Tretinoin synergistically enhances the antitumor effect of combined BRAF, MEK, and EGFR inhibition in BRAFV600E colorectal cancer [RKO samples]

Patients with BRAF-mutated colorectal cancer (BRAFV600E CRC) are currently treated by a combination of BRAF inhibitor and anti-EGFR antibody with or without MEK inhibitor. A fundamental problem in treating patients with BRAFV600E CRC is intrinsic and/or acquired resistance to this combination therapy. By screening 78 compounds, we identified tretinoin, a retinoid, as a compound that synergistically enhances the antiproliferative effect of a combination of BRAF inhibition and MEK inhibition with or without EGFR inhibition on BRAFV600E CRC cells. This synergistic effect was also exerted by other retinoids. Tretinoin, added to BRAF inhibitor and MEK inhibitor, upregulated PARP, BAK, and p-H2AX. When either RARα or RXRα was silenced, the increase in cleaved PARP expression by the addition of TRE to ENC/BIN or ENC/BIN/CET was canceled. Our results suggest that the mechanism of the synergistic antiproliferative effect involves modulation of the Bcl-2 family and the DNA damage response that affects apoptotic pathways, and this synergistic effect is induced by RARα- or RXRα-mediated apoptosis. Tretinoin also enhanced the antitumor effect of a combination of BRAF inhibitor and anti-EGFR antibody with or without MEK inhibitor in a BRAFV600E CRC xenograft mouse model. Our data provide a rationale for developing retinoids as a new combination agent to overcome resistance to the combination therapy for patients with BRAFV600E CRC. Microarray analysis was performed to confirm whether tretinoin induced different gene expression changes depending on the presence or absence of encorafenib/binimetinib. RKO cells were incubated with DMSO, tretinoin, encorafenib/binimetinib, and tretinoin/encorafenib/binimetinib for 24 hours. After incubation, total RNA of the cells was extracted. We compared between the genes differentially expressed by DMSO- and TRE-treated cells, and the ones differentially expressed by ENC/BIN- and TRE/ENC/BIN-treated cells.