BRAFV600E induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via AP-1 activation

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 BRAF^V600E inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation. To investigate the biological role of BRAF^V600E in B-cell leukemia, we generated a CLL-like B-cell line, OSUCLL, with doxycycline-inducible BRAF^V600E expression. Microarray and real-time PCR analysis showed that ABCB1 mRNA is upregulated in these cells, and P-glycoprotein (P-gp) expression as well as function were confirmed by immunoblot and rhodamine exclusion assays. Additionally, pharmacological inhibition of BRAF^V600E and MEK alleviated the BRAF^V600E-induced ABCB1/P-gp expression. ABCB1 reporter assays and gel shift assays demonstrated that AP-1 activity is crucial in this mechanism. This study, uncovers a pathological role for BRAF^V600E in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAF^V600E and MEK can be used to delay disease progression and occurrence of resistance.

Keywords: ABCB1; B-cell; BRAF; Leukemia; Lymphoma; P-glycoprotein; Vemurafenib.

Figure 1. Effects of transfected BRAF^V600E on the MAPK pathway and cell growth

A. Immunoblot analysis of wild-type and mutant BRAF expression and MAPK signaling in transfected OSUCLL cells following 24 hr incubation without or with dox (1 µg/ml). B. Proliferation in OSUCLL-Tet, OSUCLL-BRAF, and OSUCLL-BRAF^V600E cells without or with dox treatment (1 µg/ml) for 24 hr. BrdU was added for the last 4 hr of the incubations. Data are shown relative to the OSUCLL-Tet control cell line without dox treatment (N=4, *p<0.05; **p<0.005). C. OSUCLL-Tet, OSUCLL-BRAF, and OSUCLL-BRAF^V600E cells were incubated 48 hr with or without dox, then stained with anti-CD69 or isotype antibodies and assessed by flow cytometry. Data are representative of three independent experiments.

Figure 2. BRAF^V600E induces the expression of ABCB1 and functional P-gp in OSUCLL cells

A. ABCB1 mRNA levels were assessed in OSUCLL cells using real-time RT-PCR (N=3, **p<0.001). B. Immunoblot of normal BRAF, mutant BRAF, and P-gp in OSUCLL cells incubated 48 hr without or with dox. C. OSUCLL cells cultured 48 hr with dox were incubated 1 hr with the fluorescent P-gp substrate rhodamine 123, then transferred to rhodamine-free media with or without 10 µM verapamil. Retained rhodamine was assessed by flow cytometry after 90 min. Parental 697 cells and drug-resisant 697-R cells were included as controls. Results were averaged from three identical experiments (**p<0.005) and are shown as percent rhodamine-positive cells relative to vehicle-treated OSUCLL-Tet (far left). D. OSUCLL-BRAF^V600E cells were incubated 48 hr with or without dox, vincristine, and/or verapamil, and mitochondrial activity was evaluated by MTS assay. Results shown are averaged from three identical experiments. At each concentration of vincristine, comparisons were made between the dox and no dox conditions, as well as between the verapamil and control conditions in the presence of dox (**p<0.001).

Figure 3. BRAF^V600E and MEK inhibition block ABCB1/P-gp expression in OSUCLL cells

A. Real-time RT-PCR analysis of ABCB1 expression in OSUCLL cells in the presence or absence of BRAF^V600E or MEK inhibitors. Cells were incubated without or with dox 24 hr, then inhibitors (2 µM vemurafenib and/or 1 µM CI-1040) were added for an additional 16 hr. Inhibitor comparisons in the presence of dox were performed versus BRAF^V600E + dox (N=5, *p<0.05; **p<0.001). B. Immunoblot analysis of P-gp, normal BRAF, and BRAF^V600E expression in OSUCLL cells treated as in A. Results shown are representative of three individual experiments.

Figure 4. BRAF^V600E enhances ABCB1 promoter activity via MAPK and AP-1

A. HEK293T cells were transiently co-transfected with 1 µg ABCB1 reporter construct (pTL-MDR1) and 1 µg empty vector (pBabepuro) or mutant BRAF plasmid (pBabepuro-BRAF^V600E). After 8 hr, inhibitors were added as in Figure 3A. After an additional 16 hr, luciferase activity was assessed in total cell lysates. Results are shown normalized to the amount of lysate; each inhibitor was compared to control, and in addition, the control was compared to empty vector (N=3, *p<0.05; **p<0.001). B. The immunoblot from Figure 3B was additionally analyzed for c-Fos and c-Jun proteins. C. Nuclear extracts were prepared from OSUCLL cells incubated 24 hr with or without dox, then mixed with ³²P-labeled wild-type (wt) or mutant (mut) AP-1 probes, 100× cold probe (*), and antibodies as indicated: A. c-Fos; B. c-Jun; C. JunB; D. JunD; E. MEK (irrelevant control). The final lane is free probe without nuclear extract. Mixtures were separated by native acrylamide electrophoresis and bands detected by autoradiography. Results shown are representative of three individual experiments.