ALBERT

Description: Hematopoietic progenitor cells of myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit constitutive activation of JAK-STAT5/3 and NFkB signaling. Transformation of MPN-MF to AML (post-MPN sAML) occurs in up to 15% of patients with MPN-MF. Standard induction anti-AML chemotherapy and the JAK1 & 2 inhibitor (JAKi) ruxolitinib are ineffective in post-MPN sAML. BET protein BRD4 is a non-oncogene addiction target in AML, and treatment with acetyl-lysine mimetic BET protein inhibitor (BETi) disrupts binding of BRD4 to acetylated chromatin and transcription factors (TFs). This attenuates transcription of super-enhancer regulated oncogenes, including MYC, Bcl-xL, PIM1 and CDK4/6, inhibiting growth and survival of post-MPN sAML blasts. BETi treatment also inhibits binding of BRD4 to acetylated RELA (NFkB-p65), inhibiting its transcriptional activity and attenuating levels of its target cytokines. However, BETi treatment induces BRD4, potentially reducing BETi activity in repressing oncogenes. Preclinical and clinical studies have demonstrated that innate or adaptive BETi-resistance is common in sAML cells. To model BETi-resistance, we repeatedly exposed (10 times) secondary (s) AML SET2 and HEL92.1.7 (HEL) cells to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, thus generating BETi persister-resistant (BETi-P/R) SET2-P/R and HEL-P/R cells. These cells showed 〉 10-fold resistance to OTX015 and cross-resistance to other BETis. Compared to the parental controls, BETi-P/R cells lacked additional genetic alterations or altered levels of TRIM33, SPOP, DUB3 or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). However, ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses demonstrated that, as compared to their parental controls, BETi-P/R cells showed gain of peaks and active enhancers with enrichment of STAT5, MYC, PU.1 and GATA2 binding sites. Newly gained peaks were in the enhancers of JAK1/2, RUNX1, PU.1, MYC and BCL2L1. RNA-Seq determined mRNA level alterations, included induction of gene-sets involving MYC/MAX, STAT5, NFkB and TCF7L2 targets. QPCR and Western analyses confirmed increase in the mRNA and protein levels of TCF7L2, JMJD6, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Expression of the arginine demethylase JMJD6, recruited by BRD4 to regulate enhancer-mediated transcriptional pause-release, was also increased. This was associated with increased expression of the nuclear β-catenin-TCF7L2 targets, including Cyclin D1, TERT, survivin, c-Myc and PU.1. Patient-derived human AML blasts that exhibited innate resistance ex vivo to BETi, also demonstrated increased expression of TCF7L2, JMJD6 and c-Myc. We next probed the mechanistic role of the β-catenin-JMJD6-TCF7L2-MYC axis in conferring BETi-resistance. CRISPR-Cas9-mediated knockout of TCF7L2 or JMJD6 significantly reversed BETi-resistance in BETi-P/R sAML cells (p 〈 0.001). Conversely, ectopic overexpression of TCF7L2 or JMJD6 significantly conferred BETi-persister-resistance in HEL and SET2 cells (p 〈 0.001). Notably, confocal microscopy demonstrated increased binding of β-catenin with TBL1 and TCF7L2 in the nucleus of BETi-P/R sAML cells. BC2059, which disrupts binding of nuclear β-catenin with TBL1 and TCF7L2, depleted β-catenin levels and exerted similar lethality in BETi-P/R sAML and control sAML cells. shRNA-mediated knockdown of BRD4 and treatment with BRD4-PROTAC (proteolysis-targeting chimera) ARV-771 (Arvinas, Inc.) that degrades BRD4/3/2, also induced similar levels of apoptosis in BETi-P/R and control sAML cells. Co-treatment with ARV-771 and BC2059 synergistically induced lethality in BETi-P/R sAML cells as well as in patient-derived, CD34+ sAML BPCs (combination indices 〈 1.0). This was associated with marked attenuation of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL levels. Also, compared to each agent alone, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW per week) for 3 weeks, significantly reduced sAML burden and improved survival of NSG mice engrafted with HEL-P/R cells (p 〈 0.01). Collectively, these findings underscore that increased levels and activity of β-catenin-TCF7L2-JMJD6-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BRD4 degrader and β-catenin-TCF7L2 inhibitor is a promising therapeutic strategy against BETi-P/R sAML BPCs. Disclosures Bhalla: Beta Cat Pharmaceuticals: Consultancy. Verstovsek:Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding.

Description: Richter Transformation (RT) is defined as the development of aggressive DLBCL (mostly ABC-type) in up to ~15% of patients with antecedent or concurrent diagnosis of CLL. Based on the comparison of immunoglobin gene rearrangements, approximately 80% of RT-DLBCL arise due to a direct clonal evolution of the underlying CLL clone, i.e., clonally related (CLR) RT-DLBCL, which exhibit poor median survival (MS) of one year. Approximately 20% of RT-DLBCLs are clonally unrelated (CUR) to the underlying CLL, arising most likely due to branched clonal evolution from a common pre-CLL progenitor. CUR RT-DLBCLs exhibit a better MS of 5 years. Although chemo-immunotherapy and treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or anti-apoptotic BCL2 inhibitor venetoclax can induce remissions, they fail to induce prolonged disease-free survival in RT-DLBCL. Majority of patients relapse with therapy-refractory disease. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models has prevented pre-clinical testing and development of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL. Based on immunoglobulin heavy chain (IGH) clonality testing by NGS, the RT-DLBCL RT17 was CLR, RT15 was CUR and RT5 was of indeterminate clonality. The PDXs were generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. The RT-DLBCL PDXs grew in the bone marrow and spleen, causing marked splenomegaly, requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL PDX cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT17, RT15, and RT5 cells showed large numbers of genetic mutations, including mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high variant allelic frequencies. Array-CGH showed DNA copy gains or losses in multiple chromosomes, including 3, 8, 9, 11, 12, 17 and 18. A 5'-MYC amplification was detected by FISH analysis in RT5 DLBCL cells. ATAC-Seq showed increased signal intensity representing increased chromatin accessibility in the RT-DLBCL cells compared to CD34+ normal progenitors. High peak numbers were detected in specific loci, including TCF4, PAX5, IRF4, MYB, MYC, BCL2L1 and BCL-2. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities at super-enhancers/enhancers (SEs/Es), including those of TCF4, PAX5, IRF4, BCL2 (RT17 and RT15) and MYC (RT5). Western analyses showed that all three RT-DLBCL PDX cells expressed TCF4, c-Myc, and BRD4, with highest expression in RT5 cells. Accordingly, RT5 cells were more sensitive than cells RT17 and RT15 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. This was associated with greater, OTX015-mediated, depletion of c-Myc in RT5 cells. RT17 and RT15 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. Consistent with this, RT17 and RT15 cells were significantly more sensitive than RT5 cells to venetoclax-induced apoptosis (p 〈 0.01). RT17 and RT15, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT17 and RT15 cells to ibrutinib-induced apoptosis (p 〈 0.001). However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices 〈 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of all RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 also dose-dependently induced apoptosis in RT-DLBCL cells associated with depletion of c-Myc, Bcl-xL, and MCL1 protein levels. These findings highlight the activity and support further in vitro and in vivo evaluation of BETi, BET-PROTAC or CDK9i-based combinations with ibrutinib or venetoclax against genetically-profiled RT-DLBCL cells that are clonally-related or clonally-unrelated to the antecedent CLL. Disclosures Maiti: Celgene: Other: research funding. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding.

Description: LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates active enhancers and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1, mediating transcriptional repression and differentiation block in LSCs that persist in the minimal residual disease (MRD) following attainment of clinical complete remission, leading to relapse and poor outcome in AML. In AML LSCs, genetic alterations and epigenetic dysregulation of enhancers affect levels of myeloid transcriptional regulators, including c-Myc, PU.1, GATA 2 and CEBPα, and their target genes, which are involved in differentiation block in LSCs. Our present studies demonstrate that CRISPR/Cas9-mediated knockout of LSD1 in the AML OCI-AML5 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at super-enhancers and enhancers (SEs/Es) (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed CoREST, c-Myc, CDK6, and c-KIT, while inducing p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq, and protein expression by Western analyses). This led to significant growth inhibition, differentiation and loss of viability of OCI-AML5 and patient-derived AML blasts (p 〈 0.01). Similar effects were observed following exposure of OCI-AML5 (96 hours) to tet-inducible shRNA to LSD1. Knock-down of GFI1 by shRNA (by 90%) also inhibited growth and induced differentiation, associated with upregulation of PU.1, p21 and CD11b levels. Treatment with irreversible (INCB059872, 0.25 to 1.0 µM) or reversible (SP2577, 1.0 to 2.0 µM) LSD1 inhibitor (LSD1i) inhibited binding of LSD1 to CoREST, and significantly induced growth inhibition, differentiation and loss of viability (over 96 hours) of the OCI-AML5, post-myeloproliferative neoplasm (post-MPN) sAML SET2 and HEL92.1.7 cells, as well as patient-derived AML and post-MPN sAML blasts (p 〈 0.01). Co-treatment with INCB059872 and ruxolitinib synergistically induced apoptosis of the post-MPN sAML SET2 and HEL92.1.7 cells and patient-derived CD34+ post-MPN sAML blasts (combination indices 〈 1.0). Notably, pre-treatment with the LSD1i for 48 hours significantly re-sensitized ruxolitinib-persister/resistant SET2 and HEL92.1.7 cells to ruxolitinib (p 〈 0.001). We previously reported that treatment with the BET inhibitor (BETi) JQ1 or OTX015 represses SE/E-driven AML-relevant oncogenes including MYC, RUNX1, CDK6, PIM1, and Bcl-xL, while inducing p21 and p27 levels in post-MPN sAML blasts (Leukemia 2017;31:678-687). This was associated with inhibition of colony growth and loss of viability of AML and post-MPN sAML blasts (p 〈 0.01). Here, we determined that INCB059872 treatment induced similar levels of lethality in BETi-sensitive or BETi-persister/resistant AML and post-MPN sAML cells. Since BETi treatment also depleted LSD1 protein levels, co-treatment with the BETi OTX015 and LSD1i INCB059872 or SP2577 induced synergistic lethality in AML and post-MPN sAML blasts (combination indices 〈 1.0). Co-treatment with INCB059872 (1.5 mg/kg) and OTX015 (50 mg/kg) both orally for 21 days, compared to each agent alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with HEL92.1.7 or HEL92.1.7/OTX015-resistant-GFP/Luc sAML xenografts (p 〈 0.01). Collectively, these findings strongly support further in vivo testing and pre-clinical development of LSD1i-based combinations with ruxolitinib against post-MPN sAML and with BETi against AML or post-MPN sAML cells. Disclosures Bose: CTI BioPharma: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Constellation: Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Pfizer: Research Funding. Kadia:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Bioline RX: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding. Verstovsek:Ital Pharma: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; CTI BioPharma Corp: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Celgene: Consultancy, Research Funding; NS Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Sierra Oncology: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Roche: Research Funding.