ALBERT

Description: The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1.

Description: Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as “negative” by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.

Description: Overexpression of anti-apoptotic proteins MCL1 and Bcl-xL are frequently observed in many cancers. Inhibitors targeting MCL1 are in clinical development, however numerous cancer models are intrinsically resistant to this approach. To discover mechanisms underlying resistance to MCL1 inhibition, we performed multiple flow-cytometry based genome-wide CRISPR screens interrogating two drugs that directly (MCL1i) or indirectly (CDK9i) target MCL1. Remarkably, both screens identified three components (CUL5, RNF7 and UBE2F) of a cullin-RING ubiquitin ligase complex (CRL5) that resensitized cells to MCL1 inhibition. We find that levels of the BH3-only pro-apoptotic proteins Bim and Noxa are proteasomally regulated by the CRL5 complex. Accumulation of Noxa caused by depletion of CRL5 components was responsible for re-sensitization to CDK9 inhibitor, but not MCL1 inhibitor. Discovery of a novel role of CRL5 in apoptosis and resistance to multiple types of anticancer agents suggests the potential to improve combination treatments.

Description: Disruption of the intrinsic apoptotic pathway by the aberrant expression of the BCL2 family members are frequent events in multiple myeloma (MM). The anti-apoptotic protein myeloid cell leukemia-1 (MCL1) is highly expressed in MM and plays a crucial role in disease progression. Inhibition of MCL1, thus, represents a unique therapeutic opportunity for the control of the disease. Currently, there is no FDA-approved drug with the ability to selectively target MCL1. Because of its pivotal role in MM, MCL1 is considered a high-value therapeutic target in the clinic. In this report, we use a selective small-molecule inhibitor of MCL1, AZD5991, to examine the therapeutic consequences of MCL1 inhibition in MM. AZD5991 treatment resulted in dose-dependent cytotoxicity with EC50 values ranging from 64 and 417 nM at 24 hours for MCL1-sensitive cell lines (H929, MM.1S, RPMI-8226, U266, LP-1 and ANBL6VR). Two cell lines DOX40 and KMS-12-PE remained resistant to MCL1 inhibition. For the MCL1-sensitive MM.1S and H929 cells, the decrease in cell viability upon MCL1 inhibition was due to an increase in apoptosis. AZD5991 treatment led to 40-82% decrease in viability of CD138+ cells isolated from relapsed and refractory MM patients at a dose of 300 nM at 24 hours. These results indicate that AZD5991 has promising single-agent activity, but it would be prudent to study it in combination with other anti-MM therapies. The BM microenvironment enhances tumor cell growth and survival in MM. We found that soluble factors produced during the MM-BMSC interaction reduced the sensitivity of MM cells to AZD5991, and direct MM-BMSC contact blunted the cytotoxic effect of AZD5991. A comprehensive cytokine array analysis revealed an enrichment of a panel of pro-survival cytokines and growth factors, with the cytokines IL-6, IL-8 and GROα/β/γ being among the most highly up-regulated proteins, upon cell-cell contact between MM.1S cells and BMSCs. Enrichment of these cytokines in the BM milieu, at least in part, confer a protective effect on MM cells and endow them with the ability to resist MCL1 inhibition. A shift in the balance of BCL2 family members is often the primary reason for drug resistance. We found that the baseline BCL2 mRNA expression and the BCL2:MCL1 ratios in the MM cell lines examined are negatively correlated to their corresponding sensitivity to AZD5991. In other words, MM cells with a high BCL2 reservoir are more likely to circumvent cell death elicited by MCL1 inhibition. Treatment with AZD5991 alone leads to release of Bim from MCL1 and an increased Bim bound to BCL2. Venetoclax, a BH3 mimetic that selectively binds BCL2, treatment alone releases Bim from BCL2 and results in an increased Bim bound to MCL1. Bim binding to MCL1 and BCL2 was significantly diminished upon co-treatment. The free-floating Bim proteins subsequently activate the intrinsic apoptotic pathway by facilitating cytochrome c release. These results suggested that MM cells switch their survival dependency to BCL2 upon MCL1 inhibition, and that BCL2 blockade could be an effective way to overcome MCL1 resistance in MM. Based on these observations, we combined AZD5991 with Venetoclax in MM cells. A significant decrease in cell viability was observed with the combined therapy compared with both drugs used alone. Isobologram analysis confirmed greater than additive or synergistic effect upon co-treatment. The enhanced cytotoxic effect of the combined therapy retains even when the MM cells are in co-culture with BMSCs. Remarkably, the pro-survival cytokines IL-6, IL-8 and GROα/β/γ, which were expressed at high levels in the co-culture settings, were among the ones being most reduced after the combined therapy. Our results demonstrated that the combined AZD5991/Venetoclax therapy overcomes the inherent MCL1-resistance in MM via two independent mechanisms. First, the concomitant suppression of the anti-apoptotic proteins MCL1 and BCL2 prevent MM cells from escaping apoptosis by releasing Bim to trigger mitochondrial outer membrane potential and the subsequent release of cytochrome c to activate the intrinsic apoptotic pathway. Second, this combined therapy prevents MM cell growth by causing down-regulation of pro-survival cytokines and growth factors in the BM microenvironment. As a proof of concept, our data indicate combining therapeutics that selectively target the anti-apoptotic proteins MCL1 and BCL2 could be an effective therapy for MM patients. Disclosures Cidado: AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment.

Description: Mcl-1 and Bcl-2 are two major anti-apoptotic Bcl-2 proteins frequently overexpressed in malignant cells. They cooperatively support cell survival and are associated with therapy resistance. ABT-199 (venetoclax), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML as a single agent. Mcl-1 is induced by and a major resistance factor to ABT-199. Mcl-1 was recently found to also positively regulate mitochondrial oxidative phosphorylation that induces cancer stem cells and promotes chemoresistance. Mcl-1 is essential for the development of AML and for the survival of AML cells and stem cells. Increased mitochondrial oxidative phosphorylation has been demonstrated in these cells. First we found that Mcl-1 overexpressing (OE)/knockdown (KD) AML cells were markedly more resistant/sensitive to ABT-199 than were corresponding control cells, supporting the notion of Mcl-1 as a resistance factor to ABT-199. Inhibition of Mcl-1 by the selective Mcl-1 inhibitor AZD5991 or the CDK9 inhibitor AZD4573, which down-regulates short-lived proteins such as Mcl-1, induced apoptosis and showed strong synergy when combined with ABT-199 in AML cell lines, primary AML blasts, and stem/progenitor cells from patients. Importantly, combinations of AZD5991 or AZD4573 with ABT-199 synergistically induced apoptosis in OCI-AML3 and Mcl-1 OE cells intrinsically resistant to ABT-199 and in AML cell lines and primary patient cells with acquired resistance to ABT-199. Although OE/KD Mcl-1 in AML cells did not show obvious alterations in baseline cell viability, NSGS mice harboring Mcl-1 OE/KD OCI-AML3 cells survived significantly shorter/longer than those transplanted with control cells, supporting additional, non-apoptogenic functions of Mcl-1 in AML. We observed that genetic modulation of Mcl-1 alters cellular mitochondrial respiration and ROS levels. AML cells with Mcl-1 OE/KD increased/decreased O2 consumption and mitochondrial ATP and ROS generation. Consistent with this finding, inhibition of Mcl-1 by AZD5991 or AZD4573 decreased O2 consumption and ATP generation in AML cells and also in MV4-11 cells with acquired ABT-199 resistance. Mass spectrometry-based stable isotope tracing experiments using 1,2-13C-glucose showed that both genetic and pharmacological inhibition of Mcl-1 decreased flux of glucose carbon through glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting a role for Mcl-1 in cellular respiration and redox metabolism. To further assess the efficacy of combined Mcl-1 and Bcl-2 inhibition in primary AML cells resistant to ABT-199, we developed a PDX model using cells from an AML patient who initially responded to ABT-199/demethylating agent and then relapsed. NSGS mice engrafted with these PDX cells were treated with ABT-199 (50 mg/kg, oval gavage qd), AZD5991 (60 mg/kg, i.v. weekly), AZD4573 (15 mg/kg, i.p. bid with 2 h interval for two consequent days/week), ABT-199+AZD5991, or ABT-199+AZD4573 for 6 wks. Flow cytometric analysis of circulating human CD45+ cells on day 18 of therapy showed that each agent significantly decreased leukemia burden and that the combinations were significantly more effective (P

Description: Bcl-2 and Mcl-1 play critical roles in AML stem/progenitor cell survival. Venetoclax (VEN), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML. Expression of Mcl-1, a major VEN resistance factor is induced by VEN. Non-apoptotic activity of Mcl-1 has been reported but is not well understood in AML. We previously demonstrated that inhibition of Mcl-1 enhances VEN apoptogenic activity and overcomes intrinsic and acquired VEN resistance in vitro and in vivo in a PDX murine model of AML (Carter BZ et al., ASH 2018). Interestingly, CyTOF analysis of bone marrow PDX cells collected from mice treated with Bcl-2 inhibitor VEN, Mcl-1 inhibitor AZD5991, and the combination showed that AZD5991 or AZD5991+VEN, but not VEN alone greatly decreased CXCR4 in leukemia cells and stem/progenitor cell populations. Mcl-1 expression is to a large degree regulated by microenvironment clues, including CXCR4-CXCL12 axis. We found that inhibition of Mcl-1 also suppressed cell metabolic activities in AML cells suggesting that Mcl-1 may regulate leukemia-microenvironment interaction and cell metabolic functions, which may contribute to the efficacy of this combination against AML stem cells in the bone marrow microenvironment. To further investigate the roles of Mcl-1 in leukemia-stromal interactions, we determined the expression of proteins involved in cell migration and adhesion and found that Mcl-1 overexpressing (OE) or knockdown (KD) OCI-AML3 cells have increased/decreased cell surface expression of CXCR4 and CD44, both critical for leukemia-MSC (mesenchymal stroma cell) interactions. Mcl-1 OE or KD AML cells showed increased/decreased migration towards and adhesion to MSCs compared to their respective controls. Consistent with this observation, pharmacological inhibition of Mcl-1 with AZD5991 also decreased surface CXCR4 and CD44 levels in OCI-AML3 cells and decreased the interactions between leukemia and MSCs. Under the same conditions, Mcl-1 seems to exert a more profound effect on leukemia cell adhesion than migration to MSCs. Interestingly, inhibition of Bcl-2 with VEN did not significantly decrease CD44 or migration and adhesion of AML cells to MSCs, although a decrease in surface levels of CXCR4 was observed. To gain additional insight into the effects of Mcl-1 on cellular energetics and metabolism, we performed metabolomic analysis, employing a previously established method using ion chromatography-mass spectrometry (IC-MS) to trace 13C labels from 13C2-1,2-glucose and 13C5-glutamine in Mcl-1 genetically and pharmacologically modulated AML cells. Metabolomic analysis showed that overall 13C enrichment into key TCA cycle intermediates including citrate, fumarate, and malate was significantly lower in cells with Mcl-1 inhibition, by either AZD5991 or Mcl-1 KD, compared to the respective controls, both for 13C coming from glucose and glutamine, the two main carbon sources entering the TCA cycle. We observed a decrease in the total amount of secreted lactate in both the 13C2-1,2-glucose and 13C5-glutamine tracing experiments. Enrichment of 13C label in secreted lactate was mostly observed during 13C2-1,2-glucose tracing, but not during 13C5-glutamine tracing, indicating that lactate secretion is largely derived from glucose, and not from glutamine, suggesting a reduction in flux through glycolysis and/or pentose phosphate pathway (PPP). Flux through the oxidative PPP (OxPPP) is a rate limiting pathway in the generation of reductive equivalents of NADPH for ROS neutralization. Relative OxPPP flux calculation showed a decrease in OxPPP flux for both AZD5991 treatment and Mcl-1 KD. Levels of 6-Phospho-Gluconic acid (6PG) were also greatly reduced in Mcl-1 inhibited cells, in accordance with differential regulation of the OxPPP. Mcl-1 inhibition by AZD5991 or KD also decreased ATP levels. Collectively, inhibition of Mcl-1 results in a broad downregulation in cellular energetics and metabolism. Conclusion: data demonstrate that in addition to regulating apoptosis, Mcl-1 regulates leukemia-stromal interactions and metabolic activity in leukemia cells and that inhibition of Mcl-1 has anti-leukemia activity and enhance VEN activity through not only apoptosis induction, but also modulation of leukemia-stromal interactions and metabolic functions. Disclosures Carter: Ascentage: Research Funding; Amgen: Research Funding; AstraZeneca: Research Funding. Cidado:AstraZeneca: Employment. Drew:AstraZeneca: Employment. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership.

Description: Multiple myeloma is a malignancy of long-lived plasma cells of the bone marrow that is rarely curable. Thus, despite recent advances in the development of new therapies, additional approaches are required. We investigated potential molecular vulnerabilities in the BCL2 family. Using the MMRF CoMMpass (NCT0145429) study (IA13), we determined the frequency of nonsynonymous coding mutations in the BCL2 family. Analysis of baseline samples from 982 patients revealed that mutations in the BCL2 family are relatively rare events. No mutations were observed in the 3 pro-apoptotic effector genes BAX, BAK1 and BOK. Similarly, in the BH3-only genes, mutations were rare with no mutations in BCL2L11 (BIM), BAD, BID, HRK and BMF and only single mutations in BBC3 (PUMA) and PMAIP1 (NOXA). In the anti-apoptotic BCL2 genes, mutations were also rare with no mutations in BCL2, BCL2L1 (BCLX) and BCL2L10 (BCLB). A single sample had a mutation in BCL2A1 (A1) while 2 samples had mutations in BCL2L2 (BCLW). Interestingly MCL1 was mutated in 10 baseline samples (1.02%) and the frequency of the mutations in these samples was high (median 0.391, range 0.066-0.531). Therefore, we further investigated the functional consequences of the MCL1 mutant alleles. Of the 10 mutations detected, 1 was in the N-terminal region (G32R) and 4 were in the PEST domain in the N-terminal half of MCL1 that is associated with regulating protein stability (V140I, P142S, E149Q and E173K). An additional mutation was found in an uncharacterized region between the PEST and BH1 domains (L186F). We focused on the 4 mutations that lie within or near the functional BH1 (V249L and L267V) and within the BH3 (N223S, and R214Q) domains. Wild type (WT) MCL1 and the four mutant MCL1 constructs were introduced into a murine B-ALL cell line that has endogenous murine MCL1 flanked with LoxP sites and confirmed expression by western blot analysis. Human MCL1 can replace murine MCL1 in this cell model, therefore we are determining if the myeloma-derived mutants of MCL1 can complement loss of mouse Mcl1 and will report on these findings. However, we began to functionally characterize these mutations by taking advantage of an anomaly in the development of inhibitors of human MCL1. To date, no inhibitor developed against human MCL1 is as effective against murine Mcl1. Thus, differences in the activity of these inhibitors reflects changes in dependence on the introduced human MCL1. We treated cells with increasing concentrations of the MCL1 inhibitor S63845 and measured cell death (Annexin V/PI) at 24 hours. As expected, cells where the empty vector was introduced were highly resistant to S63845-induced cell death (less than 20% at 1000 nM) while cells expressing the human WT MCL1 were significantly more susceptible to the MCL1 inhibitor across a concentration range of 100 to 1000 nM. The V249L, N223S and R214Q mutations mimicked the sensitivity of the WT MCL1 suggesting they did not alter MCL1 function in the cells. In contrast the L267V mutation resulted in a dose curve that was more similar to the empty vector control suggesting this mutation either resulted in loss of function or in an MCL1 molecule that could not be inhibited by the drug. Since drug binding can stabilize MCL1 by competing for E3 ligase binding, we determined the effect of S63845 on MCL1 protein levels. We found that with all the mutants, S63845 dramatically increased human MCL1 protein expression ruling out lack of drug binding. We next performed MCL1 co-immunoprecipitation assays, and found that BIM release correlated with S63845 sensitivity. In addition to not releasing BIM, the L267V did not effectively release NOXA and BAK after S63845 treatment. Taken together, the L267V mutation does not prevent the binding of S63845 to free MCL1, rather it blocks the ability of drug to displace pro-apoptotic proteins required to induce cell death. We then tested another MCL-1 inhibitor in clinical trials, AZD-5991. The L267V mutation was completely resistant to AZD-5991-induced apoptosis despite evidence of drug binding. Interestingly the other 3 mutations also resulted in diminished killing activity when compared to cells expressing the WT MCL1, suggesting that these mutations may also influence drug function. Together these data suggest that myeloma-derived mutations in MCL1 may not necessarily influence MCL1 function, however they could alter responses to an emerging class of inhibitors where 3 drugs are currently in clinical trials. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Lonial:Amgen: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria.

Description: We and others have previously demonstrated that MM is often dependent on MCL1 or co-dependent on MCL1 and BCLXL or BCL2 for survival. Therefore, drug development targeting MCL1 has been a top priority. Here we report on AZD5991, a specific small molecule inhibitor of MCL1. We treated 17 MM cell lines with increasing concentrations of AZD5991 for 24 h and measured Annexin V staining to determine the IC50s. Nine of the cell lines tested were highly sensitive to AZD5991 with IC50 values below 100 nM, 6 lines exhibited an intermediate sensitivity (IC50 100-1000 nM), and only 2 cell lines tested were resistant (IC50 〉1000 nM). Six of the highly sensitive lines are t(11;14) and sensitive to venetoclax suggesting co-dependence on BCL2 and MCL1 for survival. We also determined the effect of the bone marrow microenvironment on the response of MM cell lines to AZD5991. We reported that IL-6 protects MM cell lines and patient samples from apoptosis by making the cells more MCL1 dependent. Based on this, we predicted IL-6 would have little to no effect on AZD5991-induced cell death. We treated 12 cell lines with AZD5991 in the presence of 1 ng/mL IL-6 or 10% Hs5 conditioned medium (CM) for 24 h and found that only 3/12 and 2/12 lines were protected from apoptosis in the presence of IL-6 and CM, respectively. Interestingly, when co-cultured with the stromal cell line Hs5, 7/11 lines tested were protected from AZD5991-induced cell death, suggesting cell-cell contact is influencing the response. This is in contrast to ABT-737 and venetoclax where cell-cell contact provided no additional protection than CM. Mechanistically apoptosis induced via MCL1 inhibition is not dependent on BIM expression as is the case with BCLXL and BCL2 inhibition. KMS26 and LP1 MM cell lines contain a bi-allelic deletion of BIM and we have reported their resistance to ABT-737. However, both cell lines respond to AZD5991 with IC50 values in an intermediate sensitivity range. Co-immunoprecipitation (CoIP) studies were employed to determine the protein bound to MCL1 that could be promoting apoptosis upon release. We found NOXA and BAK bound in KMS26 and LP1 and both were released from MCL1 in response to AZD5991. Additionally, CoIPs performed on cell lines expressing BIM showed NOXA, BIM, and BAK bound to MCL1 and released following treatment. To further investigate we used CRISPR-cas9 to generate MM cell lines lacking expression of NOXA, BAK, BAX, or BIM. In KMS26 and LP1, deletion of NOXA and BAX had little effect on AZD5991-induced cell death while the BAK deletion significantly inhibited apoptosis in both cell lines. Similar results were observed in the BIM expressing cell line OCI-My5, with no protection from AZD5991-induced apoptosis in the NOXA and BAX edited lines, significant protection in the BAK-deleted line, and an intermediate degree of protection in the BIM knockout line. In KMS18, BIM deficiency had a minimal effect on apoptosis following MCL1 inhibition, however both BAX and BAK were required for AZD-induced cell death. Additionally, we have tested 41 samples from 37 patients for sensitivity to AZD5991. Samples were treated with increasing concentrations to determine IC50 values in the same manner as the MM cell lines. The samples segregated into 4 groups based on IC50. The most sensitive group (N=3) had an IC50 below 10 nM. The largest group had an IC50 range of 50-114 nM (N=26). The last two cohorts were more resistant with a range of 500-916 nM (N=10) and 2 samples with an IC50 over 1300 nM. Since MCL1 is on 1q21, a frequently amplified region in MM, we determined if 1q21 gain was associated with sensitivity. For the 35 samples where FISH data were available, 18 had 1q21 gains by FISH while 17 were negative. There is a trend for the 1q21 gain cohort to be more sensitive (P=0.0573), with only 2/18 having an IC50 above 109 nM. In contrast for the 1q21 negative 7/17 were in the resistant groups. Thus 1q21 may be a marker of sensitivity to MCL1 inhibitors. The data reported here demonstrate that AZD5991 is effective at inducing apoptosis in MM and can overcome soluble microenvironment resistance factors that influence the response to venetoclax. This appears to be due to differential requirements for pro-apoptotic factors for BCL2 and MCL1 inhibition and suggests an underappreciated complexity in the role of BCL2 and MCL1 in cell survival. Finally these findings also suggest that 1q21 gain may be a marker for AZD5991 sensitivity. A clinical trial is currently ongoing in myeloma. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Neri:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Kaufman:Roche: Consultancy; Abbvie: Consultancy; Karyopharm: Other: data monitoring committee; Janssen: Consultancy; BMS: Consultancy. Heffner:Pharmacyclics: Research Funding; Genentech: Research Funding; ADC Therapeutics: Research Funding; Kite Pharma: Research Funding. Lonial:Amgen: Research Funding. Nooka:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive technologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Description: Apoptosis is controlled through the dynamic interactions of the Bcl2 protein family, and cancers have evolved mechanisms to hijack this pathway to evade apoptosis, often by upregulating anti-apoptotic proteins (e.g. Mcl1, Bcl2). This survival adaptation creates a dependency that could be exploited therapeutically, which is why considerable effort has been made to develop small molecule inhibitors of the anti-apoptotic Bcl2 family proteins. This class of drug was clinically validated with the approval of venetoclax, a selective Bcl2 inhibitor, for the treatment of CLL. Venetoclax is undergoing evaluation in numerous other clinical trials for predominantly hematologic malignancies. Despite impressive responses observed with venetoclax in CLL (ORR 79%), acquired resistance is beginning to emerge. Likewise, other hematologic indications are more intrinsically resistant to venetoclax, exhibiting much lower response rates in the respective Phase I clinical trials (AML = 38%, NHL = 44%, MM = 12%). Preclinical studies with venetoclax have reported increased levels of other anti-apoptotic proteins as a likely mechanism contributing to both de novo and acquired resistance. Therefore, combining cell death inducing agents that inhibit Mcl1 or BclxL could be a means of combating resistance. Inhibition of cyclin-dependent kinase 9 (CDK9), which regulates transcription elongation, has been reported to reduce protein levels of genes with short-lived transcripts and proteins, such as MCL1. AZD4573 is a novel and selective CDK9 inhibitor that shows potent single agent activity, inducing cell death in vitro and tumor regressions in vivo in a diverse set of hematologic cancers (Cidado et. al., AACR Annual Meeting 2018). AZD4573 is currently being evaluated in a Phase I clinical trial for patients with hematological malignancies (NCT03263637). This study evaluates whether combinations of AZD4573 with other cell death inducing agents could overcome de novo venetoclax monotherapy resistance. A panel of 12 AML and 6 DLBCL cell lines were treated for 6 h with venetoclax, AZD4573, and a selective BclxL probe compound (AZ'3202) either as single agents or in combinations and assayed for caspase activation. The Loewe model was used to calculate synergy scores to assess benefit over monotherapy, and combinations with scores 〉5 were deemed beneficial. Treatment with AZD4573+venetoclax and AZD4573+AZ'3202 resulted in beneficial combinations for 13/18 and 10/18 cell lines, respectively. On the other hand, venetoclax+AZ'3202 showed significant combination benefit in only two cell lines (NB4, SUDHL4), suggesting a primary dependency upon Mcl1 for most of these hematologic cancer cell lines. Interestingly, cell lines sensitive to single agent AZ'3202 (4 AML, 0 DLBCL) did not show any combination benefit when treated with AZD4573+venetoclax, highlighting the exquisite dependency of those four models upon BclxL and mutual exclusivity with Mcl1. Cell lines benefitting from the AZD4573+venetoclax combination tended to fall into one of two categories: having single agent activity to either agent that is enhanced by the combination or having no single agent activity but the combination shifts the cell line into a responder. SUDHL4 cells were sensitive to AZD4573 (caspase activation EC50 = 18 nM) but not venetoclax (EC50 = 476 nM) while OCI-AML3 was insensitive to both (EC50 〉 30 µM). In vitro biomarker kinetic analysis revealed an increase in Mcl1 levels (~2-fold) after 3 h of venetoclax treatment that was abrogated upon combination treatment, providing a mechanistic rationale for the combination benefit. Furthermore, when tested in an OCI-AML3 tumor xenograft study in mice, AZD4573 or venetoclax monotherapy exhibited minimal tumor growth inhibition (44% and 16%, respectively) while the combination led to tumor regressions (64%) with minimal effect on body weight. In a SUDHL4 tumor xenograft study, venetoclax monotherapy displayed minimal tumor growth inhibition (25%), but intermittent dosing of AZD4573 exhibited 94% tumor growth inhibition. Still, combination therapy demonstrated a clear benefit as it led to complete tumor regressions with 6/8 mice remaining tumor-free until the end of study (150 days). Together, this work presents supporting evidence that combining cell death inducing agents would be effective at overcoming de novo or acquired resistance associated with monotherapy treatments. Disclosures Cidado: AstraZeneca: Employment, Equity Ownership. Boiko:AstraZeneca: Employment. Proia:AstraZeneca: Employment. Woods:AstraZeneca: Employment. Tavana:AstraZeneca: Employment. San Martin:AstraZeneca: Employment. Tron:AstraZeneca: Employment. Shao:AstraZeneca: Employment. Drew:AstraZeneca: Employment.