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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.

Expanded, detailed methods and detailed reagent descriptions

Supplemental Figure 1. Relative activity of overexpressed SIRT2 in breast cancer cells Supplemental Figure 2. Expression of other sirtuin proteins and HDAC6 in SIRT2 O/E and knockdown cells Supplemental Figure 3. SIRT2 overexpression increases Prdx1 chaperone activity in HEK293 cells treated with H2O2. Supplemental Figure 4. Effects of SIRT2 overexpression on FOXO3A localization Supplemental Figure 5. Schematic of the zebrafish SIRT2 gene and splice-blocking exon 6 morpholino.

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.

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.

EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc-finger transcription factor. Chromosome translocation t(3;3) or inv(3) at 3q26 repositions the enhancer (E) of the tumor suppressor GATA2 to induce EVI1 over-expression, while concomitantly repressing GATA2. EVI1 over-expression promotes self-renewal and blocks differentiation of leukemia stem-progenitor cells (LSCs) and causes therapy refractoriness and inferior overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor (BETi) is active against AML cells with inv(3)/t(3;3). BETi treatment also represses EVI1 and its targets c-Myc and Bcl-xL. We recently reported that tegavivint (TV) (BC-2059), a disruptor of the nuclear TBL1/R1-β-catenin-TCF7L2 complex represses c-Myc, cyclin D1 and Survivin and inhibits growth and survival of AML LSCs. In present studies, a CRISPR screen in UCSD-AML1 cells (with 3q26 lesion and EVI1 overexpression) with gRNAs targeting epigenetic regulators highlighted BRD4 and p300 as dependencies. Treatment with BETi, e.g., OTX015, dose-dependently induced apoptosis of AML cell lines and patient-derived (PD) AML cells (AML191 and AML194) with inv(3) or t(3;3). OTX015 treatment repressed c-Myc, c-Myb, CDK4, MECOM, and MCL1, while inducing HEXIM1 and cleaved PARP levels. TV treatment also dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions, associated with depletion of nuclear β-catenin, EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but increase in p21, CD11b, BIM and cleaved PARP levels. Hi-ChIP with H3K27Ac antibody demonstrated that TV treatment abolished chromatin loops between MYC and its Es within the super-E. RNA-Seq analysis showed negative enrichment (log2-fold) of gene sets of MYC, E2F and WNT targets, DNA replication/repair and reduction of the 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization of EVI1 and β-catenin with TBL1, also confirmed by the Proximity Ligation Assay. CyTOF analysis confirmed that TV treatment reduced EVI1, c-Myc, RUNX1, β-catenin, Bcl-xL, BCL2, MCL1 and Ki67 but increased cleaved PARP levels in the phenotypically characterized LSCs (with high expression of CLEC12A, CD123, CD244 and CD99) harboring inv(3) with EVI1 overexpression. Co-treatment with OTX015 and TV or the p300 inhibitor GNE-049 synergistically induced in vitro apoptosis in the AML cell lines and the PD AML cells with 3q26.2 lesions. Notably, in the flank-implanted PDX of AML191 or tail-vein infused PDX of AML242 models in NSG mice, treatment with OTX015 and TV, compared to vehicle control or each drug alone, significantly reduced AML growth and improved survival, without any host toxicity. These findings highlight the promising pre-clinical efficacy of novel BETi-based combinations against AML models harboring 3q26 lesions and EVI1 overexpression. Citation Format: Christine E. Birdwell, Warren C. Fiskus, Tapan M. Kadia, Christopher P. Mill, John A. Davis, Kaberi Das, Stephen Horrigan, Kapil N. Bhalla. Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4900.

ATP-dependent chromatin remodeling BAF (BRG1/BRM-associated factor) complexes bind and enable transcription factors (TFs) and co-factors to gain access to enhancer/promoter DNA and modulate transcription. In BAF complexes, The BRG1 (SMARCA4) or BRM (SMARCA2) is the core ATPase. AML stem/progenitor cells (LSCs) express and are dependent on BRG1/BRM activity to maintain their dysregulated epigenome/transcriptome, especially driven by mutations in epigenetic regulators and TFs. CRISPR-dependency DepMap also shows dependency on SMARCA4 in numerous AML cell lines. FHD-286 is a selective, oral inhibitor of BRG1/BRM in early clinical development as a therapy for AML. Our present studies demonstrate that exposure to FHD-286 (10 to 100 nM) for 7 days induces differentiation followed by loss of viability of AML cell lines and PD AML cells with MLL rearrangement (r), mutant (mt) NPM1 and chromosome 3q26 lesions (with EVI1 overexpression). Treatment with FHD-286 caused whole-genome, concordant, up- or down-regulations in ATAC-Seq peaks and RNA-Seq-determined mRNA expressions of specific loci, associated with significant reduction in the gene-sets of targets of MYC, mTORC1, E2F, Interferon-gamma, IL6-JAK-STAT3, as well as of inflammatory response and oxidative phosphorylation genes. QPCR analyses determined significant reduction in mRNA expression of MYC, SPI1 and BCL2 genes. Mass spectrometry on AML cell lysates treated with FHD-286 showed log2 fold-reductions in c-Myc, SPI1, MEF2C, KMT2C and CDK4 (in MOLM13) and in EVI1, c-Myb, CDK6 and c-Myc (in AML191) cells. Co-treatment with FHD-286 and venetoclax, decitabine or BET inhibitor (OTX015) exerted synergistic in vitro lethality against AML cell lines and PD AML cells, especially those expressing MLL-r, mtNPM1 or EVI1 (Delta synergy scores > 5 by the ZIP method). Based on the known efficacy of the Menin inhibitor SNDX-50469, we also found that co-treatment with FHD-286 and SNDX-50469 was synergistically lethal against AML cell lines and PD AML cells with MLL-r or mtNPM1. Finally, in a luciferase-transduced, patient-derived xenograft (PDX) model of AML cells with MLL-AF9, FLT3, KMT2C/2D and NOTCH2 mutations, we determined that treatment with FHD-286 administered orally alone for 4 to 6 weeks was significantly effective in reducing AML burden and improving overall survival of the mice. Additionally, co-treatment with FHD-286 and venetoclax, decitabine or OTX015, as compared to each drug alone or vehicle control, significantly reduced AML burden and improved the overall survival of the NSG mice, without inducing significant toxicity. Taken together, these findings highlight the promise of FHD-286 treatment alone and in rational combinations in exerting significant anti-AML efficacy against cellular models of AML, especially those with MLL-r, mtNPM1 or chromosome 3q26 lesions and EVI1 overexpression. Citation Format: Warren C. Fiskus, Jessica Piel, Murphy Hentemann, Christopher P. Mill, Christine E. Birdwell, Kaberi Das, John A. Davis, Tapan M. Kadia, Naval Daver, Sanam Loghavi, Courtney D. DiNardo, Kapil N. Bhalla. Pre-clinical efficacy of targeting BAF complexes through inhibition of the dual ATPases BRG1 and BRM by FHD-286 in cellular models of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1140.

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.

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.

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and -active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored. Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

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.

The EVI1 gene is located on 3q26.2 and encodes a zinc fingers-containing transcription factor. Nuclear β-catenin-TBL1/R1-TCF7L2 axis activity and EVI1 expression characterize leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. EVI1 is overexpressed in AML with chromosome translocation t(3;3) or inv(3) at 3q26, where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression confers poor response to therapy and inferior relapse-free and overall survival in AML. Tegavivint (BC-2059, Iterion) targets TBL1/R1 and disrupts its binding to β-catenin and TCF7L2, repressing MYC, cyclin D1 and Survivin, leading to apoptosis of AML cells. Here, we determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines (UCSD-AML1, MUTZ3 and OCI-AML20) and patient-derived (PD) AML cells (AML191 and AML194) with 3q26.2 lesions with/without monosomy 7. This was associated with attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but upregulation of CD11b, BIM and cleaved PARP levels. Additionally, by reducing BRD4 occupancy at GATA2 enhancer and repressing EVI1, the pan-BET protein inhibitor OTX015 (100-1000 nM) also dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of inflammatory response, TNFα and interferon signaling, TGFβ and apoptosis signaling, but negative enrichment of gene sets of c-Myc, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in phenotypically characterized AML stem cells harboring t(3:3)/inv(3) and EVI1 overexpression (with high expression of CLEC12A, CD123, CD244, CD99). In vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (determined by SynergyFinder) of AML cell lines and the PD AML cells. In the tail-vein infused and engrafted PD AML191 cell model in NSG mice, treatment with TV and/or venetoclax or OTX015 for 6 weeks significantly reduced AML burden and improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. These findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 axis combined with BET protein or BCL2 inhibition is effective therapy against AML models harboring 3q26 lesions and EVI1 overexpression. Citation Format: Christine Birdwell, Warren C. Fiskus, Christopher P. Mill, John A. Davis, Qi Jin, Courtney D. DiNardo, Koichi Takahashi, Stephen Horrigan, Tapan M. Kadia, Naval Daver, Kapil N. Bhalla. Novel combination therapies against AML with 3q26 lesions and EVI1 overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2648.

In MLL1 rearranged (MLL1r) AML (~10%), N-terminus of MLL1 gene is fused to the C-terminus of a fusion partner, e.g. AF9, AF4, ENL and ELL, creating MLL1 fusion protein (MLL-FP), which increases expression of leukemogenic HOXA9 and its co-factor MEIS1. In AML with mutant (mt) NPM1 (NPM1c), MLL1 is the main driver of HOXA9, MEIS1 and FLT3, promoting self-renewal and growth of AML stem-progenitor cells (LSCs). Treatment with Menin inhibitor (MI), SNDX-50469 or SNDX-5613, disrupts binding of Menin to its binding pocket in MLL1/2 and MLL1-FP, reducing expressions of their targets and inducing differentiation and apoptosis. In the phase I/II AUGMENT-101 clinical trial, SNDX-5613 monotherapy was well tolerated and achieved objective remissions in patients with previously treated relapsed/refractory AML harboring MLL1r or NPM1c. However, majority of patients either fail to respond or eventually relapse. A minority of MLL1r AML (~9%) also exhibit a co-mutation in TP53, which is known to confer therapy resistance and poor outcome in AML. This creates the need to develop MI-based synergistic combinations with superior efficacy against patient-derived (PD) AML cells harboring MLL-FP or NPM1c. Present studies found that, following MI treatment, CyTOF analysis of PD MLL1-r and NPM1c AML cells showed decline in protein levels of Menin, MEIS1, MEF2C, PBX3, FLT3, CDK6 and BCL2 in phenotypically characterized AML LSCs expressing CLEC12A, CD123, CD244 and CD99. Notably, in vitro co-treatment with SNDX-50469 in combination with venetoclax, OTX015 (pan-BET inhibitor) or abemaciclib (CDK6 inhibitor) induced synergistic (determined by SynergyFinder V2) loss of viability in AML cell lines (MV4-11, MOLM13 and OCI-AML3), as well as in MOLM13 cells with CRISPR-Cas9-mediated knock-in of mutant or allelic loss of TP53 and in PD AML cells with MLL-r or NPM1c, but not in normal CD34+ progenitor cells or AML cells lacking MLL-FP or NPM1c. A CRISPR screen with a validated, domain-specific gRNA library against chromatin regulators revealed BRD4, p300, MOZ and KDM1A as druggable co-dependencies with treatment with MI. Consistent with this, co-treatment with SNDX-50469 and OTX015, or the p300/CBP inhibitor GNE049 was synergistically lethal in vitro in AML cells with MLL1r or NPM1c, either sensitive to MI or tolerant-resistant (induced in vitro) to MI (MITR cells). Co-treatment with SNDX-5613 and venetoclax or OTX015 compared to each drug or vehicle control, administered orally for 3 to 4 weeks to NSG mice engrafted with either MOLM13-GFP/Luciferase xenograft or with PD NPM1c and mtFLT3 AML xenograft, caused significantly greater reduction in AML burden and increased overall survival without weight loss or other toxicities (p < 0.005). These preclinical findings highlight novel MI-based combinations exhibiting superior in vitro and in vivo anti-AML efficacy against AML cells harboring MLL1-FP or NPM1c that are sensitive to MI or the MITR cells. Citation Format: Warren C. Fiskus, Christopher P. Mill, Christine Birdwell, John A. Davis, Qi Jin, Tapan M. Kadia, Courtney D. DiNardo, Koichi Takahashi, Gerard M. McGeehan, Naval Daver, Kapil N. Bhalla. Menin inhibitor-based combinations to improve efficacy and overcome resistance in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4028.

Cytoplasmic dislocation and loss of nucleolar localization of the chaperone protein NPM1c occurs in 30% of AML following a frame shift C-terminal mutation in exon 12 of NPM1 (creating NPM1c), which contributes to differentiation arrest, growth and survival of AML stem progenitor cells. In present studies, we determined that knock out (KO) of mutant (mt) NPM1 (NPM1c) in the AML OCI-AML3, utilizing targeted gRNA (compared to control) and CRISPR-Cas9, induced growth arrest, morphologic differentiation and loss of viability of the KO cells over 5 to 9 days. This was associated with depletion of protein levels of NPM1c, c-Myc, and MEIS1, but upregulation of PU.1, RUNX1, CD11b, p21 and NOXA levels. ChIP-Seq analysis with H3K27Ac antibody 5d after KO demonstrated reduced peak density at the super-enhancers of MYC, MEIS1, and HOXA9. RNA-Seq analysis showed negative enrichment of HALLMARK gene sets of mRNA expression of MEIS1/HOXA9, MYC targets, ribosome/translation and cell cycle. Compared to OCI-AML3 with NPM1c, OCI-AML3 with NPM1 KO exhibited abrogation of the Menin inhibitor SNDX-50469-induced differentiation and apoptosis. Additionally, NPM1 KO also significantly reduced sensitivity of OCI-AML3 cells to apoptosis induced by targeted agents previously shown to demonstrate efficacy against AML cells with NPM1c, including KPT-330 (XPO1 inhibitor), homoharringtonine (protein translation inhibitor), and anti-AML chemotherapeutic agents, including daunorubicin, cytarabine and etoposide. However, NPM1 KO sensitized OCI-AML3 cells to ATRA (all trans retinoic acid)-induced differentiation and loss of viability, which was associated with marked induction of p21 and CD11b. Utilizing RNA-Seq signature of NPM1c KO and interrogating the LINCS1000-CMap data set of gene expression signatures, we determined that among the top expression mimickers were several pan-HDAC inhibitors and a WEE1 kinase inhibitor. Treatment with adavosertib (WEE1 inhibitor, MK-1775) or romidepsin (HDAC inhibitor) dose-dependently induced apoptosis in OCI-AML3 cells, as well as in 5 samples of patient-derived (PD) AML cells with NPM1c. Moreover, co-treatment with adavosertib and romidepsin synergistically induced lethality in OCI-AML3 and PD AML cells with NPM1c. This was associated with marked reduction in protein expressions of c-Myc, c-Myb, MEIS1 and CDK4/6, but upregulation of TP53 and p21 levels. These findings highlight that presence of NPM1c mechanistically regulates the sensitivity of AML cells to Menin inhibitor, ATRA and anti-AML chemotherapeutic agents. They also identify the novel non-chemotherapy combination of pan-HDAC inhibitor and WEE1 inhibitor for its potential synergistic activity against AML with NPM1c. Citation Format: Christopher P. Mill, Warren C. Fiskus, John A. Davis, Courtney D. DiNardo, Christine Birdwell, Qi Jin, Koichi Takahashi, Joseph D. Khoury, Kapil N. Bhalla. Efficacy of novel combination therapy against AML cells with mutant NPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4030.

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.

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.

Whereas recent clinical studies report metastatic melanoma survival rates high as 30–50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2β) functions as a partial agonist at ErbB4. NRG2β/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z’ > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.

The N-terminus of the histone-lysine-N-methyltransferase MLL1 (KMT2A) contains the menin binding domain (MBD). Menin is a scaffold protein that binds to MBD and tethers MLL1 to chromatin. The Menin-KMT2A complex regulates expression of the leukemogenic homeobox A9 (HOXA9) gene and its co-factor MEIS1 in myeloid stem progenitor cells. In MLL-rearranged (MLL-r) AML, the N-terminus of the KMT2A gene is fused to the C-terminus of any of over 80 fusion partners, most commonly AF4, AF9, ENL and ELL. These MLL fusion partners are components of (and recruit) the super elongation complex and DOT1L to induce H3K4Me3 and H3K79Me2 marks on active chromatin, driving aberrant expression of HOXA9, MEIS1, PBX3, MEF2C and CDK6. In AML with mutant NPM1 (NPM1c), the wild type menin-KMT2A complex is the main oncogenic driver of HOXA9, MEIS1 and FLT3, promoting self-renewal of mutant leukemic blasts. KO-539 is a novel, oral investigational drug candidate targeting the Menin-KMT2A protein-protein interaction. Preclinical data show that KO-539 and close analogs induce differentiation and loss of viability of AML cells, and exert profound anti-leukemic activity in multiple PDX models harboring MLL-FP or NPM1c when dosed continuously QD for 3-6 weeks. In an ongoing phase 1/2A KOMET-001 clinical trial (NCT04067336) evaluating KO-539 in adult patients with relapsed and/or refractory AML, significant biological activity as well as complete remission lacking minimal residual disease have been observed. The present studies were focused on further elucidating biologic effects and on determining synergistic activity of KO-539 with agents targeting BCL2, BET proteins and CDK6. Treatment with KO-539 dose-dependently inhibited in vitro growth, as well as induced differentiation (by both morphology and increased CD11b expression) and loss of viability of MOLM13 (MLL-AF9 and FLT3-ITD) and OCI-AML3 (NPM1c and homozygous NRAS mutation) cells and patient-derived (PD) MLL-r or NPM1c AML cells. This was associated with repression of MEIS1, PBX3, MEF2C, FLT3, MYC, BCL2 and CDK6 transcription but increased mRNA expression of ITGAM (CD11b). KO-539 treatment also dose-dependently depleted protein levels of Menin, MEIS1, FLT3, CDK6 and BCL2, but upregulated MCL1 and CD11b proteins in MOLM13 and OCI-AML3 cells. Mass cytometry (CyTOF) analysis of PD MLL-r and NPM1c AML samples confirmed that, following KO-539 treatment there was also decline in protein levels of Menin, MEIS1, MEF2C, PBX3, FLT3, CDK6 and BCL2 in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). KO-539-mediated Menin depletion was associated with polyubiquitylation and proteasomal degradation of Menin protein, given that protein levels were restored by co-treatment with the proteasome inhibitor carfilzomib. Notably, co-treatment (in vitro) with KO-539 in combination with venetoclax, OTX015 (pan-BET inhibitor) or abemaciclib (CDK6 inhibitor) for 72 to 96 hours induced synergistic loss of viability in cultured cell lines and PD AML cells from both MLL-r and NPM1c AML but not normal CD34+ progenitor cells or AML cells lacking MLL-FP or NPM1c, as determined by the SynergyFinder algorithm. Findings of ongoing in vivo studies determining effects of treatment with KO-539 and/or venetoclax or OTX015, versus vehicle control in PDX models will be presented at the meeting. These preclinical findings highlight the molecular correlates of anti-AML efficacy of KO-539 and demonstrate potentially synergistic KO-539-based combinations with inhibitors of BCL2, BET proteins and CDK6 against MLL-r or NPM1 mutant AML. Disclosures Daver: Bristol Myers Squibb: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Novimmune: Research Funding; FATE Therapeutics: Research Funding; Genentech: Consultancy, Research Funding; Glycomimetics: Research Funding; Amgen: Consultancy, Research Funding; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Kadia: Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support; Amgen: Other: Grant/research support; BMS: Other: Grant/research support; Cure: Speakers Bureau; Genentech: Consultancy, Other: Grant/research support; Dalichi Sankyo: Consultancy; AstraZeneca: Other; Astellas: Other; Genfleet: Other; Ascentage: Other; Cellonkos: Other; Sanofi-Aventis: Consultancy; Pulmotech: Other; Jazz: Consultancy; Liberum: Consultancy; Novartis: Consultancy; Pfizer: Consultancy, Other. DiNardo: Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Leoni: Kura Oncology: Current Employment. Burrows: Kura Oncology: Current Employment.

MLL1 (KMT2A) is a transcriptional regulator and a histone-lysine-N-methyltransferase. N-terminus of MLL1 binds to the scaffold protein Menin via the menin binding domain (MBD). Menin-KMT2A complex regulates expression of the leukemogenic Homeobox A9 (HOXA9) and its co-factor MEIS1 in myeloid stem progenitor cells. In MLL1-rearranged (MLL-r) AML (~10% of AML), the N-terminus of MLL1 gene is fused to the C-terminus of any of over 80 fusion partners, including AF4, AF9, ENL and ELL (~70% of MLL-r AML), which are part of (and recruit) the super elongation complex (SEC) (including AFF1/4 and pTEFb) and DOT1L to induce H3K4Me3 and H3K79Me2 marks on active chromatin. MLL1 fusion protein (MLL-FP) dysregulates gene expression of HOXA genes and their co-factors MEIS1, PBX3, as well as of MEF2C and CDK6. In AML with mutant (mt) NPM1 (NPM1c), MLL1 is the main oncogenic driver of HOXA9, MEIS1 and FLT3, promoting self-renewal of myeloid progenitor cells. Conditional knockout of MEN1 prevents MLL-r or NPM1c AML. Treatment with orally bioavailable Menin inhibitor, SNDX-50469 or SNDX-5613, disrupts binding of Menin to its binding pocket in MLL1/2 and MLL1-FP, evicting Menin from the chromatin, reducing MLL1/2 and MLL1-FP binding to targets, as well as inducing differentiation and apoptosis in AML cells expressing MLL-FP or NPM1c. SNDX-50469 or SNDX-5613 treatment also exhibits impressive in vivo anti-AML efficacy in AML models expressing MLL-FP and NPM1c. In the phase I/II AUGMENT-101 clinical trial, SNDX-5613 monotherapy was well tolerated and achieved high rates of objective remissions in patients with previously treated relapsed/refractory AML harboring MLL-r or NPM1c. Focus of the present studies was to further elucidate epigenetic, gene-expression and anti-AML effects, as well as identify novel synergistic combinations of SNDX-50469 or SNDX-5613 with other targeted agents to achieve superior in vivo efficacy in AML expressing MLL-r or NPM1c. Utilizing ATAC and RNA-Seq in AML MOLM13 cells (expressing MLL-AF9 and FLT3-ITD), we first determined that exposure to SNDX-50469 simultaneously reduced ATAC peaks and mRNA expression of HOXA9, MEIS1, PBX3, FLT3, MEF2C and CDK6. Concurrent ChIP-Seq analysis demonstrated reduction in H3K27Ac peaks and reduced activity of super enhancers of MEIS1, PBX3, MEF2C, MYC and MYB. Single-cell RNA-Seq analysis of AML cells with MLL-AF9 and mtFLT3 also confirmed that SNDX-50469 treatment caused reduction in mRNA expression of MLL-FP targets in AML stem/progenitor cells, including HOXA9, LAMP5, PBX3, MEF2C, MEIS1, FLT3, MYB and CDK6. Consistent with this, Western analysis in MOLM13 and OCI-AML3 cells (NPM1c and homozygous NRAS mutation) following SNDX-50469 treatment showed markedly reduced protein levels of MEIS1, FLT3, CDK6, and BCL2, with increased levels of MCL1 and CD11b. Mass cytometry (CyTOF) analysis of patient-derived (PD) MLL-r or mtNPM1 AML samples also confirmed that SNDX-50469 treatment attenuated protein levels of Menin, MEIS1, MEF2C, PBX3, RUNX1, FLT3, CDK6, Bcl-xL and BCL2 in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, and CD99, but low expression of CD11b). Notably, in vitro treatment of cell lines and PD AML cells from MLL-r or NPM1c AML with SNDX-50469 or SNDX-5613 in combination with venetoclax, or OTX015 (pan-BET protein inhibitor), or abemaciclib (CDK6 inhibitor) for 72 to 96 hours induced synergistic loss of viability, as determined by SynergyFinder. No loss of viability was observed in normal CD34+ progenitor cells or AML cells lacking MLL-FP or NPM1c. We next determined in vivo anti-leukemia efficacy of SNDX-5613 alone (50 to 75 mg/kg, PO, BID) or its co-treatment with venetoclax (30 mg/kg, PO, daily) or OTX015 (30 mg/kg, PO, daily) for 3 to 4 weeks in NSG mice engrafted with either MOLM13-GFP/Luciferase xenograft or with PD NPM1c and mtFLT3-GFP/Luciferase AML xenograft. Co-treatment of engrafted mice with SNDX-5613 and venetoclax or OTX015 caused significantly greater reduction in AML burden and increased overall survival without weight loss or other toxicities than treatment with each agent alone (p< 0.005). These preclinical findings highlight the molecular correlates of anti-AML efficacy of SNDX-50469 or SNDX-5613. They also demonstrate greater in vitro and in vivo efficacy of SNDX-5613-based combinations with inhibitors of BCL2, BET proteins and CDK6 against AML harboring MLL1-FP or NPM1c. Disclosures Kadia: AstraZeneca: Other; AbbVie: Consultancy, Other: Grant/research support; Sanofi-Aventis: Consultancy; Jazz: Consultancy; Ascentage: Other; Genentech: Consultancy, Other: Grant/research support; Cellonkos: Other; Dalichi Sankyo: Consultancy; Amgen: Other: Grant/research support; Pfizer: Consultancy, Other; Astellas: Other; Liberum: Consultancy; Novartis: Consultancy; Pulmotech: Other; Genfleet: Other; Cure: Speakers Bureau; Aglos: Consultancy; BMS: Other: Grant/research support. DiNardo: Agios/Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Novartis: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Foghorn: Honoraria, Research Funding; Takeda: Honoraria; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Takahashi: Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Novartis: Consultancy; Celgene/BMS: Consultancy. McGeehan: Syndax Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Daver: Gilead Sciences, Inc.: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Novimmune: Research Funding; Trovagene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Glycomimetics: Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding.

EVI1 gene maps to the MECOM locus at chromosome 3q26.2 and encodes for a zinc finger domain-containing transcriptional regulator. EVI1 supports hematopoietic stem cell self-renewal and blocks hematopoietic differentiation. EVI1 is overexpressed in up to 10% of AML, including those harboring chromosome translocation t(3;3)(q21;q26.2) or inv(3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression due to 3q26.2 lesions in MDS and AML is frequently associated with monosomy 7 and confers poor response to therapy and inferior relapse-free and overall survival. We had previously reported the pre-clinical efficacy of targeting TBL1/R1-nuclear β-catenin-TCF7L2 by tegavivint (BC-2059, Iterion Therapeutics) against AML stem/progenitor cells. This led us to interrogate the anti-AML activity of tegavivint (TV) in AML models harboring 3q26.2 lesions, where EVI1 overexpression has been documented to drive the biology of AML stem/progenitor cells. For this, we utilized AML cell lines with 3q26.2 lesions with/without monosomy 7 (UCSD-AML1, OCI-AML20, AML191 / MUTZ-3, AML194, HNT34), as well as patient-derived (PD) AML cells with 3q26 lesions with or without monosomy 7. Treatment with TV (10 to 100 nM) dose-dependently induced apoptosis in these cellular models. This was associated with attenuation of protein levels (determined by immunoblot analyses) of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but upregulation of CD11b, BIM and cleaved PARP levels. Additionally, pan-BET protein inhibitor OTX015 (100 to 1000 nM) dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). Following TV treatment, RNA-Seq and gene set enrichment analysis in UCSD-AML1 and OCI-AML20 cells showed log2 fold-changes in gene expression and positive enrichment of pathway genes and/or reactomes of inflammatory response, TNFα and interferon signaling, TGFβ, NOTCH and apoptosis signaling, as well as negative enrichment of gene sets of c-Myc, E2F, G2M checkpoint, DNA replication and repair and chromosome maintenance (all with FDR q-values < 0.1). QPCR analysis showed repression of EVI1, MYC and KIT, but upregulation of Axin2 mRNAs. Following TV treatment, confocal microscopy showed reduction of nuclear protein levels of EVI1 and β-catenin, as well as disrupted their co-localization with TBL1. Proximity ligation assay also demonstrated that exposure to TV significantly reduced the proximity of TBL1 and EVI1 as well as of TBL1 and β-catenin. Mass cytometry (CyTOF) analysis of patient-derived (PD) AML samples with t(3:3)/inv(3) confirmed that TV treatment attenuated protein levels of EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67, but augmented protein levels of APC and cleaved PARP in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). Consistent with effects of TV on gene-expressions that regulate cell death pathways, in vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (as determined by the SynergyFinder algorithm) of AML cell lines and PD AML cells with t(3:3)/inv(3) and EVI1 overexpression. In the in vivo HNT-34 model of flank-implanted and established tumors in athymic nude mice, treatment with TV (50 mg/kg, TIW, IP) and venetoclax (30 mg/kg, PO, daily) for 5-weeks yielded more tumor growth delay than vehicle control or TV alone. Additionally, following tail vein infusion and engraftment of luciferized AML191 cells in NSG mice, treatment with TV and/or venetoclax or OTX015 (30 mg/kg, PO, daily) was evaluated for 6 weeks. Co-treatment with TV and venetoclax or TV and OTX015 significantly reduced AML growth (determined by reduction in bioluminescence by Xenogen camera) (p < 0.05), as well as improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. Collectively, these findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 by treatment with TV also inhibits EVI1 and its targets. They also demonstrate superior pre-clinical efficacy of novel TV-based combinations with BCL2 or BET protein inhibitor against AML models harboring 3q26 lesion and EVI1 overexpression. Disclosures DiNardo: Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding. Takahashi: Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Novartis: Consultancy; Celgene/BMS: Consultancy. Horrigan: Iterion Therapeutics: Current Employment. Kadia: Astellas: Other; Cure: Speakers Bureau; Genfleet: Other; Ascentage: Other; Jazz: Consultancy; Sanofi-Aventis: Consultancy; Dalichi Sankyo: Consultancy; Cellonkos: Other; Novartis: Consultancy; AstraZeneca: Other; BMS: Other: Grant/research support; Liberum: Consultancy; Amgen: Other: Grant/research support; Pulmotech: Other; Pfizer: Consultancy, Other; Genentech: Consultancy, Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support.

RUNX1 is the DNA-binding subunit of the core-binding factor complex and a master-regulator transcription factor (TF), which is involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 commonly occur in Myelodysplastic Syndromes (MDS), as well as in secondary (s) or de novo AML (~10%). The germline, intragenic mutations and deletions in RUNX1 cause the highly penetrant (~40%), autosomal dominant, Familial Platelet Disorder (RUNX1-FPD) by the median age of 33 years with a propensity to evolve into myeloid malignancy (FPD-MM), i.e., MDS or AML. Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function (LOF) mutations. Lack of specific targeted therapy, coupled with resistance to standard chemotherapy, may account for poorer outcome observed in AML or FPD-MM expressing somatic or germline mutant (mt) RUNX1, respectively. We previously reported that knockdown (KD) of RUNX1 (wild type and mtRUNX1) is significantly more lethal toward AML blasts progenitor cells (BPCs) expressing mt versus wild type (wt) RUNX1 or normal hematopoietic progenitor cells (HPCs), indicating greater dependency on the residual wtRUNX1. Based on higher occupancy of the BET protein BRD4 at the RUNX1 super-enhancer/enhancer (SE/E) and other SE-driven AML-relevant oncogenes, the BET inhibitor OTX015 evicted BRD4 from the chromatin and repressed RUNX1 and SE-driven oncogenes, mediating efficacy against AML BPCs expressing mtRUNX1. Utilizing the RNA-Seq signature of RUNX1-depleted (by shRNA) AML cells and conducting LINCS (Library of Integrated Network-based Cellular Signatures) 1000-CMap (Connectivity Mapping) analysis, we discovered several expression mimickers (EMs), including the top-ranking protein-translation inhibitor homoharringtonine (HHT), or its semi-synthetic analogue omacetaxine mepesuccinate (OM). We tested the pre-clinical efficacy of HHT or OM against AML cell lines (OCI-AML5 and Mono-Mac-1) and patient-derived primary AML BPCs expressing somatic mtRUNX1 or wtRUNX1. Treatment with HHT or OM (5 to 100 nM) induced significantly more lethality in OCI-AML5 and Mono-Mac-1, as well as primary AML BPCs expressing mutant versus wtRUNX1, or in normal bone marrow progenitor cells (p < 0.05). Notably, first-time ever, CRISPR-Cas9-mediated knock-in of mtRUNX1 (R174*) into wtRUNX1-expressing AML HL-60 and OCI-AML2 cells showed that, in this isogenic setting also, HHT or OM, or OTX015 (50 to 1000 nM) treatment induced significantly more lethality in mt versus wtRUNX1-expressing cells (p < 0.05). The differential lethal activity of HHT in cells was associated with marked HHT-mediated depletion of c-Myc, c-Myb, PU.1, mt/wtRUNX1 and MCL1 in mtRUNX1 expressing HL60-R174* and OCI-AML2-R174* cells. Possibly due to HHT-mediated MCL1 repression, co-treatment with HHT and venetoclax (V) (10 to 100 nM), synergistically induced in vitro apoptosis of OCI-AML5, HL60-R174* and OCI-AML2-R174* cells (CI < 1.0). Compared to vehicle control, whereas daily treatment with OM (1.0 mg/kg, SQ) alone for 3 weeks significantly reduced the leukemia burden, co-treatment with OM and V (20 mg/kg orally) exerted significantly greater reduction in AML burden due to luciferized OCI-AML2-R174* cells engrafted into NSG mice. Combined therapy with OM and V also improved survival of the mice engrafted with OCI-AML2-R174* cells, without inducing weight loss or any other toxicity. Treatment with HHT also exerted differentially greater in vitro lethality against HPCs from FPD-MM expressing germline mtRUNX1 along with other MDS/AML-associated co-mutations, compared to RUNX1-FPD HPCs. We recently also established in culture, first time ever, a germ-line mtRUNX1 (K194N)-expressing FPD/MM cell line (GMR-AML1) from the bone marrow aspirate cells of a patient with FPD-MM that also expressed mutations in BCOR, PHF6, SRFS2 and SF3B1. Exposure to HHT (10 to 100 nM) dose-dependently inhibited cell growth and induced lethality in GMR-AML1 cells. These studies highlight that treatment with OM could potentially revert hematopoiesis in FPD-MM back to the RUNX1-FPD status. Collectively, our preliminary findings strongly support the merit of testing co-treatment with OM and venetoclax in patients with AML expressing somatic mtRUNX1, as well as in FPD-MM prior to or instead of allogeneic SCT from an unrelated donor. Disclosures DiNardo: Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Syros: Honoraria; Jazz: Honoraria; Agios: Consultancy, Honoraria, Research Funding; ImmuneOnc: Honoraria; MedImmune: Honoraria; Calithera: Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Novartis: Consultancy.

Richter Transformation (RT) is the development of aggressive DLBCL (mostly ABC sub-type) in up to ~15% of patients with antecedent CLL. Approximately 80% of RT-DLBCLs are clonally-related (CLR) to the underlying CLL, with a median survival (MS) of one year. Alternatively, ~20% of RT-DLBCLs are clonally-unrelated (CLUR) to the underlying CLL, exhibiting a better MS of 5 years. Chemo-immunotherapy, or monotherapy with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, anti-apoptotic BCL2 inhibitor venetoclax or with anti-PD1 therapy fails to achieve prolonged disease-free survival, with a majority relapsing with therapy-refractory disease. To develop and test novel targeted therapies for RT-DLBCL, we successfully established three patient-derived xenograft (PDX) models of luciferized RT-DLBCLs, including the CLR HPRT3 and CLUR HPRT2. HPRT3 and HPRT2 cells were of ABC sub-type of DLBCL, based on positive MUM1/IRF4 and negative CD10 and BCL6 expressions. The third RT-DLBCL (HPRT1) was a rarer GCB variety of DLBCL, displaying high CD10, BCL6, Ki-67 and c-Myc expressions. FISH analysis confirmed 5' MYC amplification in HPRT1 cells. Cytogenetic and array-CGH analyses showed large numbers of karyotypic and genetic alterations in HPRT3 and HPRT2 RT-DLBCLs. Low-pass whole genome sequencing showed that HPRT3 and HPRT2, but not HPRT1, exhibit large areas of amplification of DNA on chromosome 18, with copy gains at 18q21.1 involving the TCF4 gene. NextGen sequencing (NGS) of a panel of 300 genes (L-300 panel) showed more genetic mutations (at high % VAF) in HPRT3, as compared to HPRT2 or HPRT1 cells. These genetic alterations targeted transcription factors, epigenetic regulators, DNA damage/repair enzymes, signaling enzymes and their regulators. Utilizing ATAC-Seq, ChIP-Seq with H3K27Ac and BRD4 antibodies, and single cell (sc) RNA-Seq analyses, we evaluated active chromatin and gene-expressions in the three RT-DLBCLs. ChIP-Seq-determined signal-density of H3K27Ac and BRD4, as well as ATAC-Seq-determined peak-density were increased at active SE/E of BCL2, TCF4, PAX5 and IRF4 in HPRT3 and HPRT2 cells. Notably MYC, BCL6 and CDK6 SEs were active in HPRT1 cells. scRNA-Seq generated t-SNE plots showed that HPRT3 cells exhibited highest number of transcriptionally active cell-clusters, with high mRNA expressions of TCF4, PAX5 and IRF4 in HPRT3 and HPRT2 cells. HPRT1 cells overexpressed MYC mRNA. HPRT3 and HPRT2 were relatively more sensitive to venetoclax-induced lethality, which correlated with higher BCL2, BAX, BAK and BIM protein levels. Higher Bcl-xL levels correlated with increased sensitivity of HPRT3 and HPRT2 versus HPRT1 cells to a Bcl-xL-specific inhibitor A-1155463. Conversely, higher MCL1 levels correlated with greater sensitivity of HPRT1 cells to an MCL1-specific inhibitor AZD-5991. Notably, HPRT3 and HPRT2 versus HPRT1 cells were relatively resistant to ibrutinib treatment. This was due to activation of the alternative MAP3K14 (NIK kinase)-NFkB pathway with increased processing of p100 to p52. Treatment with CDK9 inhibitor NVP2 dose-dependently induced apoptosis of the 3 RT-DLBCL cells, associated with depletion of c-Myc, Bcl-xL, and MCL1 protein levels. BET inhibitor OTX015 and BET protein degrader ARV-771 induced more lethality in HPRT1, compared to HPRT3 and HPRT2 cells, which correlated with higher levels of BRD4, c-Myc and TCF4, but markedly lower levels of IRF4 in HPRT1 cells. CRISPR-Cas9-mediated knockout (KO) of IRF4 markedly depleted nuclear levels of IRF4 and c-Myc (a target of IRF4), which significantly increased sensitivity to OTX015 in IRF4 KO RT-DLBCL cells. Co-treatment with OTX015 and ibrutinib or venetoclax was synergistically lethal in all three RT-DLBCL cell-types (CI < 1.0). Co-treatment with BET protein degrader ARV-771 and ibrutinib or venetoclax exerted synergistic lethality in all three RT-DLBCL cell-types (CI < 1.0). Finally, following tail vein infusion and engraftment of HPRT3 cells, daily treatment for three weeks with ARV-771 and venetoclax compared to each drug or vehicle control, significantly reduced the RT-DLBCL burden, as well as improved survival without inducing toxicity in the NSG mice. These findings strongly support further testing of BET protein antagonist-based combinations with BH3 mimetics, BTK inhibitors, as well as with other novel agents, utilizing pre-clinical models of RT-DLBCL. Disclosures Green: KDAc Therapeutics: Current equity holder in private company.

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.

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.

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.

Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains − I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using western blot, mass spectrometry and circular dichroism (CD) spectroscopy.

Transcriptional regulators (TFs) involved in cell-growth, differentiation and survival of AML stem/progenitor cells (LSCs) include RUNX1, PU.1, CEBPα, c-Myb and c-Myc. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2). LSD1 is part of the repressor complexes involving GFI1, CoREST and HDAC1/2, that regulate active super-enhancers/enhancers (SEs/Es) and their target genes, mediating transcriptional repression and differentiation block in LSCs. GFI1 is a zinc-finger transcriptional repressor involved in AML development and differentiation. GFI1 contains an N-terminal domain through which it binds to the CoREST/LSD1/HDAC1/2 complex to regulate differentiation in LSCs. CRISPR-suppressor scanning revealed that enzymatic activity of LSD1 was not required for LSC differentiation, instead disruption of binding of LSD1 to GFI1 and CoREST induced differentiation in LSCs. LSD1 and GFI1 expression correlates with worse prognosis in MDS/AML. In present studies, we demonstrate first-time ever that knockout (KO) or degradation of LSD1 utilizing CRISPR-Cas9 or LSD1-FKBP12(F36V) and dTAG-13, respectively, disrupted LSD1-binding to GFI1/1B and CoREST, inhibiting colony growth and inducing differentiation markers (CD86 and CD11b) and morphologic differentiation of AML and post-MPN sAML blast progenitor cells (BPCs). CRISPR-mediated knockout of LSD1 in the AML OCI-AML5 and sAML SET2 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at SEs/Es (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed DNMT1, CoREST, c-Myc, CDK6, and c-KIT, while inducing GFI1, PU.1, CEBPα, p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq and by Western analyses). This correlated with growth inhibition, % differentiation and apoptosis of AML and sAML cells. CRISPR-mediated GFI1-KO ± the irreversible LSD1 inhibitor (LSD1i) (INCB059872, INCB), repressed GFI1 levels, yet enhanced expressions of PU.1, p21 and CD11b and significantly increased % morphologic differentiation. Treatment with INCB (0.25 to 1.0 µM) also disrupted binding of LSD1 to GFI1 and to CoREST, increased GFI1/1B and PU.1 and repressed c-Myc protein levels, while significantly inhibiting colony growth, inducing differentiation and loss of viability of AML and post-MPN sAML (SET2 and HEL92.1.7) cells, as well as patient-derived AML and post-MPN sAML blasts (p < 0.01). Following INCB treatment, ATAC-Seq analysis demonstrated gained peaks in GFI1 and PU.1-target genes. Following H3K27Ac ChIP-seq analysis rank-ordering of SEs (ROSE) plot highlighted active SEs of RUNX1, GFI1, BCL2, PU.1, IRF8 and SMYD3, accompanied by increased H3K27Ac occupancy at the chromatin of GFI1 and PU.1 targets. Notably, INCB treatment also increased BRD4 occupancy, especially at the GFI1 and PU.1-target genes. RNA-Seq analysis showed that INCB treatment perturbed mRNA expressions, with positive normalized enrichment scores (NES) for interferon α, inflammatory-response, GFI1-targets and E2F-target gene-sets, and negative NES for c-Myc-targets and oxidative-phosphorylation gene-sets. RNA-Seq analyses of INCB-treated compared to untreated OCI-AML5 and SET-2 cells also demonstrated log2 fold-increase in the mRNA expressions of GFI1, PU.1 and CEBPα target-genes. Utilizing a protein domain-scanning CRISPR-Cas9 sgRNA screen followed by LSD1i treatment, present studies also demonstrate co-dependencies, including BRD4, in AML cells. BET inhibitor (BETi) treatment also depleted LSD1 protein levels, and co-treatment with the BETi OTX015 and INCB induced synergistic lethality in AML and post-MPN sAML blasts (Combination Indices < 1.0). Pre-treatment with INCB re-sensitized JAKi-resistant sAML cells to ruxolitinib-induced apoptosis and BETi-resistant post-MPN sAML cells to BETi-induced apoptosis. Notably, co-treatment with INCB (1.5 mg/kg) and ruxolitinib (20 mg/kg) or OTX015 (50 mg/kg), administered orally for 21 days, compared to ruxolitinib alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with luciferized sAML HEL92.1.7 xenografts (p < 0.01). Collectively, these findings support further pre-clinical development of LSD1i-based combinations with ruxolitinib and BETi against post-MPN sAML. Disclosures Bose: CTI BioPharma: Honoraria, Research Funding; NS Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding; Blueprint Medicines Corporation: Honoraria, Research Funding; Promedior, Inc.: Research Funding; Incyte Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kartos Therapeutics: Honoraria, Research Funding. Kadia:Incyte: Research Funding; Pulmotec: Research Funding; Cellenkos: Research Funding; Celgene: Research Funding; Amgen: Research Funding; Genentech: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Cyclacel: Research Funding; Novartis: Honoraria; Ascentage: Research Funding; Astellas: Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Astra Zeneca: Research Funding; BMS: Honoraria, Research Funding. Verstovsek:CTI Biopharma Corp: Research Funding; AstraZeneca: Research Funding; Sierra Oncology: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding; PharmaEssentia: Research Funding; Blueprint Medicines Corp: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Gilead: Research Funding; Protagonist Therapeutics: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Celgene: Consultancy, Research Funding; ItalPharma: Research Funding.

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.

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.

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.

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.

Treatment with bromodomain and extra-terminal protein inhibitor (BETi) reduces in vivo AML cell burden, inducing clinical remissions in AML. Yet, resistance to BETi treatment develops uniformly. Here, following at least 10 exposures of secondary (s) AML control (parental) SET2 and HEL92.1.7 cells to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we generated 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 controls, SET2-P/R and HEL-P/R cells lacked additional genetic alterations or altered levels of TRIM33, SPOP, DUB3 or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). However, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels of β-catenin, the transcription factor TCF7L2 and adaptor protein TBL1X (TBL1), associated with increased expression of TCF7L2 targets, including c-Myc, Cyclin D1, TERT and Survivin. ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses showed significant gain of peaks and active enhancers in HEL-P/R over HEL92.1.7 cells, with enrichment of STAT5, MYC, PU.1 and GATA2 binding sites, as well as newly gained peaks in the enhancers of JAK1/2, RUNX1, PU.1, MYC, BCL2L1 and CTNNB1. RNA-Seq analysis showed significant increase/decrease in mRNA expressions (340/247), with 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, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Also, confocal microscopy demonstrated increased binding of β-catenin with TBL1 and TCF7L2 in the nucleus in BETi-P/R sAML cells. β-catenin inhibitor BC2059, which disrupts binding of nuclear β-catenin with TBL1 and TCF7L2 and depletes β-catenin levels, exerted similar lethality in BETi-P/R sAML and control sAML cells. Both shRNA-mediated knockdown of BRD4 and BRD4-PROTAC (proteolysis-targeting chimera) ARV-771 (Arvinas, Inc.), which degrades BRD4/3/2, induced similar level 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). Thus, increased levels and activity of β-catenin-TCF7L2-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. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Steve Horrigan, Raffaella Soldi, Joseph D. Khoury, Sunil Sharma, Srdan Verstovesek, Kapil N. Bhalla. BRD4 degrader and inhibitor of beta catenin-TCF7L2 are synergistically active against human AML cells resistant to BET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3036.

Richter Transformation (RT) is the development (in up to 15%) of aggressive DLBCL in patients with antecedent or concurrent CLL. By comparing immunoglobin gene rearrangements, 80% of RT-DLBCL (mostly ABC type) are clonally related (CR) to the underlying CLL, exhibiting poor median survival (MS) of 1 year, whereas 20% of RT-DLBCLs are clonally unrelated (CUR), exhibiting MS of 5 years. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models had prevented pre-clinical testing of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL (CR: RT15 and RT17, CUR: RT5) generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. RT-DLBCL xenografts grew in the bone marrow and spleen, causing marked splenomegaly and requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT5 and RT15 cells showed mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high VAF. Array-CGH showed DNA copy gains or losses in multiple chromosomes including 3, 8, 9, 11, 12, 17 and 18. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities across all super-enhancers/enhancers (SEs/Es), with several SEs/Es exhibiting high scores. ATAC-Seq showed markedly increased signal intensity in RT-DLBCL cells compared to CD34+ normal progenitors, with high peak numbers in specific loci. Western analyses showed that RT15 and RT17 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. RT15 and RT17 cells were more sensitive than RT5 cells to anti-BCL2 venetoclax-induced apoptosis (p < 0.01). RT15 and RT17, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT15 and RT17 cells to ibrutinib-induced apoptosis. RT15 and RT17 cells were also less sensitive than RT5 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. 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 RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 dose-dependently induced apoptosis in RT-DLBCL cells which was associated with depletion of c-Myc, Bcl-xL and MCL1 protein levels. These findings highlight the activity and support further evaluation of in vitro and in vivo 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. Citation Format: Warren C. Fiskus, Abhishek Maiti, Dyana T. Saenz, Christopher P. Mill, Joseph D. Khoury, Kapil N. Bhalla. Targeted therapy evaluation in novel pre-clinical in vitro and in vivo models of Richter transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1071.

Standard AML chemotherapy and JAK inhibitor (ruxolitinib) treatments are ineffective against post-myeloproliferative neoplasm (MPN) sAML. Mutations in JAK2, MPL and calreticulin, and co-epimutations, result in a dysregulated transcriptome, which is responsible for transformation of MPN to sAML and for its therapy-refractoriness. CDK9 is the catalytic subunit of pTEFb that phosphorylates serine 2 in the heptad-repeats of the C-terminal domain of RNA pol II (RNAP2). CDK9 also phosphorylates and inactivates negative transcript elongation factors (NELF and SPT5), which induces promoter-proximal pause-release of RNAP2 to enable productive mRNA transcript elongation. The bromodomain extra-terminal (BET) protein BRD4 recruits pTEFb to the chromatin to promote RNAP2-mediated elongation of transcripts, including those of c-Myc, Bcl-xL, PIM1 and MCL1, important for cell growth and survival of post-MPN sAML cells. Here, we determined the effects of inhibiting BRD4-CDK9-RNAP2 axis in post-MPN sAML cells. Treatment with the CDK9 inhibitor (CDK9i) NVP2 or BAY1143572 (B) dose-dependently induced apoptosis of ruxolitinib-sensitive (Rux-S) sAML cultured cells lines HEL92.1.7 (HEL) and SET2, as well as of patient-derived sAML blast progenitor cells (BPCs), while sparing normal CD34+ progenitor cells (HPCs). CDK9i-induced lethality in sAML cells was associated with attenuation of protein levels of S2-phosphorylated RNAP2, as well as depletion of mRNA and protein levels of c-Myc, PIM1, MCL1, Bcl-xL and XIAP. ATAC-Seq analyses demonstrated that treatment with NVP2 or B caused marked alterations in chromatin accessibility, including large peak losses on the chromatin with binding sites of TFs such as ERG, PU.1, STAT5, c-Myc, and RELA. This was associated with attenuation of mRNA expression of cMyb, c-Myc, RUNX1, LMO2, RELB, NFKB2, c-FLIP, MCL1, CDK6 and Bcl-xL. Following engraftment of luciferase transduced HEL cells into NSG mice, daily treatment with B at 10 mg/kg for 3 wks significantly reduced sAML burden and improved survival of the NSG mice (p < 0.002). Co-treatment of NVP2 or B with ruxolitinib or BCL2/Bcl-xL inhibitor ABT263 synergistically induced apoptosis of ruxolitinib-sensitive sAML cells (CI values of < 1.0). Treatment with NVP2 or B, or with BETi OTX015, also induced apoptosis of ruxolitinib persister/resistant (Rux-P) sAML cells (which demonstrate > 10-fold higher ruxolitinib IC50 values than Rux-S cells). This was associated with attenuated protein levels of p-RNAP2, c-Myc, PIM1, Bcl-xL and XIAP. Co-treatment with OTX015 and NVP2 or B synergistically induced apoptosis of not only HEL and SET2 but also HEL-RuxP and SET-RuxP cells, as well as of patient-derived sAML BPCs (CI values < 1.0). These findings confirm that targeted inhibition of BRD4-CDK9-RNAP2 leads to high level of pre-clinical efficacy against ruxolitinib-sensitive and ruxolitinib-refractory sAML cells. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Joseph D. Khoury, Srdan Verstovsek, Kapil N. Bhalla. Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3836.

Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-alpha, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-alpha in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-alpha increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-alpha increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-alpha on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-alpha were somewhat interchangeable in their effects on EGFR signaling; however, TGF-alpha had a greater effect in vitro and AREG had a greater effect in vivo.

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.

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer.

ErbB4 is a member of the ErbB family of receptor tyrosine kinases. This family includes ErbB2 (HER2/Neu), a validated therapeutic target in breast cancer. Several studies indicate that ErbB4 functions as a tumor suppressor in breast cancer, whereas others indicate that ErbB4 functions as an oncogene. Here the authors explore the context in which ErbB4 functions as an oncogene. Silencing expression of either ErbB2 or ErbB4 in breast tumor cell lines results in reduced stimulation of anchorage independence and cell motility by the ErbB4 agonist neuregulin 2β. ErbB2 tyrosine kinase activity, but not ErbB4 tyrosine kinase activity, is required for neuregulin 2β to stimulate cell proliferation. Moreover, sites of ErbB4 tyrosine phosphorylation, but not sites of ErbB2 tyrosine phosphorylation, are required for neuregulin 2β to couple to cell proliferation. These data suggest that targeting ErbB2 expression or tyrosine kinase activity may be effective in treating ErbB4-dependent breast tumors, even those tumors that lack ErbB2 overexpression.

Bromodomain extra-terminal (BET) protein (BETP) family of chromatin reader proteins includes BRD4 that binds to acetylated lysine in the active chromatin and transcription factors (TFs) at enhancers and promoters, as well as increases RNA pol II (RNAP2) mediated mRNA transcription of active oncogenes in AML. Disruption of binding and eviction of BRD4 from chromatin results in transcriptional attenuation of pro-growth and pro-survival oncoproteins. These include c-Myc, BCL2, MCL1, CDK6, while inducing p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs). Consistent with this, first generation BETP inhibitors (BETi) (e.g., OTX015) have been shown to reduce AML burden and induce clinical remissions, albeit in a minority of patients with AML. Recently, more potent BETis such as ABBV-075 (AbbVie Inc.) have been developed and are being investigated for their clinical efficacy in AML. Venetoclax (ABT-199, AbbVie) and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis in AML BPCs. Treatment with venetoclax alone, and in combinations with other anti-AML agents, is effective in inducing clinical remissions in AML. Here, we interrogated the epigenetic mechanisms underlying transcriptional repression due to BETi treatment, as well as determined the anti-AML activity of co-treatment with BETi and venetoclax or A-1210477 against AML BPCs. ATAC-Seq analysis showed that, in BETi-treated (over untreated control) AML BPCs, BETi treatment induced significantly greater perturbations in the accessible chromatin (number of peaks gained or lost), which were especially enriched for TF-binding sites for RUNX1, c-Myc, GATA2, PU.1 and ERG, especially in the DNA of BCL2, Bcl-xL, MCL1, MYC, BIM, PIM1, CDK6, BRD2/4, HEXIM1, CDKN1A, CEBPA and ITGAM. RNA-Seq analyses, followed by confirmation with QPCR, demonstrated that BETi treatment attenuated expression of MYC, BCL2, Bcl-xL and CDK6, while inducing mRNA expression of HEXIM1 and CDKN1A. BETi (ABBV-075) treatment also dose-dependently reduced protein levels of c-Myc, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A and cleaved PARP levels in AML BPCs. This was associated with dose-dependent (10 to 250 nM) ABBV-075-induced apoptosis of cultured AML cell lines and AML BPCs. Whereas treatment with venetoclax (20 to 200 nM) or A-1210477 (1 to 10 µM) alone also induced apoptosis, co-treatment with ABBV-075 and venetoclax or A-1210477 synergistically induced apoptosis of AML BPCs, including CD34+ patient-derived AML BPCs (combination indices < 1.0). Notably, treatment with venetoclax significantly increased protein expression of MCL1 (p < 0.05), which has been recently reported as a potential mechanism of acquired-adaptive resistance to venetoclax. However, co-treatment with ABBV-075 significantly abrogated venetoclax-induced MCL-1 levels (p < 0.01), which likely contributed to the synergistic anti-AML activity of co-treatment with ABBV-075 and venetoclax against AML BPCs. We next determined the in vivo anti-AML efficacy of ABBV-075 and venetoclax in luciferase-transduced, AML BPCs (MOLM13) and patient-derived xenograft (PDX) models of AML BPCs engrafted in immune depleted (NSG) mice. Compared to treatment with either agent alone, co-treatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell burden (p < 0.001), without inducing toxicity, in AML BPC-engrafted NSG mice. Co-treatment with ABBV-075 and venetoclax also improved the median and overall survival of NSG mice engrafted with MOLM13 AML BPCs (p < 0.0001). Collectively, these pre-clinical findings elucidate the mechanistic rationale, and support evaluation of the clinical efficacy and safety of targeted co-treatment with BETi and BCL2 or MCL1 inhibitor in AML. Disclosures Kadia: Jazz: Consultancy, Research Funding; BMS: Research Funding; Novartis: Consultancy; Abbvie: Consultancy; Celgene: Research Funding; Celgene: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Abbvie: Consultancy; Amgen: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Research Funding. DiNardo:Karyopharm: Other: Advisory role; Medimmune: Other: Advisory role; Celgene: Other: Advisory role; Bayer: Other: Advisory role; Agios: Consultancy, Other: Advisory role; AbbVie: Consultancy, Other: Advisory role. Khoury:Stemline Therapeutics: Research Funding. Shen:AbbVie Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding.