BRAF- and MEK-Targeted Small Molecule Inhibitors Exert Enhanced Antimelanoma Effects in Combination With Oncolytic Reovirus Through ER Stress

Abstract

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma).

Figure 1

RT3D, PLX4720, and PD184352 are selective for melanoma relative to normal skin fibroblasts. Driving paradoxical p-ERK signaling using PLX4720 in BRAF wild-type melanoma cell lines does not enhance RT3D cell kill. (a) RT3D, PLX4720, or PD184352 was titrated along Malme 3 (normal skin fibroblast) and Malme-3M (BRAF mutant melanoma) cells, both derived from the same patient. Cell survival was measured 96 hours later by MTT assay. (b) Pictomicrograph of cytopathic effect of RT3D at a low dose (MOI 3) and high dose (MOI 350) on Malme-3 and Malme-3M cells. (c) PMWK and MeWo (wild-type BRAF/RAS), A375 and Mel624 (BRAF^V600E mutant), WM266.4 (BRAF^V600D mutant), DO4 (NRAS^Q61L mutant) and WM1791c (KRAS^Q61H mutant) melanoma cells were treated with PLX4720 (0.3 µmol/l) or PD184352 (0.001–1 µmol/l) for 6 hours before harvesting for western blot and probing for pERK1/2 downstream of RAS/MEK signaling. (d) Melanoma cells were treated with dilutions of RT3D, PLX4720, or PD184352 and cell survival was measured at 96 hours by MTT assay. (e) BRAF wild-type cells (PMWK, MeWo, DO4, WM1791c) were assessed for RT3D cytoxoicity in the presence (red) and absence (black) of PLX4720 (0.3 µmol/l), whereby p-ERK signaling is enhanced in the RAS mutant background (DO4, WM1791c). Cell survival was measured 96 hours later by MTT. Data are derived from three independent experiments ± SEM.

Figure 2

Combining RT3D with BRAF and/or MEK inhibition enhances cell kill in melanoma cell lines. BRAF mutant melanoma cells were treated with 4×, 2×, 1×, 0.5×, and 0.25× IC₅₀ doses of (a) RT3D, PLX4720, or the combination at equal ratios. (b) RT3D, PD184352, or the combination at equal ratios. (c) BRAF wild-type melanoma cell lines were treated with 4×, 2×, 1×, 0.5×, and 0.25× IC₅₀ doses of RT3D, PD184352, or the combination at equal ratios. (d) Wild-type BRAF/RAS (PMWK), BRAF^V600E mutant (A375) and BRAF wild-type/NRAS^Q61L mutant (DO4) cell lines were treated with RT3D, PLX4720 (PLX), PD184352 (PD), the combination of PLX4720 and PD184352, or the triple combination of RT3D, PLX4720, and PD184352 at 4×, 2×, 1×, 0.5×, and 0.25× IC₅₀ doses. For all experiments, cell survival was measured at 96 hours by MTT assay and data are derived from three independent experiments ± SEM. (e) BRAF^V600E mutant (A375 and Mel624) cells were treated with BRAF and/or MEK inhibitors (PLX4720 and PD184352 respectively), followed by infection with RT3D at various MOI. 48 hours later, cells were stained with crystal violet.

Figure 3

RT3D replication is not affected in the presence of BRAF/MEK inhibition; however, cleaved caspase 3/7 is enhanced in BRAF mutant melanoma. In RAS mutant melanoma, enhancing p-ERK signaling with PLX4720 prevents RT3D-induced caspase 3/7 activation. (a) RT3D replication was measured in the presence of PLX4720 (0.3 µmol/l) or PD1843522 (1 µmol/l) by one step viral growth assay in PMWK, A375 and DO4. (b) Wild-type BRAF (PMWK, DO4) were treated with PD184352 (1 µmol/l) (PD), followed by RT3D (MOI 0.1), and BRAF^V600E mutant (A375) were treated with PLX4720 (0.3 µmol/l) (PLX) followed by RT3D (MOI 0.1). At 6, 24, 30, 48, and 54 hours after infection samples were harvested and probed for cleaved caspase 3 and 7 by western blot. (c) Cells were treated with BRAF/MEK inhibition and various doses of RT3D depending on sensitivity. At 72 hours cleaved caspase 3/7 (luminescence) was analyzed by caspase glo assay. Alongside these assays, cells were treated with inhibitors in combination with RT3D at an MOI of 0.04 (PMWK, A375), or 0.01 (DO4), and at 48 hours cells were harvested and probed for cleaved caspase 3 and 7 by western blot.

Figure 4

Enhanced apoptosis observed with RT3D and BRAF/MEK inhibition is mediated by ER stress. (a) BRAF^V600E mutant (A375) and NRAS^Q61L mutant (DO4) cell lines were treated with increasing doses of RT3D in the presence of PLX4720 (0.3 µmol/l), PD184352 (1 µmol/l), or the combination of both inhibitors. At 48 hours, lysates were collected and analyzed for p-ERK and p-EIF2α by western blot. (b) Densitometry of p-EIF2α by western analysis is shown for A375 and DO4 cells treated with inhibitors and RT3D (MOI 0.1) at 48 hours. Data are averaged for three independent repeats ± SEM (c) A375 and DO4 cells were treated with PLX4720 and/or PD184352 followed by various doses of RT3D. CHOP gene expression was determined by qRT-PCR at 40 hours. Levels of mRNA were standardized to the expression of beta actin. Mean ± SEM, n = 3.

Figure 5

Salubrinal can partially rescue cells from enhanced cytotoxicity observed with RT3D and BRAF/MEK inhibition in BRAF mutant melanoma. (a) BRAF^V600E mutant (A375) cells were treated with PLX4720 (0.3 µmol/l) or PD184352 (1 µmol/l) prior to infection with RT3D, 24 hours later, cells were treated with 20 µmol/l salubrinal (as to not interfere with replication), and cell survival measured at 72 hours by MTT assay (top panel). In a similar experiment, cells were stained with crystal violet at 48 hours (bottom panel). (b) To confirm on-target effect, cells that were treated with inhibitors, followed by RT3D (MOI 0.1), and salubrinal 24 hours after infection, were also harvested at 40, 44, and 48 hours and probed with p-EIF2α by western blot. (c) A375 cells treated with inhibitors and RT3D (MOI 0.1), and at 40 hours were analyzed by qRT-PCR for PUMA and NOXA, with or without the addition of salubrinal 24 hours after infection. For all qRT-PCR analysis, levels of mRNA were standardized to the expression of beta actin. Mean ± SEM, n = 3. (d) Cells treated with inhibitors, followed by RT3D, and salubrinal 24 hours post infection were analyzed for presence of cleaved caspase 3/7 at 72 hours by caspase glo assay. (e) Cells were treated with the caspase 4 inhibitor Z-YVAD prior to treatment with PLX4720 and RT3D (MOI 3). Cell survival was then measured 72 hours later by MTT assay, or harvested 48 hours later and probed with caspase 3 to confirm on-target effect.

Figure 6

PLX4720 enhances RT3D-mediated antitumor activity in BRAF mutant tumors. (a) CD1 nude mice bearing A375 (BRAF^V600E mutant) tumors received PLX4720 (20 mg/kg) administered daily or vehicle, followed by a single intratumoral injection of 5 × 10⁷ pfu RT3D or PBS sham 3 days later (day 1). Data show tumor volumes per treatment group (n = 10 for each group) and survival rates. A log-Rank (Mantel-Cox) test was used to compare groups to the doublet therapy (RT3D plus PLX4720) (b) At day 60, tumors were analyzed for RT3D viral titre by TCID₅₀ assay. Data shown are for tumors treated with RT3D or combination treatments of RT3D with PLX4720 and are relative to tumor weight (viral titre per mg). Titres were compared by ranked Mann-Whitney test. (c) Immune-competent C57BL/6 mice bearing 4434 (BRAF mutant murine) tumors were treated daily with PLX4720 (PLX 40 mg/kg) followed by a single intra-tumoral injection of 5x10⁶ pfu RT3D or PBS sham 3 days later. Data show tumor volumes per treatment group (n = 10 for each group). (d) In vitro, 4434 cells were treated with 4×, 2×, 1×, 0.5× and 0.25× IC₅₀ doses of RT3D, PLX4720 or the combination at equal ratios. Data show surviving fractions (left panel) and photography of representative cell survival (right panel) measured at 96 hours by MTT assay where presence of purple crystals that have been solubilized denotes working mitochondria. Surviving fractions are derived from three independent experiments ± SEM.