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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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 3

In vitro upregulation of BMX under hypoxic conditions.

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In vitro upregulation of BMX under hypoxic conditions. (A) Indicated cel...
(A) Indicated cell lines were cultured in normoxia or 1% hypoxia for 24 hours followed by Western blot analysis of indicated proteins. Representative blots from at least 3 independent experiments. (B) The right panel schematically represents the BMX promoter near the HRE site and the mutant construct. HEK293 cells were transfected with an empty promoter, BMX promoter, or the BMX promoter with mutated HRE element. After 1 day, cells were transferred to a hypoxia chamber for 24 hours. Hypoxic conditions upregulated BMX transcription (*P = 0.017, Welch’s t test), which was abrogated by the deletion of the HRE element (#P = 0.025, Welch’s t test). Data shown as mean of 3 replicates and representative of 2 independent experiments. (C) HEK293 cells were transfected with control, HIF1α, or HIF2α siRNA, followed by hypoxia treatment and Western blot analysis. HIF2α knockdown abrogated hypoxia-mediated BMX upregulation. Representative blots from at least 2 independent experiments. (D) BMX expression was determined in Baf3 cells expressing indicated FLT3 constructs after cell culture in normoxic or hypoxic (24 hours) conditions. Representative blots from at least 2 independent experiments. (E) MOLM13 and MV4-11 cells were grown under hypoxic and normoxic conditions (24 hours) followed by sorafenib treatment, and cell viability was assessed by MTT assay. Representative of 3 independent experiments (18 replicates). (F) ChIP assay in MOLM13 cells showed that HIF2α can bind the BMX promoter under hypoxic conditions (*P = 0.014, Welch’s t test). Data shown as mean of 3 replicates and representative of 3 independent experiments. (G) Diagnostic FLT3-ITD+ blast samples from patient A (left) and patient B (right) were treated with sorafenib under normoxic or hypoxic conditions, and cell viability was assessed by CellTiter Glo (1 experiment, 3 replicates). These results indicate that hypoxia can cause sorafenib resistance in patient-derived primary AML cells.