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Aberrant expression of HOX and MEIS1 family genes, as seen in KMT2A-rearranged, NUP98-rearranged, or NPM1-mutated leukemias leads to arrested differentiation and leukemia development. HOX family genes are essential gatekeepers of physiologic hematopoiesis, and their expression is regulated by the interaction between KMT2A and menin. Menin inhibitors block this interaction, downregulate the abnormal expression of MEIS1 and other transcription factors and thereby release the differentiation block. Menin inhibitors show significant clinical efficacy against KMT2A-rearranged and NPM1-mutated acute leukemias, with promising potential to address unmet needs in various pediatric leukemia subtypes. In this collaborative initiative, pediatric and adult hematologists/oncologists, and stem cell transplant physicians have united their expertise to explore the potential of menin inhibitors in pediatric leukemia treatment internationally. Our efforts aim to provide a comprehensive clinical overview of menin inhibitors, integrating preclinical evidence and insights from ongoing global clinical trials. Additionally, we propose future international, inclusive, and efficient clinical trial designs, integrating pediatric populations in adult trials, to ensure broad access to this promising therapy for all children and adolescents with menin-dependent leukemias., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Abstract: BRG1 (SMARCA4) and BRM (SMARCA2) are the mutually exclusive core ATPases of the chromatin remodeling BAF (BRG1/BRM-associated factor) complexes. They enable transcription factors/cofactors to access enhancers/promoter and modulate gene expressions responsible for cell growth and differentiation of acute myeloid leukemia (AML) stem/progenitor cells. In AML with MLL1 rearrangement (MLL1r) or mutant NPM1 (mtNPM1), although menin inhibitor (MI) treatment induces clinical remissions, most patients either fail to respond or relapse, some harboring menin mutations. FHD-286 is an orally bioavailable, selective inhibitor of BRG1/BRM under clinical development in AML. Present studies show that FHD-286 induces differentiation and lethality in AML cells with MLL1r or mtNPM1, concomitantly causing perturbed chromatin accessibility and repression of c-Myc, PU.1, and CDK4/6. Cotreatment with FHD-286 and decitabine, BET inhibitor (BETi) or MI, or venetoclax synergistically induced in vitro lethality in AML cells with MLL1r or mtNPM1. In models of xenografts derived from patients with AML with MLL1r or mtNPM1, FHD-286 treatment reduced AML burden, improved survival, and attenuated AML-initiating potential of stem-progenitor cells. Compared with each drug, cotreatment with FHD-286 and BETi, MI, decitabine, or venetoclax significantly reduced AML burden and improved survival, without inducing significant toxicity. These findings highlight the FHD-286-based combinations as a promising therapy for AML with MLL1r or mtNPM1., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

AML with chromosomal alterations involving 3q26 overexpresses the transcription factor (TF) EVI1, associated with therapy refractoriness and inferior overall survival in AML. Consistent with a CRISPR screen highlighting BRD4 dependency, treatment with BET inhibitor (BETi) repressed EVI1, LEF1, c-Myc, c-Myb, CDK4/6, and MCL1, and induced apoptosis of AML cells with 3q26 lesions. Tegavivint (TV, BC-2059), known to disrupt the binding of nuclear β-catenin and TCF7L2/LEF1 with TBL1, also inhibited co-localization of EVI1 with TBL1 and dose-dependently induced apoptosis in AML cell lines and patient-derived (PD) AML cells with 3q26.2 lesions. TV treatment repressed EVI1, attenuated enhancer activity at ERG, TCF7L2, GATA2 and MECOM loci, abolished interactions between MYC enhancers, repressing AML stemness while upregulating mRNA gene-sets of interferon/inflammatory response, TGF-β signaling and apoptosis-regulation. Co-treatment with TV and BETi or venetoclax induced synergistic in vitro lethality and reduced AML burden, improving survival of NSG mice harboring xenografts of AML with 3q26.2 lesions., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Germline, mono-allelic mutations in RUNX1 cause familial platelet disorder (RUNX1-FPD) that evolves into myeloid malignancy (FPD-MM): MDS or AML. FPD-MM commonly harbors co-mutations in the second RUNX1 allele and/or other epigenetic regulators. Here we utilized patient-derived (PD) FPD-MM cells and established the first FPD-MM AML cell line (GMR-AML1). GMR-AML1 cells exhibited active super-enhancers of MYB, MYC, BCL2 and CDK6, augmented expressions of c-Myc, c-Myb, EVI1 and PLK1 and surface markers of AML stem cells. In longitudinally studied bone marrow cells from a patient at FPD-MM vs RUNX1-FPD state, we confirmed increased chromatin accessibility and mRNA expressions of MYB, MECOM and BCL2 in FPD-MM cells. GMR-AML1 and PD FPD-MM cells were sensitive to homoharringtonine (HHT or omacetaxine) or mebendazole-induced lethality, associated with repression of c-Myc, EVI1, PLK1, CDK6 and MCL1. Co-treatment with MB and the PLK1 inhibitor volasertib exerted synergistic in vitro lethality in GMR-AML1 cells. In luciferase-expressing GMR-AML1 xenograft model, MB, omacetaxine or volasertib monotherapy, or co-treatment with MB and volasertib, significantly reduced AML burden and improved survival in the immune-depleted mice. These findings highlight the molecular features of FPD-MM progression and demonstrate HHT, MB and/or volasertib as effective agents against cellular models of FPD-MM., (© 2024. The Author(s).)

Purpose: Treatment outcomes in patients with relapsed/refractory (R/R) myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) remains dismal. On the basis of both extensive preclinical data and emerging clinical data, treatment with bromodomain and extra-terminal domain inhibitors (BETi) is a potential approach for patients with high-risk myeloid malignancies., Patients and Methods: We conducted a phase I trial to study the safety and efficacy of PLX51107 (BETi) and azacitidine combination therapy in patients with R/R AML and high-risk (HR) MDS and studied mechanisms of resistance to the combination therapy., Results: Thirty-seven patients with HR R/R MDS (n = 4) and R/R AML (n = 33) were treated. Sixteen patients (43%) had MECOM gene rearrangement and 7 other patients had TP53 mutation. Median prior number of therapies was three (range 1-9); 97% had received prior hypomethylating agent and 84% prior venetoclax. Overall response rate was 8/37 (22%): complete remission with incomplete platelet recovery (n = 1); morphologic leukemia-free state (n = 2); hematologic improvement (n = 5). The most common nonhematologic toxicities were febrile neutropenia and pneumonia in 12 (32%) patients each; 6 patients (17%) had severe hyperbilirubinemia. RNA-sequencing analysis of mononuclear cells harvested on treatment (day 3) versus pretreatment showed significant changes in mRNA expressions in responders: downregulation of MYC, BCL2, IL7R, and CDK6 and upregulation of HEXIM1, CD93, DCXR, and CDKN1A. Immunoblot analyses confirmed reduction in protein levels of c-Myc, CDK6, BCL2, and BCL-xL, and induction of BRD4 and HEXIM1 protein levels in responders., Conclusions: In a heavily pretreated patient cohort with R/R MDS and AML, PLX51107+ azacitidine was well-tolerated and resulted in modest clinical benefit., (©2023 American Association for Cancer Research.)

In AML with NPM1 mutation causing cytoplasmic dislocation of NPM1, treatments with Menin inhibitor (MI) and standard AML chemotherapy yield complete remissions. However, the causal and mechanistic linkage of mtNPM1 to the efficacy of these agents has not been definitively established. Utilizing CRISPR-Cas9 editing to knockout (KO) or knock-in a copy of mtNPM1 in AML cells, present studies demonstrate that KO of mtNPM1 from AML cells abrogates sensitivity to MI, selinexor (exportin-1 inhibitor), and cytarabine. Conversely, the knock-in of a copy of mtNPM1 markedly sensitized AML cells to treatment with MI or cytarabine. Following AML therapy, most elderly patients with AML with mtNPM1 and co-mutations in FLT3 suffer AML relapse with poor outcomes, creating a need for novel effective therapies. Utilizing the RNA-Seq signature of CRISPR-edited AML cells with mtNPM1 KO, we interrogated the LINCS1000-CMap data set and found several pan-HDAC inhibitors and a WEE1 tyrosine kinase inhibitor among the top expression mimickers (EMs). Additionally, treatment with adavosertib (WEE1 inhibitor) or panobinostat (pan-HDAC inhibitor) exhibited synergistic in vitro lethal activity with MI against AML cells with mtNPM1. Treatment with adavosertib or panobinostat also reduced AML burden and improved survival in AML xenograft models sensitive or resistant to MI., (© 2023. The Author(s).)

Immune checkpoint inhibitors (PD-1 inhibitors) have shown clinical activity in Richter transformation-diffuse large B-cell lymphoma variant (RT-DLBCL), thus providing for a novel therapeutic approach. The study group consists of 64 patients with RT-DLBCL. Expression of PD-1, PD-L1, CD30, and microsatellite instability (MSI) status (hMLH1, hMSH2, hMSH6, PMS1) was assessed using immunohistochemistry. EBV-encoded RNA (EBER) was evaluated using colorimetric in situ hybridization. PD-1 and PD-L1 expression levels were categorized on the basis of tumor cell expression as follows: negative (< 5%), positive to low-positive (5-50%), or high-positive (> 50%). An "immune evasion phenotype" (IEP) was defined as RT-DLBCL cases having high-positive expression of PD-1 and/or PD-L1 on tumor cells. The level of PD1-positive tumor-infiltrating lymphocytes (TILs) was estimated as a fraction of total lymphocytes and categorized as negative/low vs. brisk (> 20%). 28/64 (43.7%) patients were characterized as IEP+ RT-DLBCL. A brisk level of PD1+ TILs was significantly more common in IEP1+ compared with IEP- tumors (17/28, 60.7% vs. 5/34, 14.7%; p = 0.001). In addition, CD30 expression was significantly more common in IEP+ compared with IEP- RT-DLBCL (6/20, 30% vs. 1/27, 3.7%; p = 0.0320). Two (2/36; 5.5%) cases were positive for EBER, both IEP+. There was no significant difference between the two groups in terms of age, sex, or time to transformation. Assessment of mismatch repair proteins demonstrated absence of microsatellite instability (MSI) in all cases (18/18; 100%). Notably, patients with brisk PD1+ TILs had a significantly better OS compared to those with a negative/low infiltrate (p = 0.0285)., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Integrated clinicopathological and molecular analyses of Richter transformation of diffuse large B-cell lymphoma subtype (RT-DLBCL) cases remain limited. This study group included 142 patients with RT-DLBCL. Morphological evaluation and immunophenotyping, using immunohistochemistry and/or multicolour flow cytometry, were performed. The results of conventional karyotyping, fluorescence in situ hybridisation analysis and mutation profiling performed using next generation sequencing were reviewed. Patients included 91 (64.1%) men and 51 (35.9%) women with a median age of 65.4 years (range 25.4-84.9 years) at the time of RT-DLBCL diagnosis. Patients had CLL for a median of 49.5 months (range 0-330 months) before onset of RT-DLBCL. Most cases (97.2%) of RT-DLBCL had immunoblastic (IB) morphology, the remainder had a high grade morphology. The most commonly expressed markers included: CD19 (100%), PAX5 (100%), BCL2 (97.5%), LEF1 (94.7%), CD22 (90.2%), CD5 (88.6%), CD20 (85.7%), CD38 (83.5%), MUM1 (83.3%), CD23 (77%) and MYC (46.3%). Most (51/65, 78.4%) cases had a non-germinal centre B-cell immunophenotype. MYC rearrangement was detected in 9/47 (19.1%) cases, BCL2 rearrangement was detected in 5/22 (22.7%) cases, and BCL6 rearrangement was detected in 2/15 (13.3%) cases. In comparison to CLL, RT-DLBCL had higher numbers of alterations involving chromosomes 6, 17, 21, and 22. The most common mutations detected in RT-DLBCL involved TP53 (9/14, 64.3%), NOTCH1 (4/14, 28.6%) and ATM (3/14, 21.4%). Among RT-DLBCL cases with mutant TP53, 5/8 (62.5%) had TP53 copy number loss, and among those, such loss was detected in the CLL phase of the disease in 4/8 (50%) cases. There was no significant difference in overall survival (OS) between patients with germinal centre B-cell (GCB) and non-GCB RT-DLBCL. Only CD5 expression correlated significantly with OS (HR=2.732; 95% CI 1.397-5.345; p=0.0374). RT-DLBCL has distinctive morphological and immunophenotypic features, characterised by IB morphology and common expression of CD5, MUM1 and LEF1. Cell-of-origin does not seem to have prognostic implications in RT-DLBCL., (Copyright © 2023 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Monotherapy with Menin inhibitor (MI), e.g., SNDX-5613, induces clinical remissions in patients with relapsed/refractory AML harboring MLL1-r or mtNPM1, but most patients either fail to respond or eventually relapse. Utilizing single-cell RNA-Seq, ChiP-Seq, ATAC-Seq, RNA-Seq, RPPA, and mass cytometry (CyTOF) analyses, present pre-clinical studies elucidate gene-expression correlates of MI efficacy in AML cells harboring MLL1-r or mtNPM1. Notably, MI-mediated genome-wide, concordant, log2 fold-perturbations in ATAC-Seq and RNA-Seq peaks were observed at the loci of MLL-FP target genes, with upregulation of mRNAs associated with AML differentiation. MI treatment also reduced the number of AML cells expressing the stem/progenitor cell signature. A protein domain-focused CRISPR-Cas9 screen in MLL1-r AML cells identified targetable co-dependencies with MI treatment, including BRD4, EP300, MOZ and KDM1A. Consistent with this, in vitro co-treatment with MI and BET, MOZ, LSD1 or CBP/p300 inhibitor induced synergistic loss of viability of AML cells with MLL1-r or mtNPM1. Co-treatment with MI and BET or CBP/p300 inhibitor also exerted significantly superior in vivo efficacy in xenograft models of AML with MLL1-r. These findings highlight novel, MI-based combinations that could prevent escape of AML stem/progenitor cells following MI monotherapy, which is responsible for therapy-refractory AML relapse., (© 2023. The Author(s).)

Purpose: The combination of venetoclax and 5-azacitidine (5-AZA) for older or unfit patients with acute myeloid leukemia (AML) improves remission rates and survival compared with 5-AZA alone. We hypothesized that the addition of venetoclax to cladribine (CLAD)/low-dose araC (low-dose cytarabine [LDAC]) alternating with 5-AZA backbone may further improve outcomes for older patients with newly diagnosed AML., Methods: This is a phase II study investigating the combination of venetoclax and CLAD/LDAC alternating with venetoclax and 5-AZA in older (≥ 60 years) or unfit patients with newly diagnosed AML. The primary objective was composite complete response (CR) rate (CR plus CR with incomplete blood count recovery); secondary end points were overall survival, disease-free survival (DFS), overall response rate, and toxicity., Results: A total of 60 patients were treated; median age was 68 years (range, 57-84 years). By European LeukemiaNet, 23%, 33%, and 43% were favorable, intermediate, and adverse risk, respectively. Fifty-six of 60 evaluable patients responded (composite CR: 93%) and 84% were negative for measurable residual disease. There was one death (2%) within 4 weeks. With a median follow-up of 22.1 months, the median overall survival and DFS have not yet been reached. The most frequent grade 3/4 nonhematologic adverse events were febrile neutropenia (n = 33) and pneumonia (n = 14). One patient developed grade 4 tumor lysis syndrome., Conclusion: Venetoclax and CLAD/LDAC alternating with venetoclax and 5-AZA is an effective regimen among older or unfit patients with newly diagnosed AML. The rates of overall survival and DFS are encouraging. Further study of this non-anthracycline-containing backbone in younger patients, unfit for intensive chemotherapy, as well as comparisons to standard frontline therapies is warranted.

The majority of RUNX1 mutations in acute myeloid leukemia (AML) are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy, AML patients expressing mtRUNX1 exhibit inferior clinical outcome than those without mutant RUNX1. Studies presented here demonstrate that as compared with AML cells lacking mtRUNX1, their isogenic counterparts harboring mtRUNX1 display impaired ribosomal biogenesis and differentiation, as well as exhibit reduced levels of wild-type RUNX1, PU.1, and c-Myc. Compared with AML cells with only wild-type RUNX1, AML cells expressing mtRUNX1 were also more sensitive to the protein translation inhibitor homoharringtonine (omacetaxine) and BCL2 inhibitor venetoclax. Homoharringtonine treatment repressed enhancers and their BRD4 occupancy and was associated with reduced levels of c-Myc, c-Myb, MCL1, and Bcl-xL. Consistent with this, cotreatment with omacetaxine and venetoclax or BET inhibitor induced synergistic in vitro lethality in AML expressing mtRUNX1. Compared with each agent alone, cotreatment with omacetaxine and venetoclax or BET inhibitor also displayed improved in vivo anti-AML efficacy, associated with improved survival of immune-depleted mice engrafted with AML cells harboring mtRUNX1. These findings highlight superior efficacy of omacetaxine-based combination therapies for AML harboring mtRUNX1., (© 2022 by The American Society of Hematology.)

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1., (© 2022. The Author(s).)

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive neoplasm derived from plasmacytoid dendritic cells. While advances in understanding the pathophysiology of the disease have been made, integrated systematic analyses of the spectrum of immunophenotypic and molecular alterations in real-world clinical cases remain limited. We performed mutation profiling of 50 BPDCN cases and assessed our findings in the context of disease immunophenotype, cytogenetics, and clinical characteristics. Patients included 42 men and 8 women, with a median age of 68 years (range, 14-84) at diagnosis. Forty-two (84%) patients had at least one mutation, and 23 (46%) patients had ≥3 mutations. The most common mutations involved TET2 and ASXL1 , detected in 28 (56%) and 23 (46%) patients, respectively. Co-existing TET2 and ASXL1 mutations were present in 17 (34%) patients. Other recurrent mutations included ZRSR2 (16%), ETV6 (13%), DNMT3A (10%), NRAS (10%), IKZF1 (9%), SRSF2 (9%), IDH2 (8%), JAK2 (6%), KRAS (4%), NOTCH1 (4%), and TP53 (4%). We also identified mutations that have not been reported previously, including ETNK1, HNRNPK, HRAS, KDM6A, RAD21 , SF3A1 , and SH2B3. All patients received chemotherapy, and 20 patients additionally received stem cell transplantation. With a median follow-up of 10.5 months (range, 1-71), 21 patients achieved complete remission, 4 had persistent disease, and 24 died. Patients younger than 65 years had longer overall survival compared to those who were ≥65 years ( p = 0.0022). Patients who had ≥3 mutations or mutations in the DNA methylation pathway genes had shorter overall survival ( p = 0.0119 and p = 0.0126, respectively). Stem cell transplantation significantly prolonged overall survival regardless of mutation status. In conclusion, the majority of patients with BPDCN have somatic mutations involving epigenetic regulators and RNA splicing factors, in addition to ETV6 and IKZF1 , which are also frequently mutated. Older age, multiple mutations, and mutations in the DNA methylation pathway are poor prognostic factors.

Richter Transformation (RT) develops in CLL as an aggressive, therapy-resistant, diffuse large B cell lymphoma (RT-DLBCL), commonly clonally-related (CLR) to the concomitant CLL. Lack of available pre-clinical human models has hampered the development of novel therapies for RT-DLBCL. Here, we report the profiles of genetic alterations, chromatin accessibility and active enhancers, gene-expressions and anti-lymphoma drug-sensitivity of three newly established, patient-derived, xenograft (PDX) models of RT-DLBCLs, including CLR and clonally-unrelated (CLUR) to concomitant CLL. The CLR and CLUR RT-DLBCL cells display active enhancers, higher single-cell RNA-Seq-determined mRNA, and protein expressions of IRF4, TCF4, and BCL2, as well as increased sensitivity to BET protein inhibitors. CRISPR knockout of IRF4 attenuated c-Myc levels and increased sensitivity to a BET protein inhibitor. Co-treatment with BET inhibitor or BET-PROTAC and ibrutinib or venetoclax exerted synergistic in vitro lethality in the RT-DLBCL cells. Finally, as compared to each agent alone, combination therapy with BET-PROTAC and venetoclax significantly reduced lymphoma burden and improved survival of immune-depleted mice engrafted with CLR-RT-DLBCL. These findings highlight a novel, potentially effective therapy for RT-DLBCL., (© 2021. The Author(s).)

The central role of β -catenin in the Wnt pathway makes it an attractive therapeutic target for cancers driven by aberrant Wnt signaling. We recently developed a small-molecule inhibitor, BC-2059, that promotes apoptosis by disrupting the β -catenin/transducin β -like 1 (TBL1) complex through an unknown mechanism of action. In this study, we show that BC-2059 directly interacts with high affinity for TBL1 when in complex with β -catenin. We identified two amino acids in a hydrophobic pocket of TBL1 that are required for binding with β -catenin, and computational modeling predicted that BC-2059 interacts at the same hydrophobic pocket. Although this pocket in TBL1 is involved in binding with NCoR/SMRT complex members G Protein Pathway Suppressor 2 (GSP2) and SMRT and p65 NF κ B subunit, BC-2059 failed to disrupt the interaction of TBL1 with either NCoR/SMRT or NF κ B. Together, our results show that BC-2059 selectively targets TBL1/ β -catenin protein complex, suggesting BC-2059 as a therapeutic for tumors with deregulated Wnt signaling pathway. SIGNIFICANCE STATEMENT: This study reports the mechanism of action of a novel Wnt pathway inhibitor, characterizing the selective disruption of the transducin β -like 1/ β -catenin protein complex. As Wnt signaling is dysregulated across cancer types, this study suggests BC-2059 has the potential to benefit patients with tumors reliant on this pathway., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

There is an unmet need to overcome nongenetic therapy-resistance to improve outcomes in AML, especially post-myeloproliferative neoplasm (MPN) secondary (s) AML. Studies presented describe effects of genetic knockout, degradation or small molecule targeted-inhibition of GFI1/LSD1 on active enhancers, altering gene-expressions and inducing differentiation and lethality in AML and (MPN) sAML cells. A protein domain-focused CRISPR screen in LSD1 (KDM1A) inhibitor (i) treated AML cells, identified BRD4, MOZ, HDAC3 and DOT1L among the codependencies. Our findings demonstrate that co-targeting LSD1 and one of these co-dependencies exerted synergistic in vitro lethality in AML and post-MPN sAML cells. Co-treatment with LSD1i and the JAKi ruxolitinib was also synergistically lethal against post-MPN sAML cells. LSD1i pre-treatment induced GFI1, PU.1 and CEBPα but depleted c-Myc, overcoming nongenetic resistance to ruxolitinib, or to BETi in post-MPN sAML cells. Co-treatment with LSD1i and BETi or ruxolitinib exerted superior in vivo efficacy against post-MPN sAML cells. These findings highlight LSD1i-based combinations that merit testing for clinical efficacy, especially to overcome nongenetic therapy-resistance in AML and post-MPN sAML.

Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we perform multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 60 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis reveals that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes is the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML.

Ecotropic viral integration site 1 (Evi1) was discovered in 1988 as a common site of ecotropic viral integration resulting in myeloid malignancies in mice. EVI1 is an oncogenic zinc-finger transcription factor whose overexpression contributes to disease progression and an aggressive phenotype, correlating with poor clinical outcome in myeloid malignancies. Despite progress in understanding the biology of EVI1 dysregulation, significant improvements in therapeutic outcome remain elusive. Here, we highlight advances in understanding EVI1 biology and discuss how this new knowledge informs development of novel therapeutic interventions. EVI1 is overexpression is correlated with poor outcome in some epithelial cancers. However, the focus of this review is the genetic lesions, biology, and current therapeutics of myeloid malignancies overexpressing EVI1.

Despite promising results with FLT3 inhibitors (FLT3i), response durations remain short. We studied pretreatment and relapse bone marrow samples from patients with FLT3 -mutated AML treated with FLT3i-based therapies (secondary resistance cohort), and pretreatment bone marrow samples from patients with no response to FLT3i-based therapies (primary resistance cohort). Targeted next generation sequencing at relapse identified emergent mutations involving on-target FLT3 , epigenetic modifiers, RAS/MAPK pathway, and less frequently WT1 , and TP53 . RAS/MAPK and FLT3- D835 mutations emerged most commonly following type I and type II FLT3i-based therapies, respectively. Patients with emergent mutations at relapse had inferior overall survival compared with those without emergent mutations. Among pretreatment RAS mutated patients, pretreatment cohort level variant allelic frequencies for RAS were higher in non-responders, particularly with type I FLT3i-based therapies, suggesting a potential role in primary resistance as well. These data demonstrate distinct pathways of resistance in FLT3 -mutated AML treated with type I versus II FLT3i., Competing Interests: Conflict of interest: MY reports research funding from Pfizer and Daiichi Sankyo. TK reports personal fees from Novartis, grants and personal fees from Pfizer, grants from BMS, grants and personal fees from Abbvie, grants and personal fees from Genentech, grants and personal fees from JAZZ, grants from Amgen, grants from AstraZeneca; Celgene: research grant; Incyte: research grant; Ascentage: research grant. CD reports grants and personal fees from Abbvie, grants and personal fees from Agios, grants and personal fees from Novartis, grants and personal fees from ImmuneOnc, grants and personal fees from Daiichi Sankyo, grants and personal fees from Celgene, personal fees from Jazz, personal fees from Notable Labs, personal fees from MedImmune, grants from Calithera, personal fees from Bayer. MK reports grants and other from AbbVie, grants and other from Genentech, grants and other from F. Hoffman La-Roche, grants and other from Stemline Therapeutics, other from Amgen, grants and other from Forty-Seven, other from Kisoji, grants from Eli Lilly, grants from Cellectis, grants from Calithera, grants from Ablynx, grants from Agios, grants from Ascentage, grants from Astra Zeneca, other from Reata Pharmaceutical, grants from Rafael Pharmaceutical, grants from Sanofi, outside the submitted work; In addition, Dr. Konopleva has a patent US 7,795,305 B2 CDDO-compounds and combination therapie with royalties paid to Reata Pharm., a patent Combination Therapy with a mutant IDH1 Inhibitor and a BCL-2 licensed to Eli Lilly, and a patent 62/993,166 combination of a MCL-1 inhibitor and midostaurin, uses and pharmaceutical compositions thereof pending to novartis. NP reports personal fees from Pacylex Pharmaceuticals, grants and other from Affymetrix, grants from SagerStrong Foundation, personal fees from Incyte, personal fees and other from Novartis, personal fees from LFB Biotechnologies, personal fees, non-financial support and other from Stemline Therapeutics, personal fees and non-financial support from Celgene, personal fees, non-financial support and other from AbbVie, personal fees and non-financial support from MustangBio, personal fees from Roche Diagnostics, personal fees from Blueprint Medicines, personal fees and non-financial support from DAVA Oncology, other from Samus Therapeutics, other from Cellectis, other from Daiichi Sankyo, other from Plexxikon. EJ reports research grants and consultancy from Abbvie, Adaptive biotechnology, Amgen, BMS, Pfizer, Takeda. NS reports personal fees from Amgen, personal fees from AstraZeneca, grants and personal fees from Takeda, grants from Astellas. GI reports grants and personal fees from Novartis, grants from Syndax, grants from Celgene. FR reports honoraria and member of advisory board from Astellas, honoraria from Daichii, honoraria and member of advisory board from Novartis. ND reports research funding from Daiichi Sankyo, Bristol-Myers Squibb, Pfizer, Karyopharm, Sevier, Genentech, Astellas, Abbvie, Genentech, Novimmune, Amgen, Trovagene, Gilead and ImmunoGen and has served in a consulting or advisory role for Daiichi Sankyo, Bristol-Myers Squibb, Pfizer, Novartis, Celgene, AbbVie, Genentech, Servier, Trillium, Syndax, Trovagene, Astellas, Gilead and Agios.

Despite major improvements in treatment outcome with novel targeted therapies, such as the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, chronic lymphocytic leukemia (CLL) remains incurable in the majority of patients. Activation of PI3K, NF-κB, and/or MYC has been linked to residual disease and/or resistance in ibrutinib-treated patients. These pathways can be targeted by inhibitors of bromodomain and extra-terminal (BET) proteins. Here we report about the preclinical activity of GS-5829, a novel BET inhibitor, in CLL. GS-5829 inhibited CLL cell proliferation and induced leukemia cell apoptosis through deregulation of key signaling pathways, such as BLK, AKT, ERK1/2, and MYC. IκBα modulation indicates that GS-5829 also inhibited NF-κB signaling. GS-5829-induced apoptosis resulted from an imbalance between positive (BIM) and negative regulators (BCL-X L ) of the intrinsic apoptosis pathway. The antileukemia activity of GS-5829 increased synergistically in combinations with B-cell receptor signaling inhibitors, the BTK inhibitor ibrutinib, the PI3Kδ inhibitor idelalisib, and the SYK inhibitor entospletinib. In cocultures that mimic the lymph node microenvironment, GS-5829 inhibited signaling pathways within nurselike cells and their growth, indicating that BET inhibitors also can target the supportive CLL microenvironment. Collectively, these data provide a rationale for the clinical evaluation of BET inhibitors in CLL.

The promising activity of BET protein inhibitors (BETi's) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin-TCF7L2-JMJD6-c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin-TCF7L2-JMJD6-c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells., (​© 2020 by The American Society of Hematology.)

RUNX1 transcription factor regulates normal and malignant hematopoiesis. Somatic or germline mutant RUNX1 (mtRUNX1) is associated with poorer outcome in acute myeloid leukemia (AML). Knockdown or inhibition of RUNX1 induced more apoptosis of AML expressing mtRUNX1 versus wild-type RUNX1 and improved survival of mice engrafted with mtRUNX1-expressing AML. CRISPR/Cas9-mediated editing-out of RUNX1 enhancer (eR1) within its intragenic super-enhancer, or BET protein BRD4 depletion by short hairpin RNA, repressed RUNX1, inhibited cell growth, and induced cell lethality in AML cells expressing mtRUNX1. Moreover, treatment with BET protein inhibitor or degrader (BET-proteolysis targeting chimera) repressed RUNX1 and its targets, inducing apoptosis and improving survival of mice engrafted with AML expressing mtRUNX1. Library of Integrated Network-based Cellular Signatures 1000-connectivity mapping data sets queried with messenger RNA signature of RUNX1 knockdown identified novel expression-mimickers (EMs), which repressed RUNX1 and exerted in vitro and in vivo efficacy against AML cells expressing mtRUNX1. In addition, the EMs cinobufagin, anisomycin, and narciclasine induced more lethality in hematopoietic progenitor cells (HPCs) expressing germline mtRUNX1 from patients with AML compared with HPCs from patients with familial platelet disorder (FPD), or normal untransformed HPCs. These findings highlight novel therapeutic agents for AML expressing somatic or germline mtRUNX1., (© 2019 by The American Society of Hematology.)

Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear β-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of β-catenin or treatment with BC2059 that disrupts binding of β-catenin to TBL1X (TBL1) depleted nuclear β-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of β-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of β-catenin and BETP antagonists against post-MPN sAML BPCs.

First-generation bromodomain extra-terminal protein (BETP) inhibitors (BETi) (e.g., OTX015) that disrupt binding of BETP BRD4 to chromatin transcriptionally attenuate AML-relevant progrowth and prosurvival oncoproteins. BETi treatment induces apoptosis of AML BPCs, reduces in vivo AML burden and induces clinical remissions in a minority of AML patients. Clinical efficacy of more potent BETis, e.g., ABBV-075 (AbbVie, Inc.), is being evaluated. Venetoclax and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis. BETi treatment is shown here to perturb accessible chromatin and activity of enhancers/promoters, attenuating MYC, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A expressions and apoptosis of AML cells. Treatment with venetoclax increased MCL1 protein levels, but cotreatment with ABBV-075 reduced MCL1 and Bcl-xL levels. ABBV-075 cotreatment synergistically induced apoptosis with venetoclax or A-1210477 in patient-derived, CD34+ AML cells. Compared to treatment with either agent alone, cotreatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell-burden and improving survival, without inducing toxicity, in AML-engrafted immune-depleted mice. These findings highlight the basis of superior activity and support interrogation of clinical efficacy and safety of cotreatment with BETi and BCL2 or MCL1 inhibitor in AML.

The outcome of patients with myelodysplastic syndromes (MDSs) after failure of hypomethylating agents (HMAs) failure is poor with a median overall survival (OS) of only 4-6 months. Omacetaxine mepesuccinate (OM) is safe and effective in myeloid malignancies but has not been studied in MDS with HMA failure. We conducted a phase II study of OM in patients with MDS or chronic myelomonocytic leukemia (CMML) who had previously failed or been intolerant to HMAs. Patients received OM at a dose of 1.25 mg/m 2 subcutaneously every 12 hours for 3 consecutive days on a 4- to 7-week schedule. The primary endpoints were the overall response rate (ORR) and OS. A total of 42 patients were enrolled with a median age of 76 years. The ORR was 33%. Patients with diploid cytogenetics were more likely to respond to OM than were those with cytogenetic abnormalities (58% vs 23%, respectively; P = .03). Overall, the median OS was 7.5 months and 1-year OS rate was 25%. Patients with diploid cytogenetics had superior OS to those with cytogenetic abnormalities (median OS 14.8 vs 6.8 months, respectively; P = .01). Two patients had ongoing response to OM of 2 years or longer (both MDS with diploid cytogenetics and RUNX1 mutation). The most common grade ≥ 3 adverse events were infections in 11 patients (26%), febrile neutropenia in 4 (10%), and hemorrhage in 3 (7%). Overall, OM was safe and active in patients with MDS or CMML who experienced HMA failure. These results support the further development of OM in this setting., (© 2018 Wiley Periodicals, Inc.)

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4 + T cell DNA methylation and the downstream tuberculosis (TB)-specific and bacillus Calmette-Guérin-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA-methylated genes, with 72% hypermethylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3 , ID2 , STAT5A , IRF5 , PPARg , RUNX2 , IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1 , TNFS11 , RELT (TNF receptor), IL12RB2, and IL12B ( p < 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypomethylation of the IL-4 pathway and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p < 0.05). In S. haematobium -infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth specific., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Introduction: Venetoclax (VEN), a selective BCL2 inhibitor, has single-agent activity in relapsed and refractory (R/R) acute myeloid leukemia (AML), and efficacy in lower intensity combinations for treatment-naïve elderly AML patients. VEN treatment combinations in R/R AML have not been previously reported., Methods: All R/R myeloid patients (including AML, myelodysplastic syndrome (MDS), and blastic plasmacytoid dendritic cell neoplasm (BPDCN)) treated with VEN combinations in the salvage setting were reviewed., Results: Forty-three patients with median age 68 (range, 25-83) were treated for AML (91%), MDS (5%), or BPDCN (5%). Most (n = 36, 84%) were ≥ salvage-2 treatment status, including prior hypomethylating agent (HMA) in 77%. In combination with VEN, most patients received HMA therapy (n = 31, 72%); eight (19%) received low-dose cytarabine (LDAC). Patients received a median of 2 treatment cycles (range, 1-4). Objective response was observed in 9 (21%) patients, including 2 complete responses (CR), 3 CRi, and 4 morphologic leukemia-free state (MLFS). Median survival was 3.0 months (range, 0.5-8.0), and estimated 6-month survival was 24%. Responses were observed in five (24%) of 21 patients with intermediate-risk cytogenetics, 3 (27%) of 11 IDH1/2-mutant, and 4 (50%) of 8 RUNX1-mutated patients. Two (20%) of 10 TP53-mutated patients responded; both had concurrent RUNX1 mutations. Of the 3 (15%) responding patients with adverse cytogenetics, all had concurrent RUNX1 mutations., Conclusion: Low-intensity chemotherapy, including HMAs or LDAC, in combination with VEN is a viable salvage option, even in multiply relapsed/refractory patients with AML, MDS, and BPDCN. Notable responses were identified in patients with diploid/intermediate cytogenetics, RUNX1, and/or IDH1/2 mutations., (© 2017 Wiley Periodicals, Inc.)

Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11-Rad51-DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair 'addiction' and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

Purpose: Suberoylanilide hydroxamic acid (SAHA; vorinostat), a small molecule inhibitor of histone deacetylase, attenuates signaling pathways known to confer trastuzumab resistance. A combination of SAHA and trastuzumab may be a promising strategy to improve the efficacy of trastuzumab against breast cancer. In this Phase I/II study, we evaluated the toxicity and response rate after treatment with SAHA and trastuzumab in patients with HER2-overexpressing metastatic breast cancer with trastuzumab-resistant progressive disease., Methods: In Phase I, the SAHA dose was modified in cohorts of 3-6 patients to find the dose level at which 0 or 1 patients experienced a dose-limiting toxicity (DLT) during the first cycle of therapy. In the Phase II study, response to the recommended dose identified in Phase I was based on the response evaluation criteria in solid tumors. Overall survival and time to progression were also evaluated., Results: The recommended dose was determined to be 200 mg twice a day on days 1-14 and IV trastuzumab 6 mg/kg on day 1 of a 21-day cycle (n = 6). The Phase II study (n = 10) was terminated when the pre-planned efficacy evaluation found that none of the patients in the primary analysis set responded to combination SAHA and trastuzumab treatment., Conclusions: In patients with HER2-positive metastatic breast cancer who had relapsed or progressed during trastuzumab therapy, we observed no DLTs with SAHA 200 mg twice daily combined with trastuzumab; however, there was insufficient statistical evidence that adding SAHA reversed trastuzumab resistance in these patients.

Chromatin packaging of DNA provides a framework for transcriptional regulation. Modifications to DNA and histone proteins in nucleosomes lead to conformational changes, alterations in the recruitment of transcriptional complexes, and ultimately modulation of gene expression. We provide a focused review of control mechanisms that help modulate the activation and deactivation of gene transcription specifically through histone acetylation writers and readers in cancer. The chemistry of these modifications is subject to clinically actionable targeting, including state-of-the-art strategies to inhibit basic oncogenic mechanisms related to histone acetylation. Although discussed in the context of acute leukemia, the concepts of acetylation writers and readers are not cell-type-specific and are generalizable to other cancers. We review the challenges and resistance mechanisms encountered to date in the development of such therapeutics and postulate how such challenges may be overcome. Because these fundamental cellular mechanisms are dysregulated in cancer biology, continued research and in-depth understanding of histone acetylation reading and writing are desired to further define optimal therapeutic strategies to affect gene activity to target cancer effectively.

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry 'CyTOF' analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

Secondary acute myeloid leukemia (s-AML) includes therapy-related AML and AML evolving from antecedent hematological disorder (AHD). s-AML arising after treating AHD likely represents a prognostically distinct, high-risk disease category. In this study, treated s-AML (ts-AML) was defined by: (1) prior diagnosis of myelodysplasia, myeloproliferative neoplasm, or aplastic anemia and (2) at least 1 therapy for that diagnosis. ts-AML was categorized by age (< or ≥60 years), and each cohort assessed for response rates and overall survival (OS) on various treatment regimens. Survival outcomes were compared against other high-risk prognostic subsets. Results showed that complete response and 8-week mortality rates were 32% and 27% in the younger, and 24% and 19% in the older age groups, respectively. There was a significant OS difference within s-AML based on prior treatment of AHD (ie, ts-AML vs s-AML with untreated AHD, 4.2 vs 9.2 months; P < .001). Survival in ts-AML was poor across both cohorts (younger and older, 5 and 4.7 months, respectively). In younger AML, survival was significantly inferior in ts-AML when compared with deletion 5/7, TP53, 3q abnormality, and therapy-related AML groups (median, 5 vs 7.9, 7.8, 7.9, and 11.2 months, respectively; P < .01). Additional adverse karyotype within ts-AML was associated with even worse outcomes (OS range, 1.6-2.8 months). ts-AML represents a very high-risk category, even in younger AML patients. s-AML should be further classified to describe ts-AML, an entity less responsive to currently applied treatment approaches. Future AML trial designs should accommodate ts-AML as a distinct subgroup., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

This corrects the article DOI: 10.1038/leu.2014.119.

Myeloproliferative neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling that responds to treatment with the JAK1 and 2 kinase inhibitor (JAKi) ruxolitinib. However, MPN-MF often progresses (~20%) to secondary acute myeloid leukemia (sAML), where standard induction chemotherapy or ruxolitinib is relatively ineffective, necessitating the development of novel therapeutic approaches. In the present studies, we demonstrate that treatment with BET (bromodomain and extraterminal) protein inhibitor (BETi), for example, JQ1, inhibits growth and induces apoptosis of cultured and primary, patient-derived (PD), post-MPN sAML blast progenitor cells. Reverse-phase protein array, mass-cytometry and Western analyses revealed that BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, whereas it concomitantly induced the levels of HEXIM1, p21 and BIM in the sAML cells. Co-treatment with BETi and ruxolitinib synergistically induced apoptosis of cultured and PD sAML cells, as well as significantly improved survival of immune-depleted mice engrafted with human sAML cells. Although BETi or heat shock protein 90 inhibitor (HSP90i) alone exerted lethal activity, cotreatment with BETi and HSP90i was synergistically lethal against the ruxolitinib-persister or ruxolitinib-resistant sAML cells. Collectively, these findings further support in vivo testing of BETi-based combinations with JAKi and HSP90i against post-MPN sAML cells.

A vailable tyrosine kinase inhibitors for chronic myeloid leukemia bind in an adenosine 5'-triphosphate-binding pocket and are affected by evolving mutations that confer resistance. Rebastinib was identified as a switch control inhibitor of BCR-ABL1 and FLT3 and may be active against resistant mutations. A Phase 1, first-in-human, single-agent study investigated rebastinib in relapsed or refractory chronic or acute myeloid leukemia. The primary objectives were to investigate the safety of rebastinib and establish the maximum tolerated dose and recommended Phase 2 dose. Fifty-seven patients received treatment with rebastinib. Sixteen patients were treated using powder-in-capsule preparations at doses from 57 mg to 1200 mg daily, and 41 received tablet preparations at doses of 100 mg to 400 mg daily. Dose-limiting toxicities were dysarthria, muscle weakness, and peripheral neuropathy. The maximum tolerated dose was 150 mg tablets administered twice daily. Rebastinib was rapidly absorbed. Bioavailability was 3- to 4-fold greater with formulated tablets compared to unformulated capsules. Eight complete hematologic responses were achieved in 40 evaluable chronic myeloid leukemia patients, 4 of which had a T315I mutation. None of the 5 patients with acute myeloid leukemia responded. Pharmacodynamic analysis showed inhibition of phosphorylation of substrates of BCR-ABL1 or FLT3 by rebastinib. Although clinical activity was observed, clinical benefit was insufficient to justify continued development in chronic or acute myeloid leukemia. Pharmacodynamic analyses suggest that other kinases inhibited by rebastinib, such as TIE2, may be more relevant targets for the clinical development of rebastinib ( clinicaltrials.gov Identifier:00827138 )., (Copyright© Ferrata Storti Foundation.)

SIRT2 is a protein deacetylase with tumor suppressor activity in breast and liver tumors where it is mutated; however, the critical substrates mediating its antitumor activity are not fully defined. Here we demonstrate that SIRT2 binds, deacetylates, and inhibits the peroxidase activity of the antioxidant protein peroxiredoxin (Prdx-1) in breast cancer cells. Ectopic overexpression of SIRT2, but not its catalytically dead mutant, increased intracellular levels of reactive oxygen species (ROS) induced by hydrogen peroxide, which led to increased levels of an overoxidized and multimeric form of Prdx-1 with activity as a molecular chaperone. Elevated levels of SIRT2 sensitized breast cancer cells to intracellular DNA damage and cell death induced by oxidative stress, as associated with increased levels of nuclear FOXO3A and the proapoptotic BIM protein. In addition, elevated levels of SIRT2 sensitized breast cancer cells to arsenic trioxide, an approved therapeutic agent, along with other intracellular ROS-inducing agents. Conversely, antisense RNA-mediated attenuation of SIRT2 reversed ROS-induced toxicity as demonstrated in a zebrafish embryo model system. Collectively, our findings suggest that the tumor suppressor activity of SIRT2 requires its ability to restrict the antioxidant activity of Prdx-1, thereby sensitizing breast cancer cells to ROS-induced DNA damage and cell cytotoxicity. Cancer Res; 76(18); 5467-78. ©2016 AACR., (©2016 American Association for Cancer Research.)

Mantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL., (© 2015 by The American Society of Hematology.)

The canonical wingless-type MMTV integration site (WNT)-β-catenin pathway is essential for self-renewal, growth and survival of acute myeloid leukemia (AML) stem/blast progenitor cells (BPCs). Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and co-factor activity of β-catenin with the transcriptional regulator T-cell factor (TCF) 4/lymphoid enhancer factor 1 in AML BPCs. Here, we determined the pre-clinical anti-AML activity of the anthraquinone oxime-analog BC2059 (BC), known to attenuate β-catenin levels. BC treatment disrupted the binding of β-catenin with the scaffold protein transducin β-like 1 and proteasomal degradation and decline in the nuclear levels of β-catenin. This was associated with reduced transcriptional activity of TCF4 and expression of its target genes, cyclin D1, c-MYC and survivin. BC treatment dose-dependently induced apoptosis of cultured and primary AML BPCs. Treatment with BC also significantly improved the median survival of immune-depleted mice engrafted with either cultured or primary AML BPCs, exhibiting nuclear expression of β-catenin. Co-treatment with the pan-histone deacetylase inhibitor panobinostat and BC synergistically induced apoptosis of cultured and primary AML BPCs, including those expressing FLT3-ITD, as well as further significantly improved the survival of immune-depleted mice engrafted with primary AML BPCs. These findings underscore the promising pre-clinical activity and warrant further testing of BC against human AML, especially those expressing FLT3-ITD.

The histone demethylase LSD1 (KDM1A) demethylates mono- and di-methylated (Me2) lysine (K) 4 on histone H3. High LSD1 expression blocks differentiation and confers a poor prognosis in acute myeloid leukemia (AML). Here, treatment with the novel LSD1 antagonist SP2509 attenuated the binding of LSD1 with the corepressor CoREST, increased the permissive H3K4Me3 mark on the target gene promoters, and increased the levels of p21, p27 and CCAAT/enhancer binding protein α in cultured AML cells. In addition, SP2509 treatment or LSD1 shRNA inhibited the colony growth of AML cells. SP2509 also induced morphological features of differentiation in the cultured and primary AML blasts. SP2509 induced more apoptosis of AML cells expressing mutant NPM1 than mixed-lineage leukemia fusion oncoproteins. Treatment with SP2509 alone significantly improved the survival of immune-depleted mice following tail-vein infusion and engraftment of cultured or primary human AML cells. Co-treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) and SP2509 was synergistically lethal against cultured and primary AML blasts. Compared with each agent alone, co-treatment with SP2509 and PS significantly improved the survival of the mice engrafted with the human AML cells, without exhibiting any toxicity. Collectively, these findings show that the combination of LSD1 antagonist and pan-HDI is a promising therapy warranting further testing against AML.

Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34(+) human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34(+) normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI-resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD-expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI-resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI., (©2014 American Association for Cancer Research.)

There is an unmet need to develop new, more effective and safe therapies for the aggressive forms of triple negative breast cancers (TNBCs). While up to 20% of women under 50 years of age with TNBC harbor germline mutations in BRCA1, and these tumors are sensitive to treatment with poly(ADP) ribose polymerase inhibitors, a majority of TNBCs lack BRCA1 mutations or loss of expression. Findings presented here demonstrate that by attenuating the levels of DNA damage response and homologous recombination proteins, pan-histone deacetylase inhibitor (HDI) treatment induces 'BRCAness' and sensitizes TNBC cells lacking BRCA1 to lethal effects of PARP inhibitor or cisplatin. Treatment with HDI also induced hyperacetylation of nuclear hsp90. Similar effects were observed following shRNA-mediated depletion of HDAC3, confirming its role as the deacetylase for nuclear HSP90. Furthermore, cotreatment with HDI and ABT-888 induced significantly more DNA strand breaks than either agent alone, and synergistically induced apoptosis of TNBC cells. Notably, co-treatment with HDI and ABT-888 significantly reduced in vivo tumor growth and markedly improved the survival of mice bearing TNBC cell xenografts. These findings support the rationale to interrogate the clinical activity of this novel combination against human TNBC, irrespective of its expression of mutant BRCA1.