Declined Presentation Inhibition of carbonic anhydrase IX as a novel strategy to target FLT3/ITD mutated aml cells under hypoxic conditions

Abstract

Recent data suggests that the hypoxic properties of the bone marrow niche play a key role in the evolution of drug resistance in FLT3/ITD+AML, thus indicating a role for hypoxia-specific drugs in AML therapy. However, the mechanisms by which low oxygen (O2) levels distort drug responses of FLT3/ITD+AML cells are mostly unknown. Here we investigated the cytotoxic activity of classic and novel agents under hypoxic (1%) and normoxic (21%) conditions against FLT3/ITD+AML. Molm14 (M14) and primary cells from relapsed/refractory FLT3/ITD+AML patients were treated with Cytarabine (C), Quizartinib (Q) or FC531 (FC; a selective inhibitor of the hypoxia responsive gene carbonic anhydrase IX [CA-IX]) under 21% and 1% O2. After 48h, proliferation and apoptosis induction were assessed per MTT and FACS assays. Cells were also assessed for FLT3 protein and Hypoxia inducible factor (HIF) target gene expression by western blot and PCR arrays. The growth inhibitory effects of C and Q against FLT3/ITD+ cells were significantly dampened under 1% compared to 21% O2. Similarly, hypoxia weakened the pro-apoptotic effects of either drug. In contrast, Q potently inhibited FLT3 activity under both 1% and 21% O2. We next sought to identify tractable pro-survival genes induced by hypoxia, and to assess their expression in response to therapy. Our data shows that the HIF pathway is upregulated in response to 1% O2 and remains widely functional in C or Q treated cells. CA-IX, a druggable HIF target, was significantly upregulated in FLT3/ITD+ cells under 1% O2. When we exposed M14 cells to FC, we found that cell growth was significantly inhibited under 1% but not 21% O2. Further, FC was significantly more effective than C or Q in inducing apoptosis under hypoxia. 1) Hypoxia blunts the cytotoxic effects of C and Q in FLT3/ITD+AML cells. 2) The HIF signaling pathway remains largely functional under hypoxic conditions, despite treatment. 3) FC is significantly more cytotoxic than C or Q under hypoxic conditions. 4) Further investigation of combined therapies between standard AML agents and FC in FLT3/ITD+AML is warranted.