Abstract 2427: Targeting intracellular pH regulation as a novel approach to overcome hypoxia-mediated drug resistance in FLT3/ITD mutated AML

Abstract

Abstract Background: FLT3/ITD+ AML is a highly aggressive form of leukemia that carries a dismal prognosis. Evidence suggests that this poor outcome is due to the survival of a subset of leukemic cells (LCs) that elude therapy and survive in hypoxic niches of the bone marrow (BM). Effective targeting of LCs under hypoxic conditions is thus critical to cure AML. Our study examines the anti-leukemic potential of targeting Carbonic anhydrase-IX (CA9), a transmembrane protein which functions to maintain a neutral intracellular pH (pHi) under hypoxic stress conditions, against AML cells. Targeting CA9 is of particular interest as its expression is confined to only a few normal tissues and may therefore show relatively few side effects compared to standard therapies. Methods: Molm14 (M14) and primary cells (PC) from FLT3/ITD+ AML patients were incubated under normoxic (21% O2) and hypoxic (1% O2) conditions in the presence or absence of Quizartinib (Q), Cytarabine (Cy) or the CA9 inhibitor FC531 (FC). After 48h proliferation and apoptosis were determined per MTT assays, annexin V/PI staining and FACS analysis. Cells were assessed for CA9 mRNA expression as well as pHi levels via RT-PCR and fluorescent imaging, respectively. FLT3/ITD+ AML xenografts were generated by injecting M14 cells in the tail vein of NSG mice. Tissue samples were fixed in 10% NBF, embedded in paraffin and stained with H&E and CA9 for pathologic evaluation. Results: Cy and Q were significantly less effective against M14 cells under 1% compared to 21% O2. CA9 mRNA was upregulated in M14 cells (4.5±0.9-fold; n=4, p<.01) and in FLT3/ITD+ AML PCs from newly diagnosed (n=5) and relapsed/ refractory (n=3) patients (up to 529- and 73-fold, respectively) under 1% compared to 21% O2. Immuno-histochemical staining of the BM and spleen obtained from AML xenografts showed multifocal CA9 staining of LCs that was localized on the cell membrane. Under hypoxic conditions, FC but not Cy or Q, significantly induced apoptosis and showed dose-dependent growth inhibitory effects against M14 cells (n=3). In addition, treatment of M14 cells with FC under 1% O2 resulted in strong pHi acidification (ΔpH=-2.7±0.6, p<.05, n=3). Moreover, FC combined with Cy synergistically inhibited M14 cell growth in vitro and effectively eliminated LCs from the BM and spleen compared to Cy only treated mice. Accordingly, spleen size and weight were significantly reduced in mice treated with FC and Cy compared to single agent Cy (n=3). Conclusions: 1. Hypoxia blunts the effects Cy and Q in FLT3/ITD+ AML. 2. CA9 is induced in FLT3/ITD+ AML cells under hypoxic conditions. 3. The CA9 inhibitor FC confers anti-leukemic activity against FLT3/ITD+ AML cells under hypoxic conditions via pHi acidification and acts synergistically with Cy. 4. CA9 inhibition has the potential to target LCs residing in hypoxic niches and may be of value as an adjunct to chemotherapy for FLT3/ITD+AML therapy. Citation Format: Fangli Chen, Adriana L. Rogozea, Xue Wu, George Sandusky, Mircea Ivan, Heiko Konig. Targeting intracellular pH regulation as a novel approach to overcome hypoxia-mediated drug resistance in FLT3/ITD mutated AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2427.