Abstract
Tyrosine kinase inhibitors (TKI) have improved CML response rates, and some are effective against resistance-promoting point mutations in BCR-ABL1. However, in the absence of point mutations, resistance still occurs. Here, we identify a novel pathway mediating resistance which connects p47phox, the organizer subunit of NADPH oxidase-2 (NOX2), with early growth response-1 (Egr-1) and the Src family kinase Fyn. We found up-regulation of p47phox, Egr-1, and Fyn mRNA and protein using paired isogenic CML cell lines and mined data. Isolation of CD34+ cells and tissue microarray staining from blast crisis CML patients confirmed in vivo over-expression of components of this pathway. Knockdown studies revealed that p47phox modulated reactive oxygen species and Egr-1 expression, which, in turn, controlled Fyn expression. Interestingly, Fyn knockdown sensitized TKI-resistant cells to dasatinib, a dual BCR-ABL1/Src inhibitor. Egr-1 knockdown had similar effects, indicating the utility of targeting Fyn expression over activation. Pointedly, p47phox knockdown also restored TKI-sensitivity, indicating that targeting the NOX2 complex can overcome resistance. The NOX2/Egr-1/Fyn pathway was also conserved within TKI-resistant EGFRΔIII-expressing glioblastoma and patient-derived glioblastoma stem cells. Thus, our findings suggest that targeting the NOX2/Egr-1/Fyn pathway may have clinical implications within multiple cancer types; particularly where efficacy of TKI is compromised.
Highlights
The advent of tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the primary oncogene associated with chronic myeloid leukemia (CML), has dramatically increased patient survival rates [1, 2]
To understand how alterations of the redox milieu are associated with TKI-resistance in CML, we first measured the levels of reactive oxygen species (ROS) within two cell line models of acquired mutation-independent resistance
Of the seven NADPH oxidase (NOX) complex family members, NOX1, NADPH oxidase-2 (NOX2), NOX4, NOX5, and DUOX2 have all been identified as isoforms present in CML [27, 33, 35]
Summary
The advent of tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the primary oncogene associated with chronic myeloid leukemia (CML), has dramatically increased patient survival rates [1, 2]. TKI-resistance is a looming clinical problem as the number of patients with detectable disease burden continues to grow [2]. This issue underscores a need for new approaches to treat refractory patients. Single point mutations in BCR-ABL1 are known determinants of TKI-sensitivity [3,4,5,6]. Persistence of CML stem cells (LSC), which are less dependent on BCR-ABL1 rendering them less sensitive to TKI, are another example of point mutation-independent resistance [8]. As stem cell populations are commonly less sensitive to inhibition of oncogenic tyrosine kinases, the identification of novel targetable pathways within those populations is of vital importance
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