Exploring the efficacy and mechanism of action of combined pan-Raf and MEK inhibition in halting the growth of non-V600 BRAF mutated tumors.

3117 Background: Class 2 & 3 non-V600 BRAF mutations mediate RAF dimerization to hyperactivate the MAPK signaling pathway. Encorafenib (Enco; BRAF monomer inhibitor) + Binimetinib (Bini; MEK inhibitor) elicit responses in <15% of patients with non-V600E BRAF mutations (NCT03839342). We hypothesized that Belvarafenib (Belva), a novel pan-RAF dimer inhibitor, is more potent than Enco in inhibiting the growth of Class 2 & 3 non-V600 BRAF mutated tumors. Methods: We performed in vitro colonogenic assays to compare the growth inhibitory effect of 5 independent doses of individual inhibitors (Bini, Belva, or Enco) in parallel to 25 different combinations of either Belva+Bini or Enco+Bini in 6 non-V600 BRAF mutated melanoma (WM3629, HMV-II), colorectal (CRC) (NCI-H508, HT55), and lung (NCI-H1666, NCI-H2087) cancer cells. Low nanomolar doses (10-1000 nM) were used to investigate the synergistic potential of either combination using the SynergyFinder tool. Belva+Bini (15 mg/kg each) and Enco+Bini (75 mg/kg + 15 mg/kg) combinations were assessed in 4 non-V600 BRAF (3 Class 3, 1 Class 2) metastatic CRC patient-derived xenograft (PDX) models. The inhibitory effect of Belva and Enco on MAPK activity in the outlined 6 cell lines was assessed by immunoblotting. Transcriptomic (RNA-Seq) analysis was performed on PDXs. Results: Belva+Bini was 2-6-fold more effective than Enco+Bini in inhibiting the growth of the 6 cell lines. Belva+Bini achieved overall higher synergy scores in the 6 cell lines and was synergistic in 5/6 cell lines (synergy score > 10) vs. Enco+Bini that was synergistic in 1/6 cell lines. In the 6 cell lines, Belva inhibited MAPK activity more robustly than Enco (assessed by pERK levels). In vivo , Belva+Bini was significantly more effective than vehicle or Enco+Bini in halting the growth of 3 out of 4 PDXs. Both Belva+Bini and Enco+Bini significantly inhibited MAPK activity vs. vehicle (as assessed by the transcriptional MAPK Pathway Activity Score). However, there was no statistically significant difference between both combinations. Gene Set Enrichment Analysis revealed that Belva+Bini significantly downregulated genes mediating the interconnected mTORC1 pathway activity and cholesterol metabolism dynamics in the 3 PDXs where Belva+Bini had anti-growth effect. Specifically, among the top downregulated genes by Belva+Bini was PCSK9 , a druggable key regulator of cholesterol metabolism. Conclusions: These results from 10 preclinical models, tested so far, put forward combined Pan-Raf and MEK inhibition as a potential effective treatment choice for patients with non-V600 BRAF mutated tumors to be investigated in clinical trials. In parallel, they unravel novel insights into the mechanism of action of this therapeutic approach and in return the druggable vulnerabilities of the non-V600 BRAF mutated tumors, a notion we are further investigating in the outlined models.