Mcl-1/CDK9 Targeting By AZD5991/AZD4573 Overcomes Intrinsic and Acquired Venetoclax Resistance in Vitro and In Vivo in PDX Model of AML through Modulation of Cell Death and Metabolic Functions

Abstract

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<0.01) than each single agent. CyTOF analysis of BM cells (day 25) showed that both combinations markedly reduced (P<0.001) human CD45+ cells and, more importantly, human CD34+CD38+/CD38- and CD34+CD38+/CD38-CD123+ cells. Those combination treatments also decreased Mcl-1, Bcl-2, b-catenin, c-Myc, and FAK protein expression in CD34+CD38-CD123+ cells. Interestingly, AZD5991, AZD4573, or their combinations with ABT-199 greatly decreased CXCR4 in all cell populations. Ultimately, each single agent only marginally prolonged survival, whereas ABT-199+AZD4573 and even more so ABT-199+AZD5991 markedly improved survival in this highly ABT-199 resistant PDX model (Fig).Conclusion: we demonstrate that Mcl-1 has metabolic functions in AML and that inhibition of Mcl-1 enhances ABT-199 apoptogenic activity and overcomes intrinsic and acquired ABT-199 resistance in vitro and in vivo in a PDX murine model of AML, suggesting that inhibition of Mcl-1 improves the efficacy of ABT-199, and overcomes established resistance to Bcl-2 inhibition. Suppressing metabolic activity and CXCR4 inhibition may also contribute to the efficacy of this combination against AML stem cells in the BM microenvironment. DisclosuresCarter:AstraZeneca: Research Funding; novartis: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties. Cidado:AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment. Andreeff:AstraZeneca: Research Funding.