MCL-1 selective MS-1 peptide, while S63845 caused increased mitochondrial susceptibility to the BCL-2 and BCL-XL selective BAD peptide. There was a correlation between % of priming caused by MS-1 and BAD peptide with Loewe synergy score (Spearman r=0.79, p We next investigated combination MCL-1 and BCL-2 antagonism in vivo in venetoclax resistant AML PDXs. We transplanted NSG mice with human AML cells and started them on venetoclax (100mg/kg, PO) until leukemia relapsed (N=6 models). We subjected resistant myeloblasts to BH3 profiling and identified dependency on BCL-2 and MCL-1 to guide therapeutic choices. As a single agent, S63845 showed limited activity in venetoclax resistance models (p Next, we applied the dynamic BH3 profiling technique that measures drug-induced changes in mitochondrial priming to discover additional agents that may overcome venetoclax resistance. We compared mitochondrial priming signals of 40 targeted agents in isolated parental and resistant cells. Although venetoclax resistant cells gained cross resistance to most of the agents, inhibitors of the FLT-3 pathway, including quizartinib, crenolanib, and gilteritinib retained similar priming responses as in parental cells. To validate this, we re-transplanted venetoclax resistant cells and found that quizartinib treatment maintained anti-leukemia efficacy compared to venetoclax or vehicle-treated mice (p In conclusion, our study illustrates the power of mitochondrial measurements as a predictive biomarker for BH3 mimetic based therapy. We provide an evidence for the persistent activity of FLT-3 inhibitors in venetoclax resistance settings, a discovery made possible by dynamic BH3 profiling. Disclosures Ryan: Vivid Biosciences: Consultancy. Erick:Novartis: Employment. Weinstock:Celgene: Research Funding. Garcia:Abbvie: Research Funding; Genentech: Research Funding. Letai:Novartis: Research Funding; Flash Therapeutics: Equity Ownership; Abbvie: Consultancy, Research Funding; Vivid Bioscience: Equity Ownership; Astrazeneca: Research Funding.
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- 2019
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9. Enhancer Rewiring Dependent Switch from BCL2 to MCL1 Dependency Predicts NOTCH1 Inhibition Response in T-ALL
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Alexandria Van Scoyk, Jake A. Kloeber, Antonis Kokkalis, Johannes M. Waldschmidt, Birgit Knoechel, Valeriya Dimitrova, Jens G. Lohr, Sayalee Potdar, Julia Frede, Jon C. Aster, Shruti Bhatt, Praveen Anand, Monica S. Nair, Huiyoung Yun, David M. Weinstock, and Anthony Letai
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Oncogene ,medicine.diagnostic_test ,Combination therapy ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Flow cytometry ,medicine.anatomical_structure ,dBZ ,In vivo ,Acute lymphocytic leukemia ,medicine ,Cancer research ,Bone marrow ,Enhancer - Abstract
Introduction: Acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and adolescents that is associated with high rates of treatment failure and early relapse. T-ALL patients frequently harbor NOTCH1 activating mutations as the driving oncogene in this disease. A multitude of strategies preventing NOTCH1 cleavage and activation, such as Gamma-secretase inhibitors (GSIs) have been developed. Despite promising pre-clinical data, the rapid development of Notch1 inhibitor resistance in early clinical trials, prevented the translation of these inhibitors into the clinical setting. In previous work, our group demonstrated that T-ALL resistant to NOTCH1 inhibition carry altered epigenetic states conferring unique dependency on epigenetic modifiers, such as BRD4. The goal of this study was to study enhancer rewiring in Notch1 inhibitor resistant T-ALL in vivo and its relationship to apoptotic priming. Methods: After reaching 5% of circulating leukemic blasts, five established T-ALL PDX models with aberrant NOTCH1 expression were divided into to two treatment groups each (8 mice per group). 1 group received the Notch inhibitor DBZ (Dibenzazepine; 10 μM/kg intraperitoneal every other day) and the other group was treated with vehicle. Short-term effect of DBZ in vivo was assessed after 1 week of treatment, when 3 mice per group were sacrificed and leukemic blasts were isolated from spleen and bone marrow. The remaining 5 mice were monitored for disease burden (by flow cytometry staining for human CD45+) and followed for survival. After reaching moribund state, animals were sacrificed, spleens and bone marrows were collected and prepared for further analyses. To assess DBZ efficacy in vivo, the presence of active NOTCH1 (ICN1) in spleen and bone marrow was analyzed by Immunohistochemistry analysis (IHC). Enhancer landscapes were identified by chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) for Histone 3 Lysine 27 acetylation (H3K27ac). A custom computational pipeline that incorporates algorithms for demultiplexing, alignment, normalization, peak calling, and computation of signal intensities within peaks was used to call differential peaks and intersect with RNA-sequencing results. BH3 profiling was performed on leukemic blasts isolated from spleen to measure overall mitochondrial priming and to identify anti-apoptotic dependencies. Results: In four out of five T-ALL PDX models, IHC analysis of spleen and bone marrow demonstrated a drastic downregulation of active NOTCH1 upon DBZ treatment, validating the efficacy of the used inhibitor. Weak ICN1 staining that remained unchanged upon DBZ treatment, was observed in 1 of the models, resulting in the exclusion of this strain from further functional analysis. Survival analysis of the four T-ALL PDX models expressing ICN1, revealed the presence of two Notch inhibitor sensitive and two refractory strains. The latter strains developed DBZ resistance rapidly after starting treatment (less than 10 days). One sensitive strain eventually developed resistance, while the second showed long-term disease control. Transcriptional profiling (bulk RNA-seq) of Notch inhibitor refractory strains versus sensitive identified the intrinsic apoptotic pathway as one of the most deferentially deregulated GSEA signatures. H3K27ac ChIPseq analysis at pretreatment (baseline), showed increased signal intensity of H3K27ac peaks at BCL2 and MCL1 enhancers in the refractory strains compared to sensitive. Upon DBZ treatment, while the enhancer state in refractory T-ALL remained unchanged, in the sensitive strains the signal intensity of H3K27ac peaks within the BCL2 and MCL1 loci decreased. Mitochondrial BH3 profiling at baseline demonstrated BCL-2 dependency (measured via BAD peptide) in sensitive strains and MCL-1 dependency (measured via MS1 peptide) in refractory strains. Upon DBZ treatment, sensitive strains showed a decrease in BCL-2 dependency and compensatory switch to MCL1-dependency, while dependency profile remained unchanged in refractory T-ALL. Conclusions: Our results suggest that enhancer rewiring near anti-apoptotic genes is critical for Notch inhibitor resistance. Combining BH3 profiling with enhancer profiling may allow to predict drug responses in vivo and may contribute to the identification of novel therapeutic targets for combination therapy in resistant disease. Disclosures Letai: Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Weinstock:Celgene: Research Funding. Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding.
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- 2019
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10. Interleukin 21 - its potential role in the therapy of B-cell lymphomas
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Kristopher A. Sarosiek, Izidore S. Lossos, and Shruti Bhatt
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Cytotoxicity, Immunologic ,Cancer Research ,Lymphoma, B-Cell ,medicine.medical_treatment ,Drug Evaluation, Preclinical ,Biology ,Immunomodulation ,03 medical and health sciences ,Interleukin 21 ,0302 clinical medicine ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Immunologic Factors ,STAT3 ,Multiple myeloma ,B cell ,Clinical Trials as Topic ,Interleukins ,Interleukin ,Hematology ,medicine.disease ,Lymphoma ,medicine.anatomical_structure ,Cytokine ,Treatment Outcome ,Oncology ,030220 oncology & carcinogenesis ,Immune System ,Immunology ,biology.protein ,Receptors, Interleukin-21 ,CD8 ,030215 immunology ,Signal Transduction - Abstract
Interleukin-21 (IL-21), a member of IL-2 cytokine family, has pleotropic biological effects on lymphoid and myeloid cells. During the past 15 years, since the discovery of IL-21, great advances have been made regarding its biological activity and the mechanisms controlling IL-21-mediated cellular responses, especially in hematological malignancies. Preclinical studies have shown that IL-21R is expressed on healthy and neoplastic B-cells and exogenous IL-21 can induce direct apoptosis of IL-21R expressing B-cell non-Hodgkin lymphomas (NHL), making it a potentially attractive anti-lymphoma therapy. However, in some hematological malignancies such as multiple myeloma, Hodgkin lymphoma and Burkitt lymphoma, IL-21 can induce proliferation of neoplastic B-cells. In NHL, the underlying mechanism of cell death was found to be different between the various subtypes, including activation of different JAK/STAT signal transduction pathways or other factors. Immunomodulatory effects of IL-21 have also been reported to contribute to its anti-tumor effects as described by earlier studies in solid tumors and B-cell associated malignancies. These effects are predominantly mediated by IL-21's ability to activate cytolytic activities by NK-cells and CD4+/CD8+ T-cells. In this review, we provide an overview of IL-21's effects in NHL, results from clinical trials utilizing IL-21, and propose how IL-21 can be therapeutically exploited for treating these lymphomas.
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- 2016
11. Pathophysiological significance and therapeutic targeting of germinal center kinase in diffuse large B-cell lymphoma
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Jennifer R. Chapman, Tyzoon K. Nomanbhoy, Matthew P. Patricelli, Hwan Geun Choi, Elena M. Cortizas, John Matthews, Nathanael S. Gray, Dita Gratzinger, Robert Tibshirani, Daxing Zhu, Ramiro E. Verdun, Ragini Shyam, Ranjana H. Advani, Jianming Zhang, Li Tan, Andrew J. Gentles, Yasodha Natkunam, Ezequiel Martinez, Xiaoyu Jiang, Sara J. Buhrlage, Izidore S. Lossos, Shruti Bhatt, and Shideh Chinichian
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0301 basic medicine ,Immunology ,Apoptosis ,Biology ,Protein Serine-Threonine Kinases ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Germinal Center Kinases ,Pathogenesis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,RNA interference ,immune system diseases ,hemic and lymphatic diseases ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Kinase activity ,neoplasms ,Lymphoid Neoplasia ,Kinase ,Germinal center ,Cell Biology ,Hematology ,Cell Cycle Checkpoints ,medicine.disease ,Lymphoma ,Neoplasm Proteins ,Gene expression profiling ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer research ,Heterografts ,Lymphoma, Large B-Cell, Diffuse ,Diffuse large B-cell lymphoma ,Neoplasm Transplantation - Abstract
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, yet 40% to 50% of patients will eventually succumb to their disease, demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNAs that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that germinal center kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell-cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Because the majority of DLBCL tumors (∼80%) exhibit activation of GCK, this therapy may be applicable to most patients.
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- 2016
12. Identification of LMO2 transcriptome and interactome in diffuse large B-cell lymphoma
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Shruti Bhatt, Xiaoyu Jiang, Aharon G. Freud, Shuchun Zhao, Jose A. Martinez-Climent, Andrew J. Gentles, Chuanxin Huang, Izidore S. Lossos, Xiaoqing Lu, Isidro Sánchez-García, Elena Cubedo, Isabel Romero-Camarero, Yasodha Natkunam, Carlos E. Bacchi, Ari Melnick, Ministerio de Economía y Competitividad (España), Fundación Alfonso Martín Escudero, National Institutes of Health (US), Dwoskin Family Foundation, Ministerio de Ciencia e Innovación (España), and European Commission
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LMO2 ,Molecular Sequence Data ,Immunology ,Biology ,Biochemistry ,Interactome ,Cell Line ,Transcriptome ,Transferases ,immune system diseases ,Cell Line, Tumor ,Proto-Oncogene Proteins ,hemic and lymphatic diseases ,medicine ,Humans ,Promoter Regions, Genetic ,Mitosis ,Adaptor Proteins, Signal Transducing ,Cell Proliferation ,Centrosome ,Regulation of gene expression ,B-Lymphocytes ,Lymphoid Neoplasia ,Base Sequence ,Tumor Suppressor Proteins ,Germinal center ,Cell Biology ,Hematology ,LIM Domain Proteins ,medicine.disease ,Gene Expression Regulation, Neoplastic ,Cancer research ,RNA, Long Noncoding ,Lymphoma, Large B-Cell, Diffuse ,Diffuse large B-cell lymphoma ,TAL1 - Abstract
LMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis. © 2012 by The American Society of Hematology., E.C. is partially supported by the Fundacion Alfonso Martin Escudero. I.S.L. is supported by National Institutes of Health (NIH) grants CA109335, CA122105, and U56 CA112973, and the Dwoskin Family and Recio Family foundations. I.S.G. is supported by MICINN (SAF2009-08 803), and by MEC OncoBIO Consolider- Ingenio 2010 (Ref. CSD2007-0017). All Spanish funding is cosponsored by the European Union FEDER program.
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- 2012
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13. miR-155 regulates HGAL expression and increases lymphoma cell motility
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Izidore S. Lossos, Liat Nadav Dagan, Klaus Rajewsky, Shruti Bhatt, Xiaoyu Jiang, and Elena Cubedo
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RHOA ,Lymphoma ,Cellular differentiation ,Blotting, Western ,Immunology ,Cell ,Motility ,Apoptosis ,Real-Time Polymerase Chain Reaction ,Biochemistry ,Mice ,Cell Movement ,hemic and lymphatic diseases ,microRNA ,Biomarkers, Tumor ,medicine ,Animals ,Humans ,RNA, Messenger ,Luciferases ,3' Untranslated Regions ,B-Lymphocytes ,Lymphoid Neoplasia ,biology ,Microfilament Proteins ,Intracellular Signaling Peptides and Proteins ,Germinal center ,Cell Differentiation ,Cell Biology ,Hematology ,Flow Cytometry ,Germinal Center ,medicine.disease ,Neoplasm Proteins ,Mice, Inbred C57BL ,MicroRNAs ,medicine.anatomical_structure ,Mutagenesis ,biology.protein ,Cancer research ,rhoA GTP-Binding Protein ,Diffuse large B-cell lymphoma - Abstract
HGAL, a prognostic biomarker in patients with diffuse large B-cell lymphoma and classic Hodgkin lymphoma, inhibits lymphocyte and lymphoma cell motility by activating the RhoA signaling cascade and interacting with actin and myosin proteins. Although HGAL expression is limited to germinal center (GC) lymphocytes and GC-derived lymphomas, little is known about its regulation. miR-155 is implicated in control of GC reaction and lymphomagenesis. We demonstrate that miR-155 directly down-regulates HGAL expression by binding to its 3′-untranslated region, leading to decreased RhoA activation and increased spontaneous and chemoattractant-induced lymphoma cell motility. The effects of miR-155 on RhoA activation and cell motility can be rescued by transfection of HGAL lacking the miR-155 binding site. This inhibitory effect of miR-155 suggests that it may have a key role in the loss of HGAL expression on differentiation of human GC B cells to plasma cell. Furthermore, this effect may contribute to lymphoma cell dissemination and aggressiveness, characteristic of activated B cell–like diffuse large B-cell lymphoma typically expressing high levels of miR-155 and lacking HGAL expression.
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- 2012
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14. Cell Type-Specific Deregulation of Polypyrimidine Tract- Binding Proteins (PTBPs) Drive Aberrant Splicing in Multiple Myeloma (MM) and Acute Myeloid Leukemia (AML)
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James D. Griffin, Sigitas Verselis, Shruti Bhatt, Linda M. Pilarski, Sophia Adamia, David P. Steensma, Richard Stone, Michael P. Chu, Daniel G. Tenen, Teru Hideshima, Daniel J. DeAngelo, and Kenneth C. Anderson
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RNA Splicing Factors ,Immunology ,Alternative splicing ,Intron ,CD34 ,Context (language use) ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Polypyrimidine tract ,RNA splicing ,Cancer research ,Minigene - Abstract
Genome-wide transcriptome profiling detected an increased splicing alterations in MM and AML. While these malignancies are derived from different cell linages, their tumor cells acquire similar aberrant splicing (AbSp), mostly intron retentions. To delineate AbSp mechanism in MM/AML, we focused on PTBPs (1/2/3) that play a critical role in intron excision. We have previously reported deregulated expression of splicing factors (SFs) in MM/AML patient and healthy donor (HD) bone marrow (BM). As MM progressed, PTBP1/2 progressively increased, and PTBP3 gradually decreased (ASH 2017). In AML, PTBP2/3 upregulation and PTBP1 downregulation were detected in patient samples in which increased intron retentions were identified by genome-wide splicing analysis (CCR 2015). Here, these findings were validated by TaqMan assays for PTBPs in 48 MM and 325 AML patient samples, 16 MM/AML cell lines, and in plasma cells (PCs) and CD34+cells from 14 HDBM. Results were consistent with differential expressions of PTBPs in MM/AML previously analyzed. Upregulation of PTBP1/2 and PTBP2/3 proteins were detected in MM and AML cell lines, respectively. PTBP1/2 upregulation was pronounced when MM cell lines were cocultured with BM stromal cells (MMBMSC) derived from MM patients' BM. Importantly, we detected increased proliferation and decreased apoptosis in MM/AML cell lines overexpressing PTBPs. These effects were most evident after coculturing MM cell lines with MMBMSC as compared to HDBMSC. To evaluate PTBP effects on AbSp, we knocked down and/or overexpressed PTBPs in MM/AML cell lines and assessed PDL1 splicing in MM, and NOTCH2 and FLT3 splicing in AML. PDL1 is spliced in ~ 30% of 90 MM patients; while NOTCH2/FLT3/CD13 are spliced in 78%/50% of 387 AML patients, respectively. After PTBP1/2 knockdown in MM cells we detected 4- to 11-fold downregulation of PDL1 splice variants, with proportional upregulation of wild-type PDL1 levels; concurrently, MM cell proliferation was decreased and apoptosis increased. To evaluate MM specific splicing alterations in the context of the MM BM microenvironment, PDL1 splicing, and PTBP1/2 mRNA/protein expressions, were monitored in MM cell lines cocultured with MMBMSC or HDBMSC. We observed time-dependent PDL1 splice variant upregulation, and higher levels of PTBP1/2 in MM cells. We also noted time-dependent PDL1 variant expression switching in association with PTBP1/2 deregulation in the BM microenvironment. RNA-seq analysis and western blotting showed that MMBMSC culture with tumor cells increased intron retention, and altered SF expressions, including PTBPs in BMSC. We next monitored PTBP effects on splicing in AML cells by evaluating NOTCH2 and FLT3 splicing in an TF1, an AML cell line that overexpresses PTBP2/3. RT-PCR analysis showed association between PTBP3 overexpression and NOTCH2 and FLT3 AbSp in TF1 cells. For further validation, we developed an ex vivo splicing assay composed of an FLT3/CD13 splicing cassette with a GFP reporter, which allows for evaluation of splicing events by flow cytometry (FACS) and microscopy. In this assay, cells overexpressing PTBP2/3 caused FLT3/CD13 minigene splicing similar to that detected in AML patients. Also, by RT-PCR, we showed that overexpression of PTBPs caused intron retention, that was confirmed by cloning and sequencing of PCR products and consistent with the FACS analysis and microscopy. Finally, we have tested effects of PTBPs using in vivo AML models (BMT and xenograft). In the BMT model, animal median survival was 48 days post-BMT for MLL-AF9/PTBP3 and 56 days in the control group. In the xenograft model, animal median survival was 66, 94, & 106 days after injection of TF1-PTBP3, TF1-PTBP2, and TF1 cells in mice, respectively (P>0.0001). These studies suggest that PTBP3 overexpression in partnership with the MLL-AF9 promotes occurrence of AML in mice. Tumor RNA samples harvested from these animals were subjected to RNA-seq analysis, which showed increased AbSp association with PTBP3 overexpression. Our studies indicate that deregulated PTBP1/2 expression in MM and of PTBP2/3 in AML drive time-dependent AbSp (intron retention) and splice variant switching, which in MM is induced by culture with BMSC; and conversely, that analogous changes are induced in BMSC cultured with tumor cells. They define role of deregulated expression of the PTBPs in MM/AML pathogenesis, and suggest novel targets for therapeutic intervention. Disclosures Stone: Pfizer: Consultancy; Jazz: Consultancy; Fujifilm: Consultancy; Merck: Consultancy; Cornerstone: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Novartis: Consultancy, Research Funding; Sumitomo: Consultancy; Ono: Consultancy; Orsenix: Consultancy; Otsuka: Consultancy; AbbVie: Consultancy; Agios: Consultancy, Research Funding; Amgen: Consultancy; Argenx: Other: Data and Safety Monitoring Board; Arog: Consultancy, Research Funding; Astellas: Consultancy. Griffin:Astellas Pharma: Consultancy; Novartis Pharma: Other: Grant, Patents & Royalties: Royalties ; Analysis Group: Consultancy; Sun Pharmaceuticals: Consultancy; RXi Pharmaceuticals: Consultancy; Lilly Pharmaceuticals: Other: Grant; Myeloproliferative Neoplasia Foundation: Other: Grant . Anderson:C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy.
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- 2018
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15. Dynamic BH3 Profiling Predicts for Clinical Response to Lenalidomide Plus Chemotherapy in Relapsed Acute Myeloid Leukemia
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Jacqueline S. Garcia, Geoffrey Fell, Shruti Bhatt, Bruno C. Medeiros, Jack Taw, Anthony Letai, David J. Iberri, and Traci M. Blonquist
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Oncology ,medicine.medical_specialty ,Mitoxantrone ,business.industry ,Immunology ,Induction chemotherapy ,Phases of clinical research ,Cell Biology ,Hematology ,Biochemistry ,Chemotherapy regimen ,Transplantation ,Internal medicine ,Cytarabine ,medicine ,business ,Etoposide ,medicine.drug ,Lenalidomide - Abstract
Background: Early prediction of response to novel chemotherapy combinations for patients diagnosed with relapsed or refractory acute myeloid leukemia (R/R AML) may have clinical utility. We conducted a phase 1 clinical trial (NCT01904643) of lenalidomide (LEN) given prior to MEC (Mitoxantrone + Etoposide + Cytarabine) salvage chemotherapy (LEN+MEC) for patients with R/R AML. Although the clinical trial was terminated due to administrative reasons, we explored the utility of FACS-based BH3 and dynamic BH3 profiling (DBP) assays to predict for response. Standard BH3 profiling is a functional assay that uses synthetic peptides derived from the BH3 domains of pro-apoptotic BCL-2 family members to measure mitochondrial "priming" (a cell's readiness for apoptosis) (Ni Chonghaile, Science, 2011). DBP measures whether short term incubation with drug enhances priming (Montero J, Cell, 2015). Methods: Primary objective was to assess the safety/toxicity profile (DLTs and MTD) of LEN+MEC. LEN was administered 5-7 days prior to MEC and dose was escalated using an mTPI design with a target toxicity level of 30%. Dose levels (DL) included LEN given at doses of 15mgx5d (DL1), 15mgx7d (DL2), 25 mgx5d (DL3), 25mgx7d (DL4), 50mgx5d (DL5), and 50 mgx7d (DL6). Dose-escalation decisions were made based on DLTs that occurred during induction chemotherapy. DLT was defined as persistent cytopenias in absence of disease, gr 3 or higher non-hematologic toxicity not due to underlying disease or intolerable gr 2 toxicities attributable to LEN. Blood/bone marrow samples were collected at pre-treatment, post LEN only, and post LEN+MEC therapy. Clinical response was defined per ELN guidelines. For correlative studies, standard BH3 and DBP assays were used to measure overall mitochondrial priming and individual anti-apoptotic protein dependencies. DBP was performed after overnight (~16h) treatment with LEN at various concentrations on pretreatment leukemic blasts. Delta priming, utilized as a readout, represents the change in mitochondrial outer membrane depolarization after LEN treatment as measured by cytochrome C release following exposure to BH3 peptides. Data analysis was performed blinded to clinical response. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off level for respective clinical end points. Results: 17 total patients with R/R AML received LEN+MEC. Median age was 55 years (range: 22-72) with 12% Fav, 47% Int and 41% Adv risk by ELN risk stratification. 60% were heavily pre-treated. DLTs were observed in DL2 (n=1, gr 4 sepsis due to bacteremia), DL3 (n=1, gr 5 septic shock), and DL5 (n=1, gr3 bacteremia). MTD was not yet reached due to early study termination (although patients were treated at all 6 dose levels). No GVHD was observed among post-transplant patients. Composite remission rate among the 17 patients (CR + PR) was 35% (5 CR and 1 PR). Of the 5 patients who achieved CR, 3 of them eventually progressed. Median PFS and OS for the entire cohort was 2.30 (90% CI 1.84-3.47) and 4.60 (90% CI 2.84-22.10) months, respectively. To assess whether mitochondrial priming might correlate with clinical response, we first performed standard BH3 profiling with a series of peptides on 16 pretreatment bone marrow samples. Mitochondrial cytochrome c release in response to BIM-BH3 correlated weakly with clinical response. DBP on pretreatment myeloblasts in response to BIM (activator) or PUMA (sensitizer) peptides were able to stratify responders based on an increase in mitochondrial priming caused by ex vivo LEN treatment (P Conclusions: LEN+MEC was tolerable and had modest efficacy in this small study. Correlative studies showed that leukemic blasts briefly treated ex vivo with LEN which then underwent DBP were predictive of in vivo response among a heterogeneous patient population to LEN+MEC treatment. This study is an example of how a powerful biomarker can potentially identify those who are more likely to respond to novel therapies to avoid undesirable toxicity in those who would otherwise have less chance to benefit. Further validation is required to confirm the utility of DBP as a predictive biomarker to targeted agents given with chemotherapy. Disclosures Garcia: Celgene: Consultancy. Letai:AbbVie: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; Flash Therapeutics: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report. Medeiros:Genentech: Employment; Celgene: Consultancy, Research Funding.
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- 2018
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16. Efficacy of bortezomib in a direct xenograft model of primary effusion lymphoma
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Enrique A. Mesri, Juan Carlos Ramos, Kristopher A. Sarosiek, Andrew J. Gentles, Lucas E. Cavallin, Ngoc L. Toomey, Wilfredo Blasini, Shruti Bhatt, Yasodha Natkunam, and Izidore S. Lossos
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DNA Replication ,Male ,medicine.medical_treatment ,Down-Regulation ,Apoptosis ,Nod ,medicine.disease_cause ,Bortezomib ,Proto-Oncogene Proteins c-myc ,Mice ,Fatal Outcome ,immune system diseases ,In vivo ,Lymphoma, Primary Effusion ,hemic and lymphatic diseases ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Doxorubicin ,Kaposi's sarcoma-associated herpesvirus ,Vero Cells ,Aged, 80 and over ,Chemotherapy ,Multidisciplinary ,business.industry ,Cell Cycle ,NF-kappa B ,Virion ,virus diseases ,Biological Sciences ,medicine.disease ,Boronic Acids ,Survival Analysis ,Xenograft Model Antitumor Assays ,Lymphoma ,Gene Expression Regulation, Neoplastic ,Treatment Outcome ,E2F3 Transcription Factor ,Pyrazines ,Herpesvirus 8, Human ,Immunology ,Unfolded Protein Response ,Cancer research ,Primary effusion lymphoma ,business ,medicine.drug - Abstract
Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma most commonly diagnosed in HIV-positive patients and universally associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Chemotherapy treatment of PEL yields only short-term remissions in the vast majority of patients, but efforts to develop superior therapeutic approaches have been impeded by lack of animal models that accurately mimic human disease. To address this issue, we developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells. We used this model to show that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions. The proapoptotic effects of bortezomib are not mediated by inhibition of the prosurvival NF-κB pathway or by induction of a terminal unfolded protein response. Transcriptome analysis by genomic arrays revealed that bortezomib down-regulated cell-cycle progression, DNA replication, and Myc-target genes. Furthermore, we demonstrate that in vivo treatment with either bortezomib or doxorubicin induces KSHV lytic reactivation. These reactivations were temporally distinct, and this difference may help elucidate the therapeutic window for use of antivirals concurrently with chemotherapy. Our findings show that this direct xenograft model can be used for testing novel PEL therapeutic strategies and also can provide a rational basis for evaluation of bortezomib in clinical trials.
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- 2010
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17. miR-181a negatively regulates NF-κB signaling and affects activated B-cell-like diffuse large B-cell lymphoma pathogenesis
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Ari Melnick, Shruti Bhatt, Xiaoyu Jiang, Goldi A. Kozloski, Francisco Vega, Rita Shaknovich, Jose F. Ruiz, and Izidore S. Lossos
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0301 basic medicine ,Immunology ,Mice, SCID ,Biology ,Lymphocyte Activation ,Biochemistry ,03 medical and health sciences ,Mice ,Mice, Inbred NOD ,hemic and lymphatic diseases ,Cell Line, Tumor ,microRNA ,medicine ,Animals ,Humans ,B cell ,Regulation of gene expression ,B-Lymphocytes ,NF-kappa B ,Germinal center ,Cell Biology ,Hematology ,medicine.disease ,NFKB1 ,Xenograft Model Antitumor Assays ,Lymphoma ,Gene Expression Regulation, Neoplastic ,MicroRNAs ,030104 developmental biology ,medicine.anatomical_structure ,Cell Transformation, Neoplastic ,HEK293 Cells ,Cancer research ,Female ,Lymphoma, Large B-Cell, Diffuse ,Signal transduction ,Diffuse large B-cell lymphoma ,HeLa Cells ,Signal Transduction - Abstract
Distinct subgroups of diffuse large B-cell lymphoma (DLBCL) genetically resemble specific mature B-cell populations that are blocked at different stages of the immune response in germinal centers (GCs). The activated B-cell (ABC)-like subgroup resembles post-GC plasmablasts undergoing constitutive survival signaling, yet knowledge of the mechanisms that negatively regulate this oncogenic signaling remains incomplete. In this study, we report that microRNA (miR)-181a is a negative regulator of nuclear factor κ-light-chain enhancer of activated B-cells (NF-κB) signaling. miR-181a overexpression significantly decreases the expression and activity of key NF-κB signaling components. Moreover, miR-181a decreases DLBCL tumor cell proliferation and survival, and anti-miR-181a abrogates these effects. Remarkably, these effects are augmented in the NF-κB dependent ABC-like subgroup compared with the GC B-cell (GCB)-like DLBCL subgroup. Concordantly, in vivo analyses of miR-181a induction in xenografts results in slower tumor growth rate and prolonged survival in the ABC-like DLBCL xenografts compared with the GCB-like DLBCL. We link these outcomes to relatively lower endogenous miR-181a expression and to NF-κB signaling dependency in the ABC-like DLBCL subgroup. Our findings indicate that miR-181a inhibits NF-κB activity, and that manipulation of miR-181a expression in the ABC-like DLBCL genetic background may result in a significant change in the proliferation and survival phenotype of this malignancy.
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- 2015
18. Chlamydophila psittaci-negative ocular adnexal marginal zone lymphomas express self polyreactive B-cell receptors
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S. Alvarez Cubela, Hendrik Veelken, Shruti Bhatt, Xiaoqing Lu, Francisco Vega, Jennifer R. Chapman-Fredricks, Daxing Zhu, D. K. Hsu, Fu-Tong Liu, Kranthi Kunkalla, I. S. Lossos, and Fengjie Guo
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Cancer Research ,Spectrometry, Mass, Electrospray Ionization ,B-cell receptor ,Blotting, Western ,Protein Array Analysis ,Fluorescent Antibody Technique ,Receptors, Antigen, B-Cell ,Enzyme-Linked Immunosorbent Assay ,Immunofluorescence ,Autoantigens ,Immunoenzyme Techniques ,Antigen ,medicine ,Tumor Cells, Cultured ,Humans ,Immunoprecipitation ,B cell ,Chromatography, High Pressure Liquid ,B-Lymphocytes ,medicine.diagnostic_test ,biology ,Eye Neoplasms ,breakpoint cluster region ,Hematology ,Lymphoma, B-Cell, Marginal Zone ,Psittacosis ,Marginal zone ,medicine.disease ,Recombinant Proteins ,Lymphoma ,medicine.anatomical_structure ,Oncology ,Chlamydophila psittaci ,Immunology ,biology.protein ,Antibody - Abstract
The pathogenesis of Chlamydophila psittaci-negative ocular adnexal extranodal marginal zone lymphomas (OAEMZLs) is poorly understood. OAEMZLs are monoclonal tumors expressing a biased repertoire of mutated surface immunoglobulins. Antigenic activation of the B-cell receptor (BCR) may have a role in the pathogenesis of these lymphomas. We have analyzed the reactivity of recombinant OAEMZL immunoglobulins. OAEMZL antibodies reacted with self-human antigens, as demonstrated by enzyme-linked immunosorbent assays, HEp-2 immunofluorescence and human protein microarrays. All the analyzed recombinant antibodies (rAbs) exhibited polyreactivity by comprehensive protein array antibody reactivity and some rAbs also demonstrated rheumatoid factor activity. The identity of several reactive antigens was confirmed by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry. The tested rAbs frequently reacted with shared intracellular and extracellular self-antigens (for example, galectin-3). Furthermore, these self-antigens induced BCR signaling in B cells expressing cognate surface immunoglobulins derived from OAEMZLs. These findings indicate that interactions between self-antigens and cognate OAEMZL tumor-derived BCRs are functional, inducing intracellular signaling. Overall, our findings suggest that self-antigen-induced BCR stimulation may be implicated in the pathogenesis of C. psittaci-negative OAEMZLs.
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- 2015
19. Direct and immune-mediated cytotoxicity of interleukin-21 contributes to antitumor effects in mantle cell lymphoma
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Xiaoyu Jiang, Izidore S. Lossos, Shruti Bhatt, Salma Parvin, John Matthews, Kristopher A. Sarosiek, Elif Isik, Dekuang Zhao, and Anthony Letai
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CD4-Positive T-Lymphocytes ,STAT3 Transcription Factor ,medicine.medical_treatment ,Immunology ,Apoptosis ,Lymphoma, Mantle-Cell ,Biochemistry ,Proto-Oncogene Proteins c-myc ,Interleukin 21 ,Cyclin D1 ,Cell Line, Tumor ,medicine ,Cytotoxic T cell ,Animals ,Humans ,Immunologic Factors ,Cytotoxicity ,bcl-2-Associated X Protein ,Chemistry ,Interleukins ,Interleukin ,Cell Biology ,Hematology ,Immunotherapy ,medicine.disease ,Killer Cells, Natural ,Mice, Inbred C57BL ,STAT protein ,Cancer research ,Mantle cell lymphoma ,Female ,Receptors, Interleukin-21 ,Signal Transduction - Abstract
Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin D1 in 95% of patients. MCL patients experience frequent relapses resulting in median survival of 3 to 5 years, requiring more efficient therapeutic regimens. Interleukin (IL)-21, a member of the IL-2 cytokine family, possesses potent antitumor activity against a variety of cancers not expressing the IL-21 receptor (IL-21R) through immune activation. Previously, we established that IL-21 exerts direct cytotoxicity on IL-21R–expressing diffuse large B-cell lymphoma cells. Herein, we demonstrate that IL-21 possesses potent cytotoxicity against MCL cell lines and primary tumors. We identify that IL-21–induced direct cytotoxicity is mediated through signal transducer and activator of transcription 3-dependent cMyc upregulation, resulting in activation of Bax and inhibition of Bcl-2 and Bcl-XL. IL-21–mediated cMyc upregulation is only observed in IL-21–sensitive cells. Further, we demonstrate that IL-21 leads to natural killer (NK)-cell–dependent lysis of MCL cell lines that were resistant to direct cytotoxicity. In vivo treatment with IL-21 results in complete FC-muMCL1 tumor regression in syngeneic mice via NK- and T-cell–dependent mechanisms. Together, these data indicate that IL-21 has potent antitumor activity against MCL cells via direct cytotoxic and indirect, immune-mediated effects.
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- 2015
20. A Functional Approach to Precision Medicine Identifies Targeted Therapies for Acute Myeloid Leukemia
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Sophia Adamia, Vineeth Kumar Murali, Anthony Letai, Holly Zhu, David M. Weinstock, Amanda L. Christie, Shruti Bhatt, and Jackie S Garcia
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business.industry ,Venetoclax ,Genetic heterogeneity ,Immunology ,Priming (immunology) ,Myeloid leukemia ,Cell Biology ,Hematology ,Precision medicine ,Biochemistry ,Chemotherapy regimen ,chemistry.chemical_compound ,chemistry ,Cancer research ,Medicine ,Biomarker (medicine) ,business ,Quizartinib - Abstract
Identification of genetic heterogeneity in acute myeloid leukemia (AML) has provided a unique opportunity for the greater individualization of therapy. However the implementation of new therapies has lagged far behind the ability to initially recognize operationally important genetic lesions. Until we have further bridged this gap between the identification of genetic lesions and the resultant knowledge of effective therapies, alternative strategies for rapidly identifying candidate therapies can become an important tool for precision medicine. Since most agents, regardless of whether "cytotoxic" or "targeted" ultimately function by activating the mitochondrial apoptotic pathway, we hypothesized that a tool that measures mitochondrial sensitivity may serve as a broadly predictable biomarker. We developed a dynamic BH3 profiling (DBP) technique that measures early death signaling within 8-16 hours after exposure to drugs. Increased cell death signaling is reflected by increased mitochondrial sensitivity (i.e. increased priming) to standardized BH3 peptides mimicking pro-apoptotic proteins. To develop a personalized therapy for AML using DBP, we utilized 20 independent patient derived xenograft (PDX) models, established from de novo, primary refractory or relapsed (R/R) patients (available at http://www.PRoXe.org). Human myeloblasts from spleen and bone marrow of xenotransplanted NSG mice were exposed to 30 targeted and 3 standard of care drugs to determine mitochondrial responses. Unsupervised hierarchical clustering of ex-vivo DBP measurements across AML PDXs revealed several distinct clusters. Majority of targeted agents with an ability to induce priming in selective PDXs were enriched within a cluster, including kinase inhibitors, epigenetic modifiers, SMAC mimetic and chemotherapy drugs. In contrast, a discrete subcluster of drugs showed sensitivity across majority of PDXs, including BH3 mimetics, CDK9 inhibitors and HDAC inhibitors. Drugs with identical mechanism of action showed similar priming patterns across PDXs. Of note, 3 non-myeloid PDXs clustered distinctly from AML, an indication of differential priming responses owing to their cells of origin. AML PDXs developed from treatment naïve patients clustered adjacently and showed greater priming responses to a large number of drugs as opposed to PDXs from R/R patients that formed a discrete cluster. These data reveal that mitochondrial priming can stratify AML PDXs according to its predicted sensitivity to targeted agents. Next, we validated ability of DBP to predict in-vivo responses of single agent birinapant (SMAC mimetic), JQ-1 (BRD-4 inhibitor), quizartinib (FLT-3 inhibitor), and venetoclax (BCL-2 inhibitor) across 6 AML PDX models, prioritized based on their greatest range of priming responses. We found that birinapant was most efficacious nonetheless as expected from ex-vivo DBP studies, responses varied between different PDX models. Myeloblasts of those PDXs that showed the greatest drug-induced changes in apoptotic priming were indeed the PDXs with the highest in-vivo responses. When we compared the ability of DBP to identify sensitive PDXs with additional precision medicine tools such as genomics, we found that DBP was able to accurately predict quizartinib activity in PDXs expressing WT FLT-3, which would have been predicted to be unresponsive based on genomic analysis. Collectively, priming responses obtained from ex-vivo DBP was able to rank different PDX models according to their sensitivities to targeted agents (AUC of ROC curve 0.8731, p In summary, our findings highlight that mitochondria-based measurements could identifying individualized therapy for a heterogeneous population and may serve as a as a powerful biomarker to identify the best responders to patient therapies. Disclosures Letai: AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding.
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- 2017
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21. Statins Potentiate the Cytotoxic Effect of ABT-199 in Diffuse Large B Cell Lymphoma
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Thanh-Trang Vo, Shruti Bhatt, Jonathan H. Schatz, Anthony Letai, Jong-Hoon Scott Lee, and David A. Fruman
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Statin ,Venetoclax ,medicine.drug_class ,Chronic lymphocytic leukemia ,Immunology ,Cell Biology ,Hematology ,Pharmacology ,Biology ,medicine.disease ,Biochemistry ,Lymphoma ,Leukemia ,chemistry.chemical_compound ,chemistry ,Cancer cell ,medicine ,Protein prenylation ,Diffuse large B-cell lymphoma - Abstract
BCL-2 is a key pro-survival protein that is highly expressed in many leukemias and lymphomas. ABT-199 (venetoclax) is a small molecule inhibitor of BCL-2 that has demonstrated impressive responses in chronic lymphocytic leukemia (CLL) leading to FDA approval for second line treatment of patients with 17p deletion. However, other hematologic malignancies are less responsive to ABT-199 as a single agent, suggesting that combinations of targeted therapies may be required to elicit more promising responses. We have investigated the potential of combining ABT-199 with HMG-CoA reductase (HMGCR) inhibitors (statins), which have known anti-cancer potential in hematologic malignancies. Using multiple chemically distinct statin compounds, we observed profound synergistic induction of apoptosis when combined with ABT-199 in both human diffuse large B cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML) cell lines. This synergy was also seen in primary murine B lymphoma cells over-expressing MYC and BCL-2. Importantly, addition of exogenous mevalonate completely rescued cells from the combination, confirming on-target efficacy of HMGCR inhibition. Using BH3 profiling, we found that simvastatin significantly primed lymphoma cells for undergoing apoptosis (termed mitochondrial priming). Notably, the degree of priming correlated with its ability to synergize with ABT-199, suggesting that BH3 profiling may be used to predict patient responses. The combination did not synergize to kill normal human peripheral blood mononuclear cells from healthy donors, suggesting that statins may selectively prime cancer cells for apoptosis. Mechanistic studies support the hypothesis that statins synergize with ABT-199 by suppressing protein prenylation, particularly protein geranylgeranylation. In support, the addition of exogenous geranylgeranyl pyrophosphate (GGPP) completely rescued cells from the effects of simvastatin. Furthermore, selective inhibition of protein geranylgeranyl transferase (GGT) increased priming and was sufficient to recapitulate the effects of simvastatin in combination with ABT-199. Statins and GGT inhibitors increased the mitochondrial abundance of a subset of BH3-only pro-apoptotic proteins. Lastly, we have identified Rap1A de-prenylation as a marker of pharmacodynamic response to statins in vivo. Thus, this project highlights a novel combination for use in aggressive lymphomas, establishes its efficacy and tolerability using preclinical models, and provides proof-of-concept to warrant investigation of its clinical potential. Disclosures Letai: AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.
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- 2016
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22. FLT3 Splice Variant (FLT3Va) As a Potential Immunotherapeutic Target in Patients with Acute Myeloid Leukemia (AML)
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David M. Dorfman, Daniel J. DeAngelo, Ilene Galinsky, Sarah R. Walker, David P. Steensma, Sophia Adamia, Shruti Bhatt, James D. Griffin, William Lento, Ricardo Attar, David M. Weinstock, Martha Wadleigh, Natalie I Voeks, Anthony Letai, Jeffrey A. Nemeth, David A. Frank, and Richard Stone
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0301 basic medicine ,Myeloid ,medicine.drug_class ,Immunology ,CD33 ,Monoclonal antibody ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,Medicine ,biology ,business.industry ,Cell Biology ,Hematology ,Molecular biology ,Transplantation ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,biology.protein ,Antibody ,Stem cell ,Clone (B-cell biology) ,business - Abstract
Alternative pre-mRNA splicing (AS) is a normal epigenetic phenomenon, a key regulator of gene expression, yields multiple transcripts and thus a variety of proteins from a single gene. Mutations in the spliceosome components resulting in aberrant splicing isoforms are common in AML, and other myeloid neoplasms, and may generate leukemia-specific neoantigens targetable with an antibody-drug conjugates (ADCs) or blocking antibodies. Our previous studies revealed that the FLT3 cell surface receptor is one of the most commonly misspliced genes in AML (54-63% of ~400 AML patients). We conducted cloning and sequencing analyses in AML cells and identified multiple aberrant splice-variants of FLT3 that resulted from either skipping of one or more exons or activation of cryptic splicing sites. Transfection of cDNA with three of these variants in TF-1 (AML cell line) cells resulted in expression of Flt3 variant proteins on the cell surface. We successfully generated rabbit polyclonal antiserum against a unique peptide sequence present in the most commonly expressed abnormal splice variant, which we termed Flt3Va. Immunoblots performed with the polyclonal antibody identified a ~160 kDa protein expressed by TF-1 cells transfected with FLT3Va, and the antibody did not react with untransfected TF-1 cell lysate. Using standard techniques, we generated rabbit hybridomas and evaluated the clones by flow cytometry and western blotting experiments. Based on these data, we selected one antibody clone (15-7) for further experiments. The 15-7 anti-Flt3Va rabbit monoclonal antibody identified Flt3Va protein expressed on the cell surface and within the cytoplasm of transfected TF-1 cells by flow cytometry and western blotting. However, no Flt3Va protein was detected in untransfected TF-1 cells or normal CD34+ bone marrow cells. The 15-7 antibody bound to 26 of 52 primary AML samples and 5 of 10 primagraft samples (PDX models) of human AML. Immunoblotting analyses of PDX models and patient samples confirmed binding to a protein of the expected size (130-160 kDa). Additionally, multi-parameter flow cytometry in 10 PDX models and 52 primary demonstrated that putative AML stem cells (as defined by the CD45dim, CD34, CD38, CD33, c-Kit cell surface expression) co-expressed Flt3Va antigen in 50% samples evaluated. An analysis of Flt3Va protein localization by live cell imaging showed a punctate distribution of Flt3Va on the cell surface. Furthermore, we observed that overexpression of Flt3Va in TF-1 cells led to GM-CSF growth factor independence. Analysis of TF-1 cells in the absence of GM-CSF and Flt3 ligand demonstrated constitutive activation of STAT5, an important mediator of Flt3 signaling, in Flt3Va overexpressing cells. In addition, Erk1/2 phosphorylation was also increased in Flt3Va overexpressing cells, another downstream effector of Flt3. In an effort to determine if Flt3Va+ cells had tumor repopulating ability, we sorted 0.3X10^6 Flt3Va+ and Flt3Va- cells from a PDX sample and injected the sorted populations or unsorted bulk tumor cells into NSG mice. The human cell engraftment in the mice was detected by the expression of human CD45, CD33, CD34, CD38, and c-kit antigens in the peripheral blood. In two experiments, mice injected with Flt3Va+ cells had detectable circulating leukemic cells by ~18 days after injection, while those injected with Flt3Va- cells had detectable circulating leukemic cells after the 4th week. These results suggest both Flt3Va+ and Flt3Va- cell populations are able to reconstitute leukemia after transplantation in NSG mice. However, Flt3Va+ may be expressed by an aggressive AML clone that facilitate early tumor engraftment. Overall, these studies suggest that Flt3Va is a leukemia-specific neoantigen and is an attractive potential immunotherapeutic target in AML. Proteins such as Flt3Va generated by alternative splicing are common in AML and may be targets for of novel blocking antibodies or ADCs, minimizing effects on normal tissues. Disclosures Adamia: Janssen: Research Funding. Nemeth:Janssen: Employment. Attar:Janssen: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Steensma:Millenium/Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Ariad: Equity Ownership; Genoptix: Consultancy. Weinstock:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Baxter: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy. Stone:Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Juno Therapeutics: Consultancy; Roche: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Merck: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Seattle Genetics: Consultancy. Griffin:Janssen: Research Funding; Novartis: Consultancy, Research Funding.
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- 2016
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23. Abstract 418: Mitochondrial perturbations as a novel approach to personalized medicine
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Shruti Bhatt, David M. Weinstock, and Anthony Letai
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Cancer Research ,business.industry ,Cell ,Myeloid leukemia ,Priming (immunology) ,Cancer ,medicine.disease ,medicine.anatomical_structure ,Oncology ,Apoptosis ,Cancer cell ,Immunology ,Cancer research ,Medicine ,Biomarker (medicine) ,Personalized medicine ,business - Abstract
Recent progress in cancer research has been marked by the continued development of exciting drugs that selectively target vulnerabilities present only in cancer cells, so-called targeted therapies. Too often, however, we are lacking the proper predictive biomarkers to identify which patients will benefit most from individual targeted therapies. Since most chemotherapeutic agents ultimately function by inducing the mitochondrial apoptotic pathway, we hypothesized that a tool that measures mitochondrial sensitivity may serve as a broadly predictable biomarker. Hence to address the unmet need for predictive biomarkers, we have developed a tool called Dynamic BH3 Profiling (DBP). In this approach, we briefly treat cancer cells (14h) from patients with targeted drugs and measure the apoptotic threshold of a cell “priming” by treatment with peptides derived from BH3 domains of pro-apoptotic proteins. Previous studies from our laboratory demonstrated that when a patient's cells exhibit robust death signaling in the DBP assay in response to a particular drug, it predicts a good clinical response to that drug in patients. Using xenotransplantation of primary AML samples into NSG mice (PDX models), we validated the utility of DBP to personalize acute myeloid leukemia therapy. We established 12 independent AML PDX models and determined the mitochondrial response of splenic myeloblasts from each PDX to 25 targeted agents. Ex-vivo DBP measurement demonstrated heterogeneous responses; while some drugs induced priming in all PDXs, others increased priming in only selective PDX models. For instance, JQ-1 (BRD-4 inhibitor) and birinapant (SMAC mimetic) demonstrated inverse correlation of their activity among 3 different PDXs. These suggested that mitochondrial priming can stratify AML PDX models according to predicted sensitivity to targeted agents. When we compared DBP results with cell death assay, we found that myeloblasts from most of the PDXs failed to sustain long-term culture (72h), a requirement for cytotoxicity measurements. However, myeloblasts from 3 of the PDXs with high starting viability showed strong correlation between mitochondrial priming and cell death, implying that cells with increased mitochondrial response are destined to commit apoptosis. By testing functional state of the apoptotic pathway in myeloblasts from spleen, bone-marrow and peripheral blood with BH3 profiling, we found that splenic blasts were relatively more primed for apoptosis in 8 out of 10 PDXs. Correlatively, drug induced priming also varied between myeloblasts from these 3 different sites. Finally, we are directly testing the performance of DBP as a predictive biomarker by treating a variety of PDX models with drugs, including drugs predicated to be active and drugs predicated to be inactive. Collectively, mitochondria based measurements can predict good and bad response to individual targeted agents and may serve as a powerful biomarker to choose patient therapy. Citation Format: shruti bhatt, david weinstock, Anthony Letai. Mitochondrial perturbations as a novel approach to personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 418.
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- 2016
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24. Interleukin-21 Potently Induces Direct and Indirect Cytotoxicity of Mantle Cell Lymphoma
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Kristopher A. Sarosiek, John Matthews, Dekuang Zhao, Izidore S. Lossos, Shruti Bhatt, and Salma Parvin
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Programmed cell death ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,Interleukin 21 ,Cyclin D1 ,Cell culture ,medicine ,Cytotoxic T cell ,Mantle cell lymphoma ,Cytotoxicity ,Diffuse large B-cell lymphoma - Abstract
Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphomacharacterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the t(11;14)(q13;q32) chromosomal translocation. This incurable lymphoma is highly chemoresistant, with short response duration, frequent relapses and eventual death, even with the most aggressive chemotherapeutic regimens. Interleukin 21 (IL-21), a member of the IL-2 cytokine family, possesses potent anti-tumor activity against a variety of cancers not expressing IL-21 receptor (IL-21R) through activation of the immune system. Previously, we established that apart from its immuno-stimulatory effects, IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B cell lymphoma (DLBCL) cells (Sarosiek KA et al.Blood, 2010). Herein we carried out a comprehensive study to delineate the effects of IL-21 on MCL cell lines and primary tumors. Flow-cytometric analysis revealed that all MCL cell lines (Mino, HBL2, Jeko1, G519, IRM2, SP53, Z138, UPN1 and L128) as well as primary tumors expressed surface IL-21R at variable levels. Treatment of Mino, HBL2 and SP53 cells with IL-21 (100ng/mL) led to a marked time-dependent decrease in cell proliferation and increased cell death. In contrast, Jeko1, IRM2, L128, Z138, UPN1 and G519 cells exhibited resistance to IL-21 treatment. Similarly, primary MCL tumors treated with IL-21 in vitro exhibited significant cell death in 4 of 5 cases expressing IL-21R. To decipher the mechanism of IL-21-induced direct cytotoxicity, responsive and resistant cell lines as well as primary tumors were utilized. Similarly to our previous study in DLBCL, IL-21 stimulation resulted in dramatic phosphorylation of STAT1 and STAT3 in IL-21 responsive cell lines (Mino, HBL-2, SP53) and a primary tumor, while minimal STAT5 phosphorylation was observed only in Mino. We have previously demonstrated that IL-21-induced cell death in DLBCL is mediated by STAT3-induced upregulation of c-Myc expression. Correspondingly, IL-21 treatment led to c-Myc upregulation only in IL-21-sensitive MCL cell lines and primary tumors but not in the resistant cell lines and primary tumors, independent of the IL-21R expression levels. Knockdown of c-Myc prevented IL-21-induced Mino cell death, whereas c-Myc overexpression in resistant MCL cell lines facilitated IL-21-induced cytotoxicity. Furthermore, IL-21 resulted in upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic proteins Bcl-XL and Bcl-2, as previously observed in DLBCL. Knockdown of STAT3 or Bax using specific siRNAs in Mino cells resulted in abrogation of the IL-21-induced cell death. In contrast to a previous report (Gelebart P et al. Leukemia, 2009), knockdown of STAT1 or overexpression of dominant negative STAT1 failed to prevent IL-21-induced Mino cell death. We also discovered that apart from its direct cytotoxic effects, IL-21 also leads to NK-cell dependent lysis of MCL cell lines resistant to direct cytotoxicity. In vivo treatment with IL-21 resulted in complete FC-muMCL1 tumor regression in syngeneic mice (p Disclosures No relevant conflicts of interest to declare.
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- 2014
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25. Germinal Center Kinase Regulates The Proliferation and Survival Of Diffuse Large B-Cell Lymphoma
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Sara J. Buhrlage, Dita Gratzinger, Jennifer R. Chapman, Tyzoon K. Nomanbhoy, Li Tan, Nathanael S. Gray, Andrew J. Gentles, Ranjana H. Advani, Ezequiel Martinez, Jianming Zhang, Shideh Chinichian, Matthew P. Patricelli, Hwan Geun Choi, Daxing Zhu, Ragini Shyam, Shruti Bhatt, Yasodha Natkunam, John Matthews, Izidore S. Lossos, and Xiaoyu Jiang
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Pathology ,medicine.medical_specialty ,Kinase ,Immunology ,Germinal center ,MAP2K4 ,Cell Biology ,Hematology ,MAP3K1 ,Biology ,medicine.disease ,Biochemistry ,Lymphoma ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Cancer research ,Neoplastic transformation ,Kinase activity ,Diffuse large B-cell lymphoma - Abstract
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). The pathogenesis of DLBCL represents a multi-step process that involves the accumulation of multiple genetic and molecular lesions. Marked advances in the understanding of DLBCL pathobiology have been made by the application of gene expression arrays, comparative genomic hybridization arrays and “next” generation sequencing. This led to the identification of previously unrecognized DLBCL subtypes (germinal center-like (GCB) and activated B cell-like (ABC)) as well as type specific-deregulation of particular signaling pathways. These approaches focused on genetic aberrations and mRNA expression profiles, whereas the crucial events transforming normal cells are executed by proteins. Kinases play an important role in neoplastic transformation. Herein, we have undertaken the task of profiling kinase activity in DLBCL to further delineate potential mechanisms of DLBCL pathogenesis and develop novel therapeutic agents. A comprehensive analysis of global kinase activity/protein expression was performed using KiNativ technology. Kinomic analysis of 8 DLBCL cell lines, as compared to non-cancerous primary B-cells, led to the discovery of 13 members of the MAPK cascade which were activated and/or overexpressed in DLBCL. Only three of the detected MAPK members were inactive or had reduced expression compared to their non-cancerous counterparts. To determine whether these findings could be extended to de novo primary human DLBCL tumors, we performed immunohistochemistry (IHC) of the proximally activated kinase, MAP4K2 or “Germinal Center Kinase” (GCK) and the phosphorylated forms of its downstream targets: MAP3K1, MAP2K4, MAP2K7, and C-jun N-terminal Kinase 1 (JNK1). Analyzed kinases were expressed and activated in more than 80% of primary DLBCL tumors, confirming the KiNativ cell line data. The kinase array data was further corroborated with classical immunoprecipitation-based JNK and p38 assays. Hierarchical clustering analysis of 36 DLBCL specimens stained for GCB and ABC markers demonstrated that GCK expression/activation is not DLBCL subtype specific. Notably, in a cohort of 151 primary DLBCL cases, we found that patients whose tumors did not express GCK had an estimated progression free survival (PFS) of 85% at 10 years of follow up, whereas those tumors expressing GCK were associated with significantly reduced PFS of 53% (p=0.04). While there was a similar trend in overall survival, it did not reach statistical significance, which may be due to the relatively small number of DLBCL cases not expressing GCK and the potential rescue of these patients with second line treatments. RNA interference studies in DLBCL cell lines confirmed the importance of GCK for the survival of these tumors, resulting in reduced viability and G0/G1 arrest. We next developed a small molecule inhibitor, HG6-64-1. KiNativ, Ambit and Invitrogen profiling of HG6-64-1 targets revealed that it potently inhibited GCK. In vitro treatment with the novel GCK inhibitor, HG6-64-1, led to cell cycle arrest and the induction of apoptosis in DLBCL cell lines and primary DLBCL tumors. G452, a DLBCL cell line minimally expressing GCK, was not affected by HG6-64-1. In vivo treatment with HG6-64-1, via intratumoral and intraperitoneal injections, significantly decreased the tumor growth rate resulting in a significantly extended lifespan of DLBCL xenograft mouse models. Overall our results identified a previously unrecognized activation of the GCK pathway which contributes to the proliferation and survival of DLBCLs and can be used as a therapeutic target using novel GCK inhibitors. Disclosures: Patricelli: ActivX Biosciences: Employment. Nomanbhoy:ActivX Biosciences: Employment.
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- 2013
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26. B Cell Receptors Of Chlamydophila Psittaci negative MALT Lymphomas Of The Ocular Adnexa Recognize Common Self-Antigens
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Shruti Bhatt, Marcus Dühren-von Minden, Jennifer R. Chapman-Fredricks, Hassan Jumaa, Izidore S. Lossos, Xiaoqing Lu, Chen Lossos, Hendrik Veelken, and Daxing Zhu
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biology ,Immunology ,B-cell receptor ,Cell Biology ,Hematology ,Biochemistry ,Virology ,Molecular biology ,Isotype ,Immunoglobulin G ,Antigen ,Methylenetetrahydrofolate dehydrogenase ,biology.protein ,Immunohistochemistry ,Rab ,Antibody - Abstract
Ocular adnexal mucosa associated lymphoid tissue lymphomas (OAMALTLs) are the most common lymphomas in the ocular adnexa. The etiology and pathogenesis of OAMALTLs are still controversial. Association with Chlamydophila (C.) psittaci infection demonstrates marked geographic differences. We have previously reported biased IGHV (IGHV4 family and IGHV4-34 gene) and IGKV (IGKV3-20) usage in C. psittaci-negative OAMALTLs (PLoS One. 2011;6(12):e29114; Am J Hematol.2013; 88:379). However, the identity and role of potential antigens (Ags) in OAMALTL pathogenesis are unknown. Herein we evaluated the reactivity of OAMALTLs derived B-cell receptors (BCRs) for bacterial and human antigens. IGHV and IGKV genes derived from 5 OAMALTLs (tumors 4726 and 4438 expressing IGHV4-34 with and without somatic mutations, respectively, both paired with IGKV3-20; tumor 4968 expressing IGHV3-30 and IGKV1-33; tumor 11274 expressing IGHV3-23 and IGKV3-20; and tumor 5334 expressing IGHV4-59 and IGKV3-1) were cloned into pAH6180 and pAN6714 plasmids, respectively. Irrespective of the original isotype, all recombinant antibodies (rAbs) representing tumor BCRs were expressed on a common IgG3 heavy chain backbone to facilitate the detection and comparability in reactivity assays and to ensure that differences in binding activity could be attributed to specific variable regions. rAbs were produced in HEK293T cells adapted to serum-free suspension cultures. rAbs did not react with bacterial proteins derived from lysates of Staphylococcus aureus, Salmonella enteridis and C. psittaci infected HeLa cells. The rAbs also did not react with I/i Ags. Rheumatoid factor (RF) enzyme-linked immunosorbent assay (ELISA) suggested that rAbs derived from tumors 4726, 4968, 5334, and 11274 exhibit rheumatoid factor activity. However, a competitive inhibition assay using increasing quantities of purified human IgG Fc fragment failed to block the reactivity, indicating non-specific RF reactivity. To test the rAbs for reactivity with self-antigens, the standard indirect immunofluorescence assay (IFA) using permeabilized human HEp-2 cells was performed. All tumor derived rAbs were reactive with HEp-2 cells and exhibited diverse staining patterns; predominantly cytoplasmic staining with rAbs 5334 and 4968 and combined nuclear and cytoplasmic staining with the others. We next examined tumor derived rAbs for polyreactivity based on the commonly used definition of reactivity with at least two of the following diverse self and non-self antigens: ssDNA, dsDNA, insulin, and LPS. Only rAb 4726 showed polyreactivity based on these standard criteria. To identify the self-antigens recognized by the other OAMALTLs derived rAbs, antibody specificity profiling was performed using ProtoArray Human Protein Microarrays v5.0 containing more than 9,000 proteins (Life Technologies), using 0.1 μg/mL and 1.0 μg/mL concentrations of each rAb. The data were processed using Invitrogen’s proprietary ProtoArray Prospector software. These analyses identified 17, 11, 9 and 25 candidate Ags for rAb4438, rAb4726, rAb5334, and rAb11274, respectively. Some of the candidate Ags were unique for specific rAbs (e.g. methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2 (MTHFD2) reactive with rAb4438) while others were recognized by more than one rAb (e.g. galectin-3/LGALS3 was reactive with rAb4438, rAb4726 and rAb11274). Follow up bidirectional co-immunoprecipitation with commercial and tumor derived rAbs confirmed recognition of these, as well as several additional protein microarray identified Ags. We specifically focused on galectin-3, which was recognized by 3 of the tumor BCRs. ELISAs performed with and without lactose confirmed rAbs reactivity with galectin-3. IFA using rAbs and a commercial galectin-3 Ab confirmed colocalization; siRNA-induced galectin-3 knockdown eliminated staining with both commercial and tumor derived rAbs. Immunohistochemistry of primary OAMALTLs showed that galectin-3 is expressed by tumor microenvironment cells but not by the tumor lymphocytes. Our findings demonstrate that the BCR of C. psittaci negative OAMALTLs exhibit oligo-self-reactivity and identified galectin-3 as a common antigen. The role of these antigens in pathogenesis of OAMALTLs is under investigation. Disclosures: No relevant conflicts of interest to declare.
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- 2013
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27. Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine
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Shruti Bhatt, Izidore S. Lossos, John M. Timmerman, Joseph D. Rosenblatt, Seung Uon Shin, Daxing Zhu, and Xiaoyu Jiang
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Antibody-dependent cell-mediated cytotoxicity ,biology ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Complement-dependent cytotoxicity ,Raji cell ,Interleukin 21 ,medicine.anatomical_structure ,Cell killing ,immune system diseases ,hemic and lymphatic diseases ,biology.protein ,medicine ,Cancer research ,Cytotoxic T cell ,Antibody ,B cell - Abstract
The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.
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- 2013
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28. Preclinical Activity of Brentuximab Vedotin (SGN-35) in Primary Effusion Lymphoma (PEL)
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Brittany Ashlock, Izidore S. Lossos, Yaso Natkunam, Shruti Bhatt, Enrique A. Mesri, and Juan Carlos Ramos
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Pathology ,medicine.medical_specialty ,CD30 ,Immunology ,Population ,Biology ,Biochemistry ,chemistry.chemical_compound ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Propidium iodide ,Brentuximab vedotin ,education ,education.field_of_study ,integumentary system ,Cell Biology ,Hematology ,medicine.disease ,Lymphoma ,Cell killing ,Monomethyl auristatin E ,chemistry ,Cancer research ,Primary effusion lymphoma ,medicine.drug - Abstract
Abstract 3728 Primary effusion lymphoma (PEL) is a distinct and aggressive subtype of non-Hodgkin lymphoma (NHL) commonly presenting with pleural, peritoneal, or pericardial malignant effusions usually without a contiguous tumor mass. PEL is most commonly diagnosed in HIV-positive patients, accounting for 4% of all NHLs in this population, yet may also develop in immunosuppressed HIV-negative individuals. While Human Herpes Virus 8 (HHV8 or Kaposi's sarcoma-associated herpesvirus) is directly implicated in the oncogenesis of this lymphoma, most PEL cases are also associated with Epstein-Barr virus and the combination of the two may facilitate transformation. The tumor cells exhibit plasmablastic features and express CD45, CD38, CD138, HHV8 and CD30. PEL is an aggressive tumor characterized by a short median survival of only 6 months with current therapeutic approaches underscoring the urgent need for development of new therapeutics. Brentuximab vedotin (SGN-35) is an antibody-drug conjugate (ADC) comprised of an anti-CD30 monoclonal antibody cAC10 conjugated by a protease-cleavable dipeptide linker to a potent cell killing agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and is transported to lysosomes, where the peptide linker is selectively cleaved allowing binding of the released MMAE to tubulin and leading to cell cycle arrest and apoptosis. Brentuximab vedotin was recently reported to have promising antitumor activity in CD30 expressing tumors, such as Hodgkin and Anaplastic large cell lymphomas. Since PEL tumors are reported to express CD30, we have hypothesized that brentuximab vedotin might be effective in the treatment of this NHL subtype. Initially, we have confirmed by flow cytometry the expression of CD30 on PEL cell lines (UM-PEL 1, UM-PEL 3, BC-1 and BC-3), and by review of immunohistochemistry and flow cytometry results in patients with previous diagnosis of PEL at our institution. To examine in vitro potency of brentuximab vedotin, UM-PEL 1, UM-PEL 3, BC-1 and BC-3 PEL cell lines were treated with brentuximab vedotin at concentration ranging from 0–100 micrograms/ml. Staining with YO-PRO and Propidium Iodide (PI) demonstrated dose dependent cell apoptosis and death in all the cell lines at 72 hours post treatment. In contrast, control IgG conjugated with MMAE failed to induce apoptosis and cell death of PEL cell lines confirming specific brentuximab vedotin cytotoxicity. Furthermore, brentuximab vedotin decreased proliferation of PEL cells at 48 hours leading to a complete proliferation arrest at 72 hours, as measured by MTS assay. These effects were absent after equivalent doses of control IgG conjugated drug treatment. Supportive to this, labeling of cells with PI to detect active DNA content by flow cytometry showed that bretuximab vedotin induced growth arrest in G2/M phase. To further establish the anti-tumor potential of brentuximab vedotin in vivo, we used the direct xenograft UM-PEL 1 model, established in our laboratory (Sarosiek, PNAS 2010), which mimics human PEL tumors. UM-PEL 1 bearing mice were injected intraperitoneally 3 times a week with brentuximab vedotin or control IgG conjugated MMAE for 4 weeks. Brentuximab vedotin treatment markedly prolonged overall survival of UM-PEL-1 bearing mice compared to controls (p Disclosures: No relevant conflicts of interest to declare.
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- 2011
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29. Synergistic Preclinical Activity of Bortezomib with Suberoylanilide Hydroxamic Acid (SAHA) in Primary Effusion Lymphoma (PEL)
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Enrique A. Mesri, Juan Carlos Ramos, Izidore S. Lossos, Brittany Ashlock, Shruti Bhatt, and Ngoc L. Toomey
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education.field_of_study ,Bortezomib ,Immunology ,Population ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Herpesviridae ,medicine.anatomical_structure ,Lytic cycle ,medicine ,Proteasome inhibitor ,Cancer research ,Primary effusion lymphoma ,education ,Vorinostat ,B cell ,medicine.drug - Abstract
Abstract 1650 Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma typically presenting as effusions in the serous body cavities without a contiguous tumor mass. PEL may develop in elderly immunosuppressed HIV-negative individuals but more commonly affects HIV-positive patients, accounting for 4% of all lymphomas in this population. Kaposi's sarcoma-associated herpesvirus (KSHV) is directly implicated in the pathogenesis of PEL, however in most patients the malignant B cells are also coinfected with Epstein-Barr virus which may facilitate transformation. Current chemotherapeutic approaches result in dismal outcome of PEL patients with a median survival of only 6 months. Consequently, development of new therapeutic approaches is urgently needed. Recently we reported development of the UM-PEL1 direct xenograft mice model reproducing human PEL (Sarosiek, PNAS 2010) in which bortezomib (BORT) induced virus lytic reactivation leading to malignant B cell death and transient remission of the PEL in vivo. Further improvement on this monotherapy is warranted. Recent studies have shown that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor is a highly effective viral lytic-cycle inducer. As herpesviruses are dependent on the proteasome for replication and mature viral production, induction of lytic replication with concomitant inhibition of the proteasome may provide a highly targeted strategy for eradicating KSHV infected cells without leading to increased viremia. Consequently, we hypothesized that combining BORT with SAHA may act synergistically in PEL tumors. Incubation of human PEL cell lines, UM-PEL1, BC1, BC3 and BC5 with BORT-SAHA resulted in increased apoptotis compared to individual treatment with BORT or SAHA, as assayed by flow cytometry using YO-PRO/PI staining. Concordantly, a statistically significant decrease in UM-PEL1 cell proliferation and viability, as examined by an MTT assay, was observed at 48 and 72 hours following combination therapy as compared to untreated cells or cells treated individually with BORT or SAHA. Cell cycle analysis demonstrated that BORT-SAHA combination induced more pronounced G1 cell cycle arrest and apoptosis as compared to individual treatments. SAHA induced a more robust KSHV lytic reactivation compared to BORT. Intriguingly, the BORT-SAHA combination led to an increased expression of the master lytic transactivator RTA and thymidine kinase, however the late lytic gene, K8.1, showed reduced mRNA expression relative to the individual SAHA treatment. These findings were further confirmed by immunofluorescence staining of the K8.1 protein suggesting that BORT could inhibit mature virion production in lytically reactivated malignant B-cells. To comprehensively examine the activity of the BORT-SAHA combination compared to individual BORT or SAHA treatments in vivo, we used UM-PEL1 direct xenograft model. Mice receiving intraperitoneal BORT-SAHA combination showed statistically significant prolonged survival compared to all the control treatments (p Disclosures: No relevant conflicts of interest to declare.
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- 2011
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30. Identification of LMO2 Transcriptome and Interactome in Diffuse Large B-Cell Lymphoma by Integrated Experimental and Computational Approach
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Jose A. Martinez-Climent, Andrew J. Gentles, Isidro Sánchez-García, Isabel Romero-Camarero, Sylvia K. Plevritis, Shruti Bhatt, Xiaoqing Lu, Chuanxin Huang, Ari Melnick, Elena Cubedo, Yasodha Natkunam, Xiaoyu Jiang, and Izidore S. Lossos
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LMO2 ,Immunology ,GATA2 ,Germinal center ,Cell Biology ,Hematology ,Transfection ,Biology ,Biochemistry ,Molecular biology ,Chromatin ,Transcriptome ,LYL1 ,hemic and lymphatic diseases ,Chromatin immunoprecipitation - Abstract
Abstract 438 The LMO2 is a cysteine-rich protein containing two zinc binding LIM domains indirectly regulating gene expression by mediating protein-protein interactions with other transcriptional factors (TFs), facilitating the formation of multipartite DNA-binding complexes. It is mainly expressed in endothelial and hematopoietic cells forming cell type specific complexes containing LDB1, TAL1, E2A and GATA2 proteins in endothelial cells and hematopoietic stem cells and LDB1, TAL-1, GATA1 and E proteins in the erythroid lineage. In these cells LMO2 is involved in angiogenesis and erythroid hematopoiesis. In T cells where LMO2 is only expressed in immature CD4/CD8 double-negative thymocytes, the LMO2 complex consist of LDB1, TAL1 and E2A, but may also bind to GATA3. Aberrant expression of LMO2 in T cells induces leukemogenesis. In the B cells, LMO2 is specifically up regulated in Germinal Center (GC) B cells. LMO2 is also expressed in GC-derived non-Hodgkin's lymphomas and is one of the most powerful survival predictors in DLBCL patients. However, its function in GC B cells and DLBCL is currently unknown. In the present study we aimed to characterize the LMO2 transcriptome and interactome in DLBCL cells. Gene expression arrays were performed in Rck8 cells expressing low levels of endogenous LMO2, in which LMO2 was stably overexpressed to levels observed in GCB-like DLBCL. A total of 311 differentially expressed genes (DEGs) between control cell lines transfected with mock vector and LMO2 stably transfected samples were identified at FDR of 0.05. Sixty-four genes were down-regulated by LMO2 transfection, while 247 were up-regulated. Prominent amongst these were 27 genes encoding proteins of the histone cluster 1, colocalized on chromosome 6 and consistently higher expressed in LMO2 expressing cell lines. At the same time, multiple cell-cycle-related genes were also up-regulated by LMO2 transfection, including centromere proteins (CENPE, CENPI, and CENPN), the kinetochore associated protein NDC80 and the mitotic protein CDC25C. Expression of several randomly selected genes (SPIC, LAX1, DLEU2, DOCK3, CHND2 and TNFRSF9) was validated by real time PCR and confirmed the observed gene expression changes upon LMO2 overexpression. To obtain a higher-level view of expression changes, we compared the list of DEGs to Gene Ontology (GO) categories. This revealed significant induction of genes involved in chromosome, nucleosome, and chromatin and protein-DNA complex assembly. We screened our microarray data against the Broad Institute Molecular Signatures Database, to identify TFs whose target genes were significantly up- or down-regulated by stable LMO2 transfection. This candidate list was filtered to include only those TFs whose target genes significantly overlapped with genes which had previously identified binding motifs for LMO2 complexes in their promoter sequences. Our analysis identified Sp1, NFAT, Elk1, and LEF1 as potential novel LMO2 co-factors, but not classical TAL1 and GATA partner proteins. We examined the expression of previously reported and new potential interaction partners of LMO2 in DLBCL cell lines and GC lymphocytes by Western blotting and analyzed the interaction of the expressed proteins with LMO2 by co-immunprecipitation. We identified that in DLBCL the LMO2 complex contains some of the traditional partners such as LDB1, E2A, HEB, lyl1 and ETO2, but not TAL1 or GATA proteins. Furthermore, we demonstrated LMO2 interaction also with SP1, ELK1, NFATc1 and LEF1 proteins. All the identified LMO2 interacting partners are also expressed in GC B lymphocytes, suggesting the presence of a similar protein complex in both normal and malignant B cells. LMO2 interaction with LEF protein affected its transcriptional activity, as measured by reporter assays. A Chromatin immunoprecipitation assay using promoter region of the DLEU2 gene, whose expression was up-regulated by LMO2 expression, confirmed the direct and specific LMO2 binding to a region harboring E2A and a SP1 binding sites. Overall, our studies identified an LMO2 transcriptome in DLBCL as well as its interactome, which contains novel previously unknown interacting partners. These findings suggest that LMO2 may affect unique and novel cellular functions in GC lymphocytes which may potentially contribute to DLBCL pathogenesis and are currently being investigated in our laboratory. Disclosures: No relevant conflicts of interest to declare.
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- 2011
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31. Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models
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Ari Melnick, Xiaoyu Jiang, Rita Shaknovich, Goldi A. Kozloski, Shruti Bhatt, and Izidore S. Lossos
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Immunology ,Germinal center ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Gene expression profiling ,DNA methylation ,microRNA ,medicine ,Cancer research ,Centroblasts ,Carcinogenesis ,Transcription factor ,Diffuse large B-cell lymphoma - Abstract
Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis. Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. Figure 1 Figure 1. Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. Disclosures No relevant conflicts of interest to declare.
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