Abstract
A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAFV600E inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation.
Cell line models to study the mechanism of BRAFV600E in B cell leukemia do not exist. Therefore, we utilized the CLL-like B-cell line OSUCLL (PLoS One 2013, 8(10):e76607) to generate cells with doxycycline (dox)-inducible BRAFV600E expression to examine transcriptional and biochemical features induced by this mutation in B-cells. We observed that BRAFV600E induction enhanced proliferation and activation of MAPK signaling in these cells. Microarray using Affymetrix U133 Plus 2.0 GeneChips demonstrated that 235 genes were up-regulated at least 2-fold, and 227 genes were down-regulated at least 2-fold. Several were confirmed by real-time RT-PCR analysis, including ABCB1 (p<0.001, vehicle versus dox-treated). Increased expression of the ABCB1 gene product, P-glycoprotein (P-gp), was also confirmed by immunoblot. This increase resulted in enhanced P-gp function as determined by rhodamine exclusion assays (p<0.005), an effect that was significantly reversed by the P-gp inhibitor verapamil (p<0.005). As an additional demonstration of P-gp function, BRAFV600E induction via dox treatment resulted in a significant increase in resistance to the P-gp substrate vincristine, and the addition of verapamil significantly reduced vincristine resistance. Importantly, pharmacological inhibition of BRAFV600E and MEK, by vemurafenib and CI-1040 respectively, diminished BRAFV600E-induced MAPK pathway activation and enhancement of ABCB1/P-gp expression. To further understand the transcriptional mechanism of BRAFV600E-induced P-gp expression, we performed luciferase assays using a vector containing 1 kb of ABCB1 promoter driving a luciferase reporter, co-transfected into HEK293 cells with either an empty vector or a vector containing BRAFV600E. These assays showed that BRAFV600E expression enhanced luciferase activity (p<0.001), and this effect was down-regulated by vemurafenib and CI-1040 (p<0.001). The role of AP-1 has been reported in ABCB1 regulation. Thus, we conducted electrophoretic mobility shift assays in the BRAFV600E-transfected OSUCLL B-cells, with or without dox treatment, to identify AP-1 factor(s) involved in ABCB1 regulation by BRAFV600E. In these experiments, a supershift in the AP-1 complex was produced by an antibody to JunD in the presence of dox treatment, demonstrating that at least JunD activity is important in this mechanism.
Based on these observations, we conclude that BRAFV600E activates AP-1 proteins including JunD to induce ABCB1/P-gp expression and drug resistance in B-cells. This study uncovers a new pathological role for BRAFV600E in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAFV600E and MEK may be beneficial in delaying disease progression and occurrence of resistance to drugs that are substrates of P-gp.
Disclosures
Byrd: Acerta Pharma BV: Research Funding.
Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor.