BET Inhibitors Plus Sunitinib Induce Early Signs of Clinical Activity in Melanoma Cells

Sunitinib (Sutent) demonstrated synergistic activity with the BET inhibitors JQ1 and NHWD-870 in melanoma cell lines, according to preclinical findings from a study that were published in Experimental & Molecular Medicine.

This study showed that BET inhibitors inhibit the BRD4/GDF15 and BRD4/IL6/STAT3/GDF15 pathways, thus sensitizing melanoma cell lines to sunitinib.

“These findings suggest that BET inhibitor–mediated GDF15 inhibition plays a critical role in enhancing sunitinib sensitivity in melanoma, indicating that BET inhibitors synergize with sunitinib in melanoma,” lead study author Furong Zeng, of the Xiangya Clinical Research Center for Cancer Immunotherapy in Changsha, Hunan, China, and colleagues, wrote in a paper of the data.

To determine which drugs from the FDA-approved drug library synergize with BET inhibitors in melanoma, the investigators screened 240 antitumor agents in combination with JQ1, a BET inhibitor developed by the investigators, in an in vitro drug combination assay. Both sunitinib and CDK4/6 inhibitors had high levels of synergy with JQ1 in A375 and SK-MEL-28 melanoma cells.

To further determine the synergy between sunitinib and JQ1, the investigators performed a dose-response experiment with increasing drug concentrations in A375 and SK-MEL-28 cells. The combination index values in both cell lines were less than 1, indicating drug synergy. Synergy in A375 and SK-MEL-28 cells was also noted between sunitinib and NHWD-870, another BET inhibitor developed by the investigators.

The investigators also conducted a colony formation assay to determine the antiproliferative effect of BET inhibitors plus sunitinib and found that combination treatment with these agents significantly suppressed the proliferation of melanoma cells, further supporting the hypothesis of synergy between sunitinib and BET inhibitors.

To clarify how this synergy occurs, the investigators performed an RNA sequencing analysis on A375 melanoma cells treated for 24 hours with control, JQ1 alone, sunitinib alone, or JQ1 plus sunitinib. A gene set enrichment analysis showed that the combination significantly inhibited cell cycle progression more than the other groups. The combination inhibited 17 cell cycle–associated genes, including CDK1, which regulates the cell cycle, and CDC6, which regulates DNA replication in eukaryotic cells. Additionally, flow cytometry demonstrated that BET inhibitors plus sunitinib induced G1 arrest more than BET inhibitors or sunitinib alone.

A gene set variation analysis showed that the combination activated autophagy and apoptosis pathways more than each agent alone. The antitumor effect of the combination was partially inhibited by Z-VAD-FMK, an apoptosis inhibitor, but not by ferroptosis, necroptosis, or autophagy inhibitors. Z-VAD-FMK also partially inhibited A375 cell death induced by JQ1 and different concentrations of sunitinib. Furthermore, the combination of a BET inhibitor and sunitinib was associated with a higher proportion of apoptotic cells than either agent alone, indicating that the combination drives apoptosis of melanoma cells.

The investigators also analyzed the associations between STAT3 signaling, part of cell cycle arrest and apoptosis, and sunitinib sensitivity. STAT3 signaling was found to be significantly activated in cells with a higher half maximal inhibitory concentration (IC50) of sunitinib. Additionally, GSEA showed that STAT3-resistant cells had an activated IL6/JAK/STAT3 pathway. STAT3 phosphorylation was also increased in generated sunitinib-resistant melanoma cells. In generated STAT3 knockdown melanoma cells, STAT3 inhibition enhanced sunitinib sensitivity. In addition, the STAT3 inhibitor stattic sensitized SK-MEL-28 and A375 cells to sunitinib.

When investigating the site of STAT3 phosphorylation that mediates the sensitivity of melanoma cells to sunitinib, the investigators ectopically expressed wild-type STAT3 or STAT3 phosphorylation mutants. They found that coexpression of wild-type STAT3 impaired shSTAT3-induced melanoma cell sensitization, but STAT3 phosphorylation mutants did not. These findings indicate that STAT3 inhibition enhances the sensitivity of melanoma cells to sunitinib through STAT3 phosphorylation suppression.

The investigators further analyzed the ability of BET inhibitors to sensitize melanoma cells to sunitinib by suppressing STAT3 signaling. In JQ1-treated cells, JQ1 inhibited c-Myc target expression. However, the targets with STAT3-induced downregulation were upregulated in the JQ1-treated cells, indicating that JQ1 inhibits IL6/STAT3 signaling activity. IL6 concentration was also reduced in the melanoma cells treated with BET inhibitors, as was STAT3 phosphorylation.

To determine which BET proteins contribute to IL6/STAT3 pathway signaling, the investigators individually silenced BRD2, BRD3, and BRD4, which showed that only BRD4 protein knockdown markedly reduced IL6 concentration and STAT3 phosphorylation. Further BRD4 silencing inhibited STAT3 signaling and reduced IL6 mRNA levels. These findings indicate that BET inhibitors use the BRD4/IL6 axis to suppress STAT3 signaling.

The investigators also evaluated whether STAT3 signaling mediates BET-inhibitor–induced sunitinib cell sensitization by incubating control and STAT3-silenced melanoma cells with sunitinib or sunitinib plus BET inhibitors. After BET inhibitor treatment, the control cells had a marked degree of sunitinib sensitization, but the STAT3-silenced cells did not. Stattic sensitized the melanoma cells to sunitinib but did not further enhance this sensitization in the presence of BET inhibitors.

When the investigators overlapped the downregulated genes after BET inhibitor treatment and BRD4 silencing, they found 35 differentially expressed genes. Of those, GDF15 and IL1A had markedly increased mRNA levels in cells resistant to sunitinib and comparable mRNA levels after long-term sunitinib withdrawal. GDF15 expression was positively associated with the sunitinib logIC50, but IL1A expression was not. A real-time polymerase chain reaction (PCR) analysis validated that BET inhibitor treatment reduced GDF15 mRNA levels and increased GDF15 expression in sunitinib-resistant cells.

Furthermore, GDF15 knockout increased cell sensitivity to sunitinib and GDF15 overexpression reduced this sensitivity, indicating that BET inhibitors regulate the sensitivity of melanoma cells to sunitinib by inhibiting both GDF15 expression and STAT3 activity.

The investigators also found that STAT3-silenced cells had reduced GDF15 expression compared with control cells, and that GDF15b expression further decreased after stattic treatment. A ChIP-qPCR analysis confirmed that in melanoma cells, STAT3 binds to the GDF15 promoter.

As BET inhibitors suppressed GDF15 more strongly than stattic treatment or STAT3 silencing, the investigators hypothesized that this suppression may occur in another way in addition to the BRD4/IL6/STAT3 axis. When seeking to determine whether BRD4 can directly regulate GDF15 expression, the investigators found that STAT-silenced cells treated with BET inhibitors did have reduced GDF15 expression.

To evaluate the therapeutic potential of sunitinib plus BET inhibitors in melanoma, the investigators created subcutaneous xenograft models by inoculating the right flanks of nude mice with A375 cells. When the tumor volume reached 50 to 100 mm3, the mice were randomized into vehicle, sunitinib, NHWD-870, or sunitinib plus NHWD-870 cohorts. All treatments occurred over 2 successive days followed by 1 day without treatment.

The tumor weight and volume were decreased in the combination cohort vs the other groups, with no significant change occurring in the body weight of the mice. Real-time PCR analysis of the treated tumors demonstrated reduced GDF15 and IL6 mRNA expression in the NHWD-870 and combination cohorts, and immunohistochemistry (IHC) staining showed decreased protein levels of GDF15, p-STAT3 (Y705) and p-STAT3 (S727) in these cohorts.

In addition, IHC staining for Ki67 showed fewer proliferative cells in the combination cohort, and a TUNEL assay showed that this cohort had an increase in the number of apoptotic cells. These findings indicate the synergy between BET inhibitors and sunitinib for inducing in vivo tumor suppression.

“These findings are potentially translatable toward novel therapies for melanoma and other diseases that can be cotreated with BET inhibitors and sunitinib,” the study authors concluded.

Reference

Zeng F, Li Y, Meng Y, et al. BET inhibitors synergize with sunitinib in melanoma through GDF15 expression. Exp Mol Med. 2023;55(2):364-376. doi:10.1038/s12276-023-00936-y