ALBERT

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

Description: Myeloproliferative Neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of the JAK-STAT signaling and often progress (~20%) to AML (sAML). As a single agent, JAK1&2 inhibitor ruxolitinib confers significant clinical benefit in MPN-MF, but exhibits modest activity in sAML, which is also incurable with standard anthracycline and Ara-C-based chemotherapy. This supports the rationale to develop and test novel combination therapies for post-MPN-MF sAML. Genetic alterations documented in sAML include those in JAK2, MPL or calreticulin (CALR) gene, as well as in TP53, TET2, ASXL1, IDH1&2, SRSF2, RUNX1, MYC, PTPN11, NRAS, and SETBP1 genes. Variant allelic frequency analyses demonstrated a common co-occurrence of JAK2V617F and mutant TP53 in the dominant clones of sAML. We and others have previously reported that treatment with BET (bromodomain and extra-terminal) protein bromodomain antagonists (BA) results in growth arrest, differentiation and apoptosis of AML cells (Mol Cancer Ther 2014,13:2315). In the present studies, we demonstrate that treatment with JQ1 but not its inactive enantiomer (R-JQ1) dose-dependently (100 to 2000 nM) mediates growth inhibition and apoptosis of the cultured (HEL92.1.7, SET2 and UKE1 cells) and primary (p) post-MPN-MF sAML cells. JQ1 treatment reduced the promoter occupancy of BRD4 and RNA polymerase II on MYC, BCL2 and IL7R promoters, attenuated the mRNA and protein expressions of BCL2, BCL-xL, MYC, CDK4/6 and PIM1, and repressed the pSTAT5 and pSTAT3 levels, while concomitantly inducing the levels of HEXIM1, p21, p27 and BIM in the sAML cells. Following engraftment of NOD/scid/IL-2Rγ null) (NSG) mice with HEL92.1.7 cell xenografts, treatment with JQ1 (50 mg/kg/day, administered IP daily x 5 days per week x 3 weeks) also significantly improved the median survival of the mice (p 〈 0.01). Compared to treatment with each agent alone, co-treatment with JQ1 and ruxolitinib (100 to 1000 nM) or pacritinib (250 to 1000 nM), which is also a clinically active JAK2, JAK2-V617F and Fms-like tyrosine kinase 3 inhibitor that does not inhibit JAK1, was synergistically lethal against the cultured sAML cells (Combination indices of 〈 1.0 on the isobologram analyses). Additionally, co-treatment with JQ1 and ruxolitinib caused a marked inhibition of pJAK2, pSTAT5, pSTAT3, MYC, CDK4/6, BCL-xL and PIM1 in the sAML cells. Co-treatment with JQ1 and the pan-PIM kinase inhibitor AZD1208 (500-3000 nM), which inhibits the PIM kinase substrates BAD, p70S6 kinase and 4EBP1, also synergistically induced apoptosis of the cultured sAML cells (CI 〈 1.0). HEL92.1.7 and SET2 cells are not only positive for the JAK2V617F mutation, they also express mutant TP53 (M133K in HEL92.1.7 and R248W in SET2 cells). Co-treatment with JQ1 and the heat shock protein (HSP) 90 inhibitor AUY922 (2.5-20 nM), which is known to down-regulate the levels of mutant-TP53, JAK2, c-RAF, pSTAT5, pSTAT3 and pAKT, is synergistically lethal against HEL92.1.7 and SET2 cells. We have isolated JAK inhibitor-resistant HEL92.1.7 cells (〉 10-fold resistant to ruxolitinib; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAK inhibitor (Clin Cancer Res 2011;17:7347). Compared to the parental HEL92.1.7, HEL/JIR cells are highly and collaterally sensitive to AUY922. HEL/JIR cells also remain sensitive to JQ1-induced apoptosis. Importantly, co-treatment with JQ1 and AUY922 is also synergistically lethal against HEL/JIR cells (CI 〈 1.0). Taken together, these findings highlight the pre-clinical activity of BA-based combination with JAK inhibitors (ruxolitinib or pacritinib), PIM kinase inhibitor (AZD1208) or HSP90 inhibitor (AUY922) against JAK kinase inhibitor-sensitive sAML, as well as of the BA-based combination with HSP90 inhibitor or pan-PIM kinase inhibitor against JAK inhibitor-resistant sAML cells. Collectively, these findings also support further in vivo testing of these BA-based combinations against sAML cells. Disclosures No relevant conflicts of interest to declare.

Description: Mantle Cell Lymphoma (MCL) exhibits genetic alterations in p53, CDK4, CDKN2A, MYC, BCL2, B cell receptor (BCR) and NFkB signaling, which confer a cell autonomous, pro-growth and pro-survival advantage on MCL cells. Treatment with the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib inhibits BCR and NFκB activity, mediating growth inhibition and apoptosis of MCL cells. Although ibrutinib induces impressive clinical responses in patients with MCL, long-term durable remissions and cure remain elusive. Ibrutinib-resistant MCL cells exhibit mutations in TRAF2/3 and MAP3K14 (NIK), activating the alternative NFκB signaling. Additionally, a cysteine-to-serine C481S mutation in BTK at the binding site of ibrutinib, which confers resistance to ibrutinib, has also been documented in MCL that relapsed while on ibrutinib. These observations suggest that ibrutinib-resistant MCL cells remain dependent for growth and survival on the NFκB-activated gene-expressions. In the ibrutinib-refractory primary MCL cells, mutations in MLL2, CREBBP, PIM1 and ERB4 were also detected. Our findings have shown that the BET (bromodomain and extra-terminal) protein BRD4 antagonists (BA), e.g., JQ1, transcriptionally repress the levels of MCL-relevant oncogenes BCL-2, c-Myc, cyclin D1, CDK6 and PIM1, while inducing the levels of HEXIM1, p21 and p27 in the MCL cells. Consistent with the reported findings that BRD4 is essential for the transcriptional activity of NFkB, JQ1 treatment also attenuated the nuclear RelA and inhibited the expression of several NFkB target genes, including XIAP, IkBα, cFLIP, cIAP2, as well as BTK. Collectively, this resulted in growth arrest and apoptosis of the cultured (MO2058, Mino, Z138 and JeKo-1) and primary MCL (pMCL) cells. We also demonstrate that compared to ibrutinib alone, co-treatment with JQ1 and ibrutinib markedly inhibits pBTK, BTK, pPLCγ2, pAKT, PIM1 and nuclear RelA levels, as well as synergistically induced apoptosis of the cultured and pMCL cells with combination indices (CI) of less than 1.0 by the median dose effect isobologram analysis. Following tail-vein infusion and engraftment of Mino cells in the bone marrow and spleen, co-treatment with JQ1 and ibrutinib, versus each agent alone, significantly improved the survival of NOD/SCID mice. By exposing parental MCL Mino or MO2058 cells to escalating levels of ibrutinib, we isolated Ibrutinib-resistant Mino/IR (〉13-fold resistant) and MO2058/IR cells (〉8-fold resistant), respectively. Sequencing of the BTK gene did not reveal any mutation in the BTK gene (e.g. C481S) in the ibrutinib-resistant cells. Notably, compared to Mino, Mino/IR cells expressed higher protein levels of BTK, AKT, CDK6, PIM1, XIAP, Bcl-xL and BCL2 levels, while simultaneously displaying lower NOXA, PUMA and p21 levels. Importantly, treatment with JQ1 inhibited the suspension culture growth of Mino/IR cells. JQ1 treatment also induced similar level of apoptosis in Mino/IR, as compared to Mino cells. JQ1-induced apoptosis of Mino/IR cells was associated with a reduction in the nuclear levels of RelA, as well as inhibition of the mRNA levels of BTK, CDK6, and BCL2. In contrast, JQ1 treatment induced the mRNA expression of BCL2L11 and HEXIM1. JQ1 treatment also markedly attenuated the protein levels of BTK, c-Myc, PIM and CDK6 in Mino/IR cells; however, a more modest inhibition of Bcl-xL, BCL2, and XIAP protein levels, and a lack of effect on the protein levels of AKT was observed. Furthermore, pre-treatment of Mino/IR cells with JQ1 enhanced their sensitivity to ibrutinib. Next, we determined that co-treatment with JQ1 and the BCL2 antagonist ABT-199, palbociclib (a CDK4 and CDK6 inhibitor) or AZD1208 (a PIM1 & 2 kinase inhibitor) was synergistically lethal against Mino and Mino/IR cells (CI 〈 1.0). Co-treatment with JQ1 and ABT199, AZD1208 or palbociclib was also synergistically lethal against pMCL cells, but not against the normal B or hematopoietic cells. These findings demonstrate that treatment with the BA JQ1 sensitizes MCL cells to not only ibrutinib but also to BCL2, PIM1 & 2 and CDK4/6 antagonists. Furthermore, co-treatment with BA and BCL2, PIM1 & 2 or CDK4/6 antagonist are promising combination therapies for testing against ibrutinib-sensitive or ibrutinib-resistant MCL cells. Disclosures Wang: Pharmacyclics, Janssen, Celgene, Oncopep, Kite, Juno: Research Funding; Janssen: Honoraria.

Description: There is a clear need to develop novel therapies that would overcome differentiation block and eliminate AML stem/progenitor cells. Genetic and epigenetic dysregulation of enhancers regulates expressions of myeloid lineage transcriptional regulators and their target genes in AML stem/progenitor cells. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates enhancer maintenance and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1 that mediate transcriptional repression and differentiation block in AML blast progenitor cells (BPCs). We had previously reported that treatment with the reversible LSD1 inhibitor (LSDi) SP2509 increases the permissive H3K4Me3 mark on the chromatin, associated with induction of p21, p27 and CEBPα levels, as well as of differentiation and loss of viability of AML BPCs (Leukemia. 2014; 28: 2155-64). In the present studies, we further evaluated the anti-AML efficacy of LSD1i-based combination with BET protein inhibitor (BETi). First, we determined that tet-inducible shRNA to KDM1A depleted protein levels of KDM1A, repressed c-Myc, but de-repressed p21, CD11b (ITGAM), CD86 and CEBPα, thereby inhibiting colony growth and modestly inducing lethality in genetically diverse cultured AML cell lines. Following sgRNA-directed, CRISPR/Cas9-mediated gene-editing of LSD1 in AML BPCs, surviving clones exhibited ~50% KDM1A levels and decreased c-Myc and DNMT1 expressions compared to the control AML BPCs. Treatment with either the reversible LSDi, SP2577 (Salarius Pharma), or with the irreversible LSDi ORY-1001, disrupted binding of KDM1A with CoREST. Following LSDi treatment, ATAC-Seq analyses demonstrated significant increase in the accessible chromatin of AML BPCs (represented by gained peaks). Gained ATAC-Seq peaks also involved the chromatin of MED11/13, LY96, CEBPB, RARA, CDKN1C and CD86 genes. ChIP-Seq analysis also showed increased H3K27Ac peaks in the chromatin of CD86, ITGAM, SAMHD1, TET2, MED12 and E2F1, and a reduction of peaks in RUNX1, CDK6, KIT, CTNNB1, HOXB5, FLT3 and MEIS1. RNA-Seq analyses after LSD1i treatment also showed significant perturbations (log2 fold-change 〉1.25 and p

Description: 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.

Description: 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.

Description: Key Points BA reduces MYC, CDK4/6, nuclear RelA, and BTK expression and is synergistically lethal with ibrutinib in MCL cells. Cotreatment with BA and inhibitor of BCL2, CDK4/6, or histone deacetylases is synergistically lethal against ibrutinib-resistant MCL cells.

Description: Hematopoietic stem/progenitor cells (HPCs) of BCR-ABL1-negative myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit mutations in JAK2, c-MPL, or calreticulin (CALR) gene and display constitutive activation of JAK-STAT signaling. In MPN-MF, transformation to AML (sAML) occurs in up to 20% of patients. Ruxolitinib (R) is a type I, ATP-competitive, JAK1 & 2 inhibitor (JAKi), which is currently used in the therapy of MPN-MF. Treatment with R confers notable clinical benefit in MPN-MF, but exhibits only modest activity and does not significantly impact the clinical outcome in post-MPN sAML. Prolonged exposure to R may also lead to a loss of response, causing the emergence of JAKi-resistant (JIR) sAML cells. Although they lack additional JAK2 mutations, JIR cells exhibit reactivation of JAK-STAT signaling due to trans-phosphorylation of JAK2 by JAK1 or TYK2 tyrosine kinases (TK). Sequential genomic assessments in pre- and post-sAML transformation have revealed mutations in TET2, ASXL1, IDH1&2, SRSF2, RUNX1, MYC, PTPN11, NRAS, SETBP1 and TP53 genes. Here, we determined that treatment with BETi, e.g., JQ1 or OTX015, which disrupts the binding of BRD4 with acetylated chromatin, significantly inhibits growth and induces apoptosis of JAKi-sensitive cultured, including those that express JAK2 V617F and mutant TP53, e.g., HEL92.1.7 and SET2, and patient derived (PD) CD34+ sAML cells. Western analyses revealed that BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, while concomitantly inducing the levels of HEXIM1, p21, NOXA and BIM in the sAML cells. Co-treatment with BETi and R synergistically induced apoptosis of cultured and PD CD34+ sAML cells. As compared to treatment with vehicle control, JQ1 or R treatment alone, co-treatment with JQ1 and R significantly improved the median survival of the immune-depleted mice engrafted with HEL92.1.7 cells. However, treatment with BETi leads to the accumulation of BRD4, which could promote the activity of c-MYC, NFkB and several other transcription factors. Therefore, we compared the anti-sAML activity of the novel BETP-PROTACs (BET protein-proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas Inc.), which degrade BETPs (including BRD4), with the BETi OTX015, against cultured and PD CD34+ sAML cells. ARV-825 and ARV-771 recruit and utilize the E3 ubiquitin ligase activities of cereblon and VHL, respectively, to effectively degrade BETPs. At equimolar concentrations (100 to 500 nM) ARV-825 and ARV-771 were significantly more potent than the BETi OTX015 in inducing apoptosis of cultured and primary sAML cells (p 〈 0.05), while sparing the CD34+ normal hematopoietic progenitor cells. Notably, whereas OTX015 treatment increased BRD4 levels, BETP-PROTACs caused efficient and prolonged depletion of the levels of BETPs, including BRD4 (〉 90%) in the sAML cells. BETP-PROTAC treatment caused more up and down regulation of mRNA and protein expressions than BETi, utilizing the RNAseq and reversed phase protein array (RPPA) analyses, respectively. Western analyses showed that BETP-PROTAC versus BETI treatment also caused greater depletion of c-MYC, JAK2, p-STAT5, STAT5, p-STAT3, STAT3, PIM1 and Bcl-xL, whereas the protein levels of p21 and p27 were upregulated. CyTOF or mass-cytometry approach showed that BETP-PROTAC, more than OTX015 treatment, reduced BRD4, c-MYC and p-RB, while inducing p21 levels in the phenotypically characterized CD34+ sAML stem/progenitor cells, based on their high expression of CD90, CD244, CD123+ and TIM3Fc+. Compared to treatment with each agent alone, co-treatment with ARV-825 and R (100 to 1000 nM) was synergistically more lethal against the cultured and PD CD34+ sAML cells. We have isolated JAKi-resistant HEL92.1.7 cells (〉 10-fold resistant to R; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAKi. Notably, compared to the parental HEL92.1.7, HEL/JIR cells were highly and collaterally sensitive to BETP-PROTAC. Additionally, co-treatment with BETP-PROTAC and HSP90 inhibitor AUY922 or BCL2/BcL-xL antagonist ABT263 is synergistically lethal against JAKi sensitive and JIR sAML cells. These findings strongly support the in vivo testing of the BETP-PROTACs alone and in combinations against post-MPN sAML. These studies are underway in our laboratory and will be presented at the ASH meetings. Disclosures Raina: Arvinas, LLC: Employment. Coleman:Arvinas, LLC: Employment. Winkler:Arvinas, LLC: Employment. Qian:Arvinas, LLC: Employment. Crew:Arvinas, LLC: Employment. Shen:Arvinas, LLC: Employment.

Description: Mantle Cell Lymphoma (MCL) exhibits pathogenetic mutations or deletion of RB1, ATM, p53, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2. Activated B cell receptor (BCR) signaling, and the ensuing downstream pro-growth and pro-survival NFkB activity, is also a notable feature of MCL. Collectively, the genetic alterations and the ensuing deregulated signaling and activity of transcription factors, including c-MYC and NFkB, creates the MCL-specific 'transcriptome' that promotes growth and survival of MCL cells. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), exhibits unprecedented single-agent activity in relapsed/refractory MCL, however approximately 40% of patients demonstrate primary refractory/resistant disease with a one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, despite ibrutinib treatment, sustain classical or alternative NFkB signaling and transcriptional activity, as well as confer resistance to ibrutinib in MCL. We previously reported that the BET protein (BETP) bromodomain inhibitors (BETis), which disrupt the binding of BRD4 with acetylated chromatin, inhibit the in vitro growth and induce apoptosis of cultured and patient-derived (PD) primary MCL cells. This was associated with BETi-mediated attenuation of c-MYC, BCL2, CDK4/6 as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, Bcl-xL, IL10, TNFα and BTK. Concomitantly, BETi treatment induced HEXIM1, p21, p27 and NOXA levels in MCL cells. Co-treatment with BETi and ibrutinib was synergistically lethal and improved the median survival of the immune-depleted mice engrafted with human MCL cells. However, treatment with BETi leads to the accumulation of BRD4, which could promote the deregulated transcriptional activity of c-MYC, NFkB and other transcription factors. Here, we compared the anti-MCL activity of the novel BETP-PROTACs (proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) that degrade BRD4 with the BETi OTX015 against cultured and primary MCL cells. ARV-825 and ARV-771 recruit and utilize the E3 ubiquitin ligase activities of cereblon and VHL, respectively, to effectively degrade BET proteins including BRD4. At equimolar concentrations (10 to 500 nM) ARV-825 and ARV-771 were significantly more potent than the BETi OTX015 in inducing apoptosis of cultured and primary MCL cells (p 〈 0.01), while sparing the CD19+ normal B and CD34+ hematopoietic progenitor cells. Notably, whereas OTX015 treatment increased, BETP-PROTACs markedly attenuated (〉 90%) the levels of BRD4 in the MCL cells. BETP-PROTAC treatment caused more profound up and down regulation of mRNA and protein expressions, utilizing the RNAseq and reversed phase protein array (RPPA) analyses, respectively. BETP-PROTAC treatment also caused greater and more sustained depletion of c-MYC, CDK4/6, PIM1, cyclin D1, as well as of the NFkB transcriptional targets Bcl-xL, XIAP, MCL1 and BTK, while concomitantly inducing the level of NOXA and p21. As compared to treatment with OTX015, ARV-771 treatment dramatically inhibited the growth and improved survival of NSG mice engrafted with luciferase-transduced, ibrutinib-resistant, Z138 MCL cells. Also, notably, co-treatment of ARV-825 or ARV-771 with ibrutinib or the BCL2-antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of cultured and primary MCL cells. We have also generated, ex vivo, ibrutinib-resistant (e.g., Mino/IR and JeKo1/IR) and ibrutinib-persister/resistant (Mino/IPR) cultured MCL cells, with 〉10-fold higher IC50 value for ibrutinib than the parental MCL cells. BETP-PROTAC treatment more potently induced lethality than BETi in the Mino/IR and Mino/IPR cells, associated with attenuation of c-MYC, BCL2, CDK4/6 and NFkB target gene expressions including BTK. BETP-PROTAC and BETi also induced synergistic lethality with venetoclax and palbociclib against the Mino/IR, Mino/IPR and Z138 MCL cells. These findings underscore the superior in vitro and in vivo activity of BETP-PROTACs versus BETi against ibrutinib-sensitive and ibrutinib-refractory MCL cells, as well as highlight a promising new class of agents to be developed for the therapy of human MCL. Disclosures Raina: Arvinas, LLC: Employment. Coleman:Arvinas, LLC: Employment. Winkler:Arvinas, LLC: Employment. Qian:Arvinas, LLC: Employment. Crew:Arvinas, LLC: Employment. Shen:Arvinas, LLC: Employment. Wang:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; BeiGene: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Onyx: Research Funding; Celgene: Research Funding.

Description: Bromodomain and extra-terminal protein (BETP) inhibitors (BETis) disrupt the chromatin binding and activity of the BETP BRD4 in facilitating RNA pol II-mediated mRNA transcription, thereby depleting levels of active oncoproteins including c-Myc, CDK6, BCL2, PIM1 and MCL1. BETi treatment also increases protein levels of p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs), including post-MPN, secondary AML (sAML) BPCs. Treatment with BETi (e.g., OTX015) has been shown to reduce AML burden and induce clinical remissions. However, BETi-refractory AML develops uniformly. Previous reports utilizing mouse AML models have highlighted that persister-resistance to BETi (BETi-P/R) in AML stem progenitor cells is observed despite BETi treatment and reduction of BRD4 occupancy on the chromatin. This is mediated by re-expression of c-Myc due to transcriptional activity of WNT-β-catenin. In the present studies, we developed human sAML models of BETi-P/R to elucidate the mechanisms and develop targeted therapies against BETi-P/R sAML BPCs. Utilizing human sAML control (parental) SET2 and HEL92.1.7 cells and subjecting them to at least 10 exposures to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we first generated the BETi-P/R SET2-P/R and HEL-P/R cells. These cells were 〉 10-fold resistant to OTX015 and exhibited cross-resistance to other BETis, including JQ1 and ABBV-075. As compared to the control sAML cells, SET2-P/R and HEL-P/R cells neither exhibited additional genetic alterations by NextGen whole-exome sequencing, nor showed altered levels of TRIM33, SPOP or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). In contrast, compared to the control, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels and binding of β-catenin to the transcription factor TCF7L2 (TCF4) and TBL1X (TBL1), associated with increased expression of TCF4 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, including enrichment of the STAT5, MYC, PU.1, GATA2 and MYB transcription factor 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 increased expression of gene-sets involving MYC/MAX, STAT5, NFkB and TCF4 targets. QPCR and Western analyses confirmed significant perturbation in gene expressions, with increase in TCF4, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Consistent with the finding that shRNA-mediated knockdown of BRD4 exerted similar lethal effects in BETi-P/R versus control cells, we also discovered that BETP-PROTAC (proteolysis targeting chimera) ARV-771 (Arvinas, Inc.) that degraded BRD4/3/2 was equipotent in inducing apoptosis of BETi-P/R and control sAML cells. Also, consistent with increased nuclear levels and binding (utilizing confocal microscopy) of β-catenin with TBL1 and TCF4 in BETi-P/R sAML BPCs, β-catenin inhibitor BC2059 (Beta-Cat Pharma), which disrupts the binding of nuclear β-catenin with TBL1 and TCF4 and depletes β-catenin levels, exerted similar lethal effects in BETi-P/R sAML and control sAML cells. Consistent with these findings, we also determined that co-treatment with ARV-771 and BC2059 exerted synergistic in vitro lethality against BETi-P/R sAML BPCs (combination indices 〈 1.0), which was associated with greater reduction in levels of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL. Co-treatment with ARV-771 and BC2059 was also synergistically lethal against 12 patient-derived samples of CD34+ sAML BPCs. Notably, compared to treatment with each agent alone or vehicle control, 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 the sAML burden and improved survival of the NSG mice engrafted with luciferase-transduced HEL-P/R cells (p 〈 0.01). These findings demonstrate that increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BETP degrader and β-catenin-TCF4 inhibitor is synergistically lethal against BETi-P/R sAML BPCs. Disclosures Soldi: Beta Cat Pharma: Employment. Bose:Astellas Pharmaceuticals: Research Funding; Celgene Corporation: Honoraria, Research Funding; Blueprint Medicines Corporation: Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; CTI BioPharma: Research Funding; Incyte Corporation: Honoraria, Research Funding. Kadia:BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Celgene: Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Celgene: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Pfizer: Consultancy, Research Funding; Abbvie: Consultancy; Abbvie: Consultancy. DiNardo:Abbvie: Honoraria; Medimmune: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Agios: Consultancy. Horrigan:Beta Cat Pharma: Employment. Khoury:Stemline Therapeutics: Research Funding. Verstovsek:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy.