These results were validated by use of dissimilar BMX shRNA constructs (Supplemental Figure 8)

These results were validated by use of dissimilar BMX shRNA constructs (Supplemental Figure 8). upregulation was observed in both AML and non-AML cell lines. Functional studies in human FLT3-ITD+ cell lines showed that BMX is Bromodomain IN-1 usually a 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 Bromodomain IN-1 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. gene did not show any mutations or copy number changes, indicating that BMX upregulation during sorafenib resistance is not likely due to Bromodomain IN-1 copy number changes. Open in a separate window Physique 1 Transcriptional upregulation of BMX in AML patients during sorafenib resistance.RNA-Seq analysis of bone marrow aspirates from 4 patients collected at initial relapse of AML (Pre-TKI) and at development of resistance to TKI therapy (TKI Res). (A) Integrative Genomics Viewer snapshot of RNA-Seq data showing genomic locus of 2 BMX transcripts. (B) BMX overexpression was confirmed using RT-PCR (in triplicate). (C) Exon junction read counts from RNA-Seq analysis for each patient representing log fold change of resistance minus diagnosis; axis shows patients 1C4 per Tec kinase. Sorafenib induces BMX upregulation in a MOLM13 FLT3-ITD mouse model. To decipher the molecular mechanisms that contribute to BMX upregulation during sorafenib resistance, we used a MOLM13 FLT3-ITD+ mouse model of sorafenib resistance. To understand the contribution of FLT3 inhibition to BMX upregulation, we also included crenolanib, another FLT3 inhibitor Pcdha10 (16). In a pilot survival study, mice bearing MOLM13 FLT3-ITD+ cells were treated with vehicle, crenolanib, or sorafenib until the development of resistance. Emerging resistance was determined by an increase in leukemic cell outgrowth decided from bioluminescence imaging (Supplemental Physique 1A). Mice treated with vehicle, crenolanib, or sorafenib survived a median of 16 days, 28 days, and 45 days, respectively (Supplemental Physique 1B). In a follow-up study, mice were given the same treatments, bone marrow was harvested on days 17, 24, and 40 in vehicle-, crenolanib-, and sorafenib-treated mice, respectively, BMX expression was determined by Western blotting, and FLT3 TKD mutations were assessed by deep amplicon sequencing. Interestingly, BMX expression was not observed in mice treated with vehicle or crenolanib, while BMX was significantly upregulated in the sorafenib-treated group (Supplemental Physique 1B). Analysis of FLT3-ITD TKD mutation status showed that 2 of 8 crenolanib-treated mice and 3 of 8 sorafenib-treated mice developed TKD mutations (Physique 2A and Supplemental Table 4). These results indicated that BMX upregulation is likely to be independent of the presence of TKD mutations and not a direct effect of FLT3 inhibition, since the crenolanib-treated group did not show any BMX upregulation. To further confirm Bromodomain IN-1 the independence of BMX upregulation from the presence of TKD mutations, we performed a short-term experiment of 5 and 10 days of sorafenib treatment, when neither an outgrowth of leukemia cells nor sorafenib resistance is observed (Supplemental Physique 1A), and found that BMX expression was already increased after 5 and 10 days of sorafenib treatment as compared with the vehicle-treated group (Physique 2B). Next, we generated a phospho-BMX antibody against the autophosphorylation site of BMX (Supplemental Physique 2), which could be used as a readout of BMX kinase activity. Indeed, Bromodomain IN-1 we found that phospho-BMX levels were elevated in bone marrow leukemic cells from sorafenib-treated mice (Physique 2C and Supplemental Physique 3A). Protein levels of other Tec kinases, including BTK, were not increased compared with samples from vehicle-treated mice. These results obtained at early time points were further confirmed in samples obtained from mice treated with sorafenib for 30 days, at the time of leukemic outgrowth (Physique 2D and Supplemental Physique 3B). Furthermore, we carried out BMX in vitro kinase assay, which showed that BMX kinase activity was elevated in the AML cells derived from sorafenib-treated mice as compared with vehicle-treated groups (Supplemental Physique 3C). To determine whether high BMX expression contributes to sorafenib resistance, bone marrow MOLM13 cells with low/absent.