Comparative profiles of BRAF inhibitors: the paradox index as a predictor of clinical toxicity

Abstract

BRAF inhibitor (BRAFi) therapy is associated with the induction of neoplasia, most commonly cutaneous squamous cell carcinoma (cuSCC). This toxicity is explained in part by "paradoxical ERK activation," or the hyperactivation of ERK signaling by BRAFi in BRAF wild-type cells. However, the rate of cuSCC induction varies widely among BRAFi. To explore this mechanistically, we profiled paradoxical ERK activation by vemurafenib, dabrafenib, encorafenib (LGX818), and PLX8394, demonstrating that vemurafenib induces ERK activation the greatest, while dabrafenib and encorafenib have higher "paradox indices", defined as the pERK activation EC80 divided by the IC80 against A375, corresponding to wider therapeutic windows for achieving tumor inhibition without paradoxical ERK activation. Our results identify differences in the paradox indices of these compounds as a potential mechanism for the differences in cuSCC induction rates and highlight the utility of using ERK activity as a biomarker for maximizing the clinical utility of BRAFi.

Keywords: BRAF; melanoma; paradoxical ERK; small molecule inhibitor; squamous cell carcinoma.

Figure 1. Paradoxical ERK activation profiles and paradox indices correlate with BRAFi clinical toxicities

a–d. MAPK stimulation in immortalized human keratinocytes (HaCaT) stably expressing HRAS^G12V was measured through quantitative western blotting of phosphorylated ERK (pERK), normalized to total ERK levels after 15 minutes of treatment. Vemurafenib strongly simulated pERK, as did dabrafenib and encorafenib at lower levels. PLX8394 did not stimulate pERK. Efficacy in melanoma was measured with cell viability assay conducted at 72 hours and quantitatively compared to pERK induction in the text. e–h. Time-course treatment of HaCaT^HRASG12V at the IC₈₀ against A375 of each compound. Treatment was refreshed at 36 hours. AUC measurements rank pERK induction strength from greatest to least: vemurafenib, dabrafenib, encorafenib, PLX8394. Summary parameters for each compound are compiled in Table 1. (*p<0.05; *** p<0.001; NS = not significant, n≥3 for all data points).

Figure 2. Paradoxical ERK activation and apoptosis suppression profiles are consistent with differences in proliferation

a. Apoptosis 24 hours after 700J/m² UV-irradiation of HaCaT^HRASG12V cells measured with AnnexinV/TMRE FACs. Only vemurafenib treatment induced significant apoptosis suppression. b–c. Colony growth assay after 12 days of continual drug treatment, quantified by total colony area (ImageJ), showed strong growth advantage for vemurafenib and a detectable advantage with encorafenib and dabrafenib versus control. Scale bar on representative images is 5 cm. All statistical tests were two-tailed, unpaired Student's t-tests. Summary parameters for each compound are compiled in Table 1. (*p<0.05; *** p<0.001; NS = not significant, n≥3 for all data points).