Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia
Jolieke G. van Oosterwijk, … , Navjotsingh Pabla, Sharyn D. Baker
Jolieke G. van Oosterwijk, … , Navjotsingh Pabla, Sharyn D. Baker
Published December 11, 2017
Citation Information: J Clin Invest. 2018;128(1):369-380. https://doi.org/10.1172/JCI91893.
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Research Article Hematology Oncology

Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia

Abstract

Oncogenic addiction to the Fms-like tyrosine kinase 3 (FLT3) is a hallmark of acute myeloid leukemia (AML) that harbors the FLT3–internal tandem duplication (FLT3-ITD) mutation. While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, we used RNA-Seq–based analysis of patient leukemic cells and found that upregulation of the Tec family kinase BMX occurs during sorafenib resistance. This upregulation was recapitulated in an in vivo murine FLT3-ITD–positive (FLT3-ITD+) model of sorafenib resistance. Mechanistically, the antiangiogenic effects of sorafenib led to increased bone marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX upregulation was observed in both AML and non-AML cell lines. Functional studies in human FLT3-ITD+ cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Taken together, our results demonstrate that hypoxia-dependent upregulation of BMX contributes to therapeutic resistance through a compensatory prosurvival signaling mechanism. These results also reveal the role of off-target drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anticancer therapeutics, resulting in drug resistance through cell-nonautonomous microenvironment-dependent effects.

Authors

Jolieke G. van Oosterwijk, Daelynn R. Buelow, Christina D. Drenberg, Aksana Vasilyeva, Lie Li, Lei Shi, Yong-Dong Wang, David Finkelstein, Sheila A. Shurtleff, Laura J. Janke, Stanley Pounds, Jeffrey E. Rubnitz, Hiroto Inaba, Navjotsingh Pabla, Sharyn D. Baker

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Figure 2

Sorafenib induces BMX upregulation in a MOLM13 FLT3-ITD+ xenograft model of sorafenib resistance.

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Sorafenib induces BMX upregulation in a MOLM13 FLT3-ITD+ xenograft model...
(A) MOLM13 cells were administered to NSG mice by tail vein injection (TVI), and 10 days after TVI mice were treated with vehicle, crenolanib, or sorafenib until the time of leukemic progression (on days 17, 24, and 40 after TVI, respectively), at which time bone marrow MOLM13 cells were isolated, FLT3 tyrosine kinase domain (TKD) mutation status was assessed by deep amplicon sequencing, and BMX protein expression was assessed by Western blotting. A schematic representation of FLT3 TKD mutation status and BMX upregulation is shown; each box represents an individual sample. (B) NSG mice engrafted with MOLM13 cells for 10 days were treated with vehicle or sorafenib for 5 or 10 days. BMX upregulation was observed after 5 days of sorafenib treatment. (C and D) After 10 (C) or 30 days (D) of sorafenib treatment, BMX is the only Tec kinase that is activated by sorafenib in comparison with vehicle-treated mice as shown by increased phospho-BMX (p-BMX); p-BTK and Tec kinase are downregulated. (B–D) Western blots are from 1 experiment. (E and F) MOLM13 cells were obtained from the bone marrow of mice treated with vehicle or sorafenib at the time of leukemic progression (day 14 after TVI for vehicle; day 40 after TVI for sorafenib). Expression of BMX and p-BMX was determined by Western blot, cells with low/absent (D, lanes 1–4) or high p-BMX expression (D, lanes 2, 5–10, 12) were pooled, and 1 × 106 cells were administered i.v. to NSG mice. Starting 7 days after TVI, mice were treated with vehicle (n = 5) or sorafenib (n = 5) for 18 days. Survival was significantly longer in mice injected with MOLM13 cells with low versus high BMX expression (34 vs. 24 days; *P = 0.0031, Kaplan-Meier analysis).