Inhibition of Carbonic Anhydrase-IX As a Novel Strategy to Overcome Hypoxia Mediated Drug Resistance in FLT3/ITD Mutated Acute Myeloid Leukemia

Background: FLT3/ITD mutated (FLT3/ITD⁺) acute myeloid leukemia (AML) is an aggressive form of blood cancer that carries a poor prognosis due to short remission durations and high relapse rates. Several studies suggest that this poor outcome is due to a small fraction of leukemic cells (LCs) that elude treatment and survive in hypoxic niches of the bone marrow (BM) where they lead to relapse. The identification of molecules capable to target LCs under hypoxic conditions is therefore of utmost importance on the way to find a cure for AML. Our study addresses the effects of pharmacological inhibition of carbonic anhydrase-IX (CA9), a transmembrane protein which functions to maintain a neutral intracellular pH - a key survival mechanism for tumor cells to escape cell death in the hypoxic environment - on AML cells. Targeting CA9 is of particular interest as its expression is confined to only a few normal tissues. CA9 inhibition may therefore show relatively few side effects compared to standard chemotherapies. Methods: Molm14 (M14), M13, OCI-AML3, THP-1 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 (Quiz), Cytarabine (Cy) or the CA9 inhibitors FC531 (FC) or SLC-0111 (SLC). After 48h proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis. Cells were also assessed for CA9 and CA12 mRNA expression as well as intracellular pH 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: CA9 mRNA was upregulated in M14 cells (3.8±1.1-fold; n=3, p<.05) and FLT3/ITD⁺ AML PCs (215.4±142.7-fold, n=7, p=NS) under 1% compared to 21% O₂. Consistently, immunohistochemical staining of M14 xenograft tissues showed multifocal CA9 staining of LCs in the BM and spleen. In M14 cells, inhibition of CA9 with FC conferred substantial, dose-dependent growth inhibitory effects under 1% O₂ (IC₅₀=4.04µM) and was significantly more effective than clinically achievable doses of Quiz or Cy in this regard. Under 1% O₂, flow cytometry revealed significant induction of apoptosis in FC, but not in Quiz or Cy treated cells (p<.01 vs Cy, p<.01 vs Quiz; n=3). Similar results were obtained in M13 cells. Treatment of M14 cells with 5µM FC under 1% O₂ resulted in substantial intracellular pH acidification (ΔpH=-2.7±0.6, p<.05 vs 1% O₂, n=3) within 48h. FC synergistically inhibited M14 cell growth when combined with Cy (CI=0.78). To investigate the effects of CA9 inhibition against non-FLT3 mutated cells we incubated OCI-AML3 and THP-1 cells to increasing doses of FC (1-10µM) under 1% O₂. We observed that both cell lines were less sensitive to FC (IC₅₀>10µM) compared to M14 and M13 cells. Interestingly, these cell lines displayed higher levels of CA12 mRNA (CA9/12 ratio=0.68 [OCI-AML3] and 0.54 [THP-1]) compared to the FC sensitive cell lines M14 and M13 (CA-9/12 ratio=2.5 and 4.6, respectively). Next, we assessed the effects Cy (1µM), Quiz (100nM) and FC (200µM) against FLT/ITD⁺ PCs. As expected, Cy and Quiz did not confer cytotoxic effects under 1% O₂. Surprisingly, FC was only mildly effective against FLT/ITD⁺ PCs, which harbored low CA9/12 ratios (<0.5). In contrast, treatment with equimolar doses of SLC, a different class CA9 inhibitor, exhibited substantial anti-leukemic activity under 1% O₂ conditions (78.8±1.9% growth inhibition, n=3, p<.05 vs FC, p<.001 vs Cy or Quiz). Conclusions: 1. CA9 is upregulated in FLT3/ITD⁺ cell lines and PCs under hypoxic conditions. 2. CA9 is expressed in LCs residing in the BM and spleen of FLT/ITD⁺ AML xenografts. 3. The CA9 inhibitor FC531 confers marked anti-leukemic activity against FLT3/ITD⁺ AML cells under hypoxic conditions via intracellular pH acidification and acts synergistically with cytarabine. 4. Upregulation of other carbonic anhydrase isoforms, such as CA12, might represent a resistance mechanism against FC531 that can be overcome by different class CA9 inhibitors. 5. Our data suggests that CA9 inhibition has the potential to specifically target LCs residing in hypoxic niches and may be of value as an adjunct to chemotherapy for FLT3/ITD⁺AML treatment. 6. Our ongoing work evaluates the effects of CA9 inhibition in AML xenografts.