BET and BRAF inhibitors act synergistically against BRAF-mutant melanoma

Abstract

Despite major advances in the treatment of metastatic melanoma, treatment failure is still inevitable in most cases. Manipulation of key epigenetic regulators, including inhibition of Bromodomain and extra-terminal domain (BET) family members impairs cell proliferation in vitro and tumor growth in vivo in different cancers, including melanoma. Here, we investigated the effect of combining the BET inhibitor JQ1 with the BRAF inhibitor Vemurafenib in in vitro and in vivo models of BRAF-mutant melanoma. We performed cytotoxicity and apoptosis assays, and a xenograft mouse model to determine the in vitro and in vivo efficacy of JQ1 in combination with Vemurafenib against BRAF-mutant melanoma cell lines. Further, to investigate the molecular mechanisms underlying the effects of combined treatment, we conducted antibody arrays of in vitro drug-treated cell lines and RNA sequencing of drug-treated xenograft tumors. The combination of JQ1 and Vemurafenib acted synergistically in BRAF-mutant cell lines, resulting in marked apoptosis in vitro, with upregulation of proapoptotic proteins. In vivo, combination treatment suppressed tumor growth and significantly improved survival compared to either drug alone. RNA sequencing of tumor tissues revealed almost four thousand genes that were uniquely modulated by the combination, with several anti-apoptotic genes significantly down-regulated. Collectively, our data provide a rationale for combined BET and BRAF inhibition as a novel strategy for the treatment of melanoma.

Keywords: BET inhibition; BRAF inhibition; JQ1; Melanoma; Vemurafenib.

Figure 1

JQ1 (J) interacts synergistically with Vemurafenib (V) in BRAF‐mutant melanoma cell lines. (A) Percentage of viability relative to DMSO‐treated control of four melanoma cell lines after 72 h exposure to increasing combinations of JQ1. (B–D) Relative viability of 451Lu (B, *P ≤ 0.0005 vs. all groups), SK‐MEL‐28 (C, *P ≤ 0.3 vs. all groups) and A375 (D, *P ≤ 0.0001 vs. all groups) treated with various concentrations of J, V, or combinations. Ranges for average relative risk rations (RRR) are shown (RRR<1 defines synergism). Error bars represent mean +/−SD.

Figure 2

Enhanced apoptosis elicited by JQ1 (J) combined to Vemurafenib (V) in BRAF‐mutant melanoma cell lines. Fold increase in apoptotic cells relative to controls in A375 (A), 451Lu (B) and SK‐MEL‐28 (C). cells treated with various concentrations of J, V or combinations. Error bars represent mean +/−SD. *P < 0.05, **P < 0.01 for comparison of combination group to all single groups. Apoptosis in control group is approximately 10% for all cell lines.

Figure 3

In vitro modulation of proteins involved in apoptosis after treatment with JQ1 alone or in combination with Vemurafenib. Protein lysates of 451Lu (A) and A375 (B) cells subjected to 48 h treatment with DMSO, JQ1 (500 nmol/L), Vemurafenib (500 nmol/L for A375, 1 μmol/L for 451Lu) or their combination were analyzed by antibody arrays for apoptotic proteins expression. Heat maps depict of log2‐transformed normalized densitometry averages of experimental replicates for the most consistently altered proteins. Log2 ratios >1 or <−0.62 defines significant up or downregulation, as per manufacturer's recommendation. CD‐40L, CD40‐ligand; FASL‐L, FAS‐ligand; CYT‐C, cytochrome C; TRAIL‐R, TRAIL receptor; IGFBP, insulin growth factor‐binding protein.

Figure 4

Enhanced activity of JQ1 (J) combined to Vemurafenib (V) in a xenograft NOG mouse model of BRAF‐mutant melanoma (A375). (A) Tumor growth curves of mice treated for 8 days with intraperitoneal J at 50 mg/kg per day, V at 25 mg/kg twice a day, or both. The combination resulted in reduced tumor growth compared to J, V and the control group since day+5 of treatment (P ≤ 0.01). (B) Kaplan–Meier survival curves. Mice in the combination group had significantly longer survival compared to all other groups starting from day+22 (P ≤ 0.03). (C) Relative Ki67 immunostaining levels on four tumors per group on day+5. (D) Representative micrographs of Ki67 immunostaining in each group.

Figure 5

Combined JQ1 (J) and Vemurafenib (V) treatment significantly impacts transcriptional programs in vivo. (A) Venn diagram indicating the number of differentially expressed genes in tumors of each group treatment compared to control. J+V group shows a high number of genes (n = 3816) that are not shared with any of the other groups. (B) Gene set enrichment (GSE) analysis of genes significantly downregulated in J+V group compared to all others.