Combinatorial treatments that overcome PDGFRβ-driven resistance of melanoma cells to V600EB-RAF inhibition

Abstract

(V600E)B-RAF mutation is found in 50% to 60% of melanomas, and the novel agents PLX4032/vemurafenib and GSK2118436 that inhibit the (V600E)B-RAF kinase achieve a remarkable clinical response rate. However, as might be expected, acquired clinical resistance to these agents arises in most melanoma patients. PLX4032/vemurafenib resistance that arises in vivo in tumor matched short-term cultures or in vitro in melanoma cell lines is not caused by acquisition of secondary mutations in (V600E)B-RAF but rather is caused by upregulating platelet-derived growth factor receptor β (PDGFRβ) or N-RAS which results in resistance or sensitivity to mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK) kinase inhibitors, respectively. In this study, we define a targeted combinatorial strategy to overcome PLX4032/vemurafenib resistance in melanoma cell lines or short-term culture where the resistance is driven by PDGFRβ upregulation, achieving synergistic growth inhibition and cytotoxicity. PDGFRβ-upregulated, PLX4032-resistant (PPRM) cell lines show dual phospho (p)-ERK and p-AKT upregulation, and their growth inhibitory responses to specific small molecule inhibitors correlated with p-ERK, p-AKT, and p-p70S6K levels. Coordinate inhibition of (V600E)B-RAF inhibition and the RTK-PI3K-AKT-mTORC axis led to functionally significant rebound signaling, illustrating a robust and dynamic network connectivity. Combined B-RAF, phosphoinositide 3-kinase (PI3K), and mTORC1/2 inhibition suppressed both immediate early and delayed compensatory signaling, resulting in a highly synergistic growth inhibitory response but less efficient cytotoxic response. In contrast, the combination of MEK1/2, PI3K, and mTORC1/2 inhibitors consistently triggered apoptosis in a highly efficient manner. Together, our findings offer a rational strategy to guide clinical testing in preidentified subsets of patients who relapse during treatment with (V600E)B-RAF inhibitors.

Figure 1

Compensatory signaling limits the growth-inhibitory responses of PPRM cell lines to co-targeting of ^V600EB-RAF with RTK, AKT or mTORC1. A, Upregulation of p-AKT (Ser 473) and p-ERK in PPRM cell lines. Western blots of lysates showing relative levels of indicated proteins in PPRM (1 µM PLX4032) and parental cell lines. NIH3T3, treated with PDGF-BB (20 ng/ml, 1 h) or PBS. Tubulin, loading control. B, Survival curves of PPRM cell lines (treated with 1 µM PLX4032, unless otherwise indicated) in response to imatinib or AKTi titration with or without 0.5 µM AZD6244 (72 h) (top; mean ± SD, n=5). Dashed line, 50% growth inhibition. Western blots showing indicated protein levels from cells treated (1 h) with DMSO, 0.5 µM imatinib (or 0.5 µM AKTi), 0.5 µM AZD6244 or both (bottom). C, Survival curves in response to sunitinib titration with or without 0.5 µM AZD6244 (72 h) (top) and Western blots showing indicated protein levels from cells treated (1 h) with DMSO, 0.2 µM sunitinib, 0.5 µM AZD6244, or both (bottom). D, Western blots showing indicated proteins levels in cells treated (1 h) with DMSO, 0.1 µM rapamycin, rapamycin + 0.5 µM AZD6244, or rapamycin + AZD6244 + 0.5 µM AKTi (top) and survival curves in response to rapamycin titration alone or in the presence of 0.5 µM AZD6244 or AZD6244 + 0.5 µM AKTi (bottom).

Figure 2

MAPK targeting strongly synergizes with co-suppression of PI3K and mTORC1/2 in growth inhibiting PPRM cell lines. A, Survival curves for PPRM cell lines titrated with the mTORC1/2 inhibitor AZD8055 with or without 1 µM PLX4032 for 72 h (top) and Western blots showing indicated proteins levels treated with DMSO, PLX4032 (1 µM), AZD8055 (0.1 µM) or both for 1 h (bottom). B, Relative synergy of indicated drug combinations (expressed as log₁₀ of CI values). C, Survival curves for PLX4032-sensitive parental lines vs. PLX4032-resistant sub-lines (1 µM PLX4032) titrated with BEZ235 (top) or for all PPRM cells titrated with BEZ235 with or without of 1 µM PLX4032 (middle). Western blots showing indicated proteins levels from PPRM cells were treated with DMSO, 0.1 µM BEZ235, 1 µM PLX4032, or both drugs for 1 h (bottom).

Figure 3

Co-targeting of specific signaling nodes attenuates AKT and ERK signal recovery. A. B-RAF targeting combined with co-suppression of PI3K and mTORC1/2 prevented delayed (24 h) p-AKT recovery. Western blots of lysates from PPRM cells treated with DMSO, 1 µM PLX4032, 0.1 µM AZD8055, PLX4032 + AZD8055, 0.1 µM BEZ235, or PLX4032 + BEZ235 for 24 h. B. Western blots showing indicated protein levels in PPRM cell lines treated with DMSO or a single dose of AZD8055 (0.1 µM) or BEZ235 (0.1 µM) for the indicated durations (h) in the presence of 1 µM PLX4032. C. MEK1/2 targeting combined with co-suppression of PI3K and mTORC1/2 prevented delayed (24 h) p-ERK recovery. Western blots showing indicated protein levels from indicated cell lines treated with DMSO, 0.1 µM BEZ235, 1 µM AZD6244, or both for 24 h. D. Western blots showing indicated protein levels in PPRM cell lines treated with DMSO or a single dose of 1 µM PLX4032 or 1 µM AZD6244, with or without co-treatment with AZD8055 (0.1 µM) or BEZ235 (0.1 µM), for the indicated durations (h).

Figure 4

MEK1/2, rather than B-RAF, targeting in combination with dual PI3K and mTORC1/2 suppression augments apoptosis induction. A. Live (green) vs. dead (orange) cells in PPRM spheroids treated with DMSO, 1 µM PLX4032, 1 µM AZD6244, 0.1 µM BEZ235 or the indicated combinations (dosed every 48 h) for 6 days. B, PPRM cells treated with DMSO (D), 1 µM PLX4032 (P), 0.1 µM BEZ235 (B) or both (P+B) for 5 days (M229 R5, Pt48 R) or 6 days (M238 R1); levels of apoptosis plotted on the right. C, Treatment with DMSO, 1 µM AZD6244 (A), 0.1 µM BEZ235, or both and apoptosis plotted.