TP53 Deficient/Mutant AMLs Are Resistant to Individual BH3 Mimetics: High Efficacy of Combined Inhibition of Bcl-2 and Mcl-1

Abstract

In spite of recent progress in AML therapy, the outcomes of TP53 mutated AML remain extremely poor. The FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents is resulting in CR/CRi rates of 70-95% and good tolerability in elderly AML patients. However, patients with TP53 mutations achieve lower response rates (CR/CRi 47%) (DiNardo CD et al., Lancet Oncol 2018; DiNardo CD et al., Blood 2019) and invariably relapse. Combined Bcl-2 inhibition and p53 activation is synthetic lethal in TP53 wild-type AML in part through targeting Mcl-1 (Pan R et al., Cancer Cell 2016). In TP53 deficient/mutant AML, direct targeting of Mcl-1 may partially compensate for the TP53 defect. We therefore postulate that combined inhibition of Mcl-1 and Bcl-2 effectively induces apoptosis in TP53 deficient/mutant AML cells. Reverse phase protein array analysis of a large cohort of newly diagnosed AML patients (n=511) enabled us to stratify patients into various prognostic groups based on p53 pathway protein expression, which included 8 core proteins: TP53, TP53pS15, MDM2, MDM4, TRIM24, SFN, IRS1.pS1101, and YWHAZ. The group with p53 pathway dysfunction, defined by high p53 protein levels, is characterized by poor outcomes (Quintas-Cardama A et al., Leukemia 2017). This group, encompassing both TP53 wild-type and TP53 mutations, had significantly lower expression of Bax (p=0.0007), the chief executioner of intrinsic apoptosis. A survey of Bcl-2 family proteins in TP53 wild-type and mutant AML showed that only Bax was significantly lower in patients with TP53 mutations (p=0.0498). We next investigated the roles of p53 in response to BH3 mimetics in TP53 wild-type and knockdown (KD) OCI-AML3 cells and in TP53 wild-type and mutant Molm13 cells, generated by long-term exposure of TP53 wild-type Molm13 cells to RG7388 (idasanutlin). Western blot analysis showed that Bax protein was consistently decreased in both TP53 KD and mutant cell lines compared to their respective controls. p53 KD OCI-AML3 and TP53 mutant Molm13 cells exhibited decreased sensitivity not only to VEN, but also to Mcl-1 inhibitor AZD5991 compared to OCI-AML3 vector control and Molm13 parental cells, respectively. To investigate if combined inhibition of Bcl-2 and Mcl-1 could counter-balance the loss of TP53 activity, p53 KD and TP53 mutated AML cells were treated with VEN and AZD5991: like in their respective control cells, the combination synergistically induced cell death in these cells (Fig. 1). The EC50 levels of VEN required for the described synergism in OCI-AML3 cells can easily be reached/exceeded clinically. The combination was also synergistic in leukemia cell lines lacking wild-type TP53 such as KG-1 and U937. Importantly, the combined inhibition was more effective than each single agent in primary AML cells and stem/progenitor cells lacking wild-type TP53 due to deletion of chromosome 17 or mutations in TP53 gene. Conclusion: AML cells deficient in functional TP53 have decreased Bax protein expression, increased apoptotic threshold and are more resistant to individual BH3 mimetics. Combined inhibition of Bcl-2 and Mcl-1 is highly synergistic in p53 deficient/mutant AML. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Cidado:AstraZeneca: Employment. Drew:AstraZeneca: Employment. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: 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; Leukemia Lymphoma Society: 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; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy.