Targeting Mcl-1 Enhances the Activity of Tyrosine Kinase Inhibitor Gilteritinib in FLT3 Mutated AML

Anti-apoptotic Bcl-2 proteins play critical roles in AML cell and AML stem/progenitor cell survival and drug resistance, hence are relevant therapeutic targets. Indeed, the combination of the selective Bcl-2 inhibitor venetoclax (VEN) with a hypomethylating agent elicits CR/CRi rates of > 65%, is well tolerated by elderly AML patients, and obtained FDA approval. However, despite of the major improvement in response rates, survival extension was limited and most patients ultimately relapsed largely due to the development of resistant disease. Molecular analysis of treated patients revealed that primary and adaptive resistance to VEN-based combinations was frequently characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS (DiNardo CD et al., Blood 2020). FLT3 is one of the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways such as RAS/MAPK, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients treated with TKIs ultimately relapse and adapt to TKI therapy by reactivating the MAPK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 expression can be induced by VEN treatment and is a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML, targeting AML cells and stem/progenitor cells. To determine if targeting Mcl-1 enhances the activity of TKIs in FLT3 mutated AML, we treated MV4-11 and Molm13 cells with Mcl-1 inhibitor AMG176 and TKI gilteritinib (GIL) and observed synergism, as defined by combination index < 1 in both cells. Mechanistic studies demonstrated that GIL markedly decreased Mcl-1 and antagonized AMG176-induced Mcl-1 induction. GIL and its combination with AMG176 also decreased Bcl-xL. Although Bcl-2 protein levels were largely not changed in MV4-11 cells, we found both single treatment and the combination greatly decreased Bcl-2 associated athanogene (BAG) proteins BAG1, BAG3, and BAG4 at the RNA level, which needs to be confirmed at the protein level. The BAG proteins are a family of chaperone regulators and BAG1 was reported to bind and enhance the activity of multiple proteins known to support cells survival, including Bcl-2 (Takayama S et al., Cell 1995). Interestingly, GIL treatment greatly diminished the levels of beta-catenin and its target protein c-Myc, consistent with our previous report that FLT3 regulates beta-catenin signaling (Xiang et al., CCR, 2018). We have generated Mcl-1 overexpressing (OE) and VEN-resistance (VEN-R) MV4-11 and Molm13 cells. The Mcl-1 OE cells are highly resistant to VEN and the VEN-R cells expressed high levels of Mcl-1. Combined inhibition of AMG176 and GIL synergistically induced cell death in Mcl-1 OE and VEN-R resistant cells. Although the expression is low in AML cells we tested, BCL2A1 is also known as a resistant factor to VEN. We generated BCL2A1 OE MV4-11 and Molm13 cells and demonstrated that combined inhibition of FLT3 and Mcl-1 was highly effective in these cells as well. Western blot analysis revealed that GIL effectively decreased Mcl-1 in Mcl-1 OE and VEN-R and BCL2A1 in BCL2A1 OE MV4-11 cells. Next, we treated FLT3 mutated AML patient samples harboring both, ITD and D835 mutations, from 2 patients who had both failed VEN-based therapy and from 1 patient with ITD mutation, with AMG176 and GIL under MSC co-culture conditions. Synergy was observed in all samples in AML blasts and AML stem/progenitor cells. Collectively, our data demonstrate that targeting Mcl-1 enhances the activity of GIL in FLT3 mutated AML, including those resistant to/relapsed from VEN-based therapy, findings that may warrant clinical evaluation. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding. Hughes:Amgen: Current Employment. Chen:Amgen: Current Employment. Morrow:Amgen: Current Employment. Andreeff:Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.