Synchronous BRAF(V600E) and MEK inhibition leads to superior control of murine melanoma by limiting MEK inhibitor induced skin toxicity

Abstract

The BRAF inhibitor (BRAFi) treatment has led to impressive responses in BRAF(V600E) mutation-positive melanomas, but responses are not durable in many patients. As most of the BRAFi escape mechanisms involve ERK reactivation, combinations with MEK inhibitors (MEKi) are currently tested to improve BRAFi-mediated response durations. Additionally, such a combination is expected to reduce MEKi-induced skin toxicities, as these drugs are thought to have antagonistic effects on ERK activation in keratinocytes. However, preclinical in vivo data exploring the combination of BRAFi and MEKi to achieve improved tumor control in the absence of skin toxicities are limited. Using a murine Tyr::CreER(T2);Pten(LoxP/LoxP);Braf(CA/+) melanoma model, we have determined the effect of BRAFi and MEKi treatment and their combination on melanoma control and occurrence of adverse events. We found that the MEKi dosed beyond the maximum tolerable dose (MTD) led to stronger control of tumor growth than did the BRAFi, but mice had to be removed from treatment because of skin toxicity. The combination of BRAFi and MEKi reduced MEKi-associated skin toxicity. This allowed high and long-term dosing of the MEKi, resulting in long-term tumor control. In contrast to previous hypotheses, the addition of a BRAFi did not restore the MEKi-mediated downregulation of pERK1/2 in skin cells. Our data describe, for the first time, the alleviation of MEKi-mediated dose-limiting toxicity by addition of a BRAFi in a mouse melanoma model. Additional clinical Phase I studies should be implemented to explore MEKi dosing beyond the single drug MTD in combination with BRAFi.

Keywords: BRAF; MEK; melanoma; skin toxicity; trametinib; vemurafenib.

Figure 1

Synchronous BRAFi and MEKi allows long-term tumor control in a murine melanoma model. Notes: 4–10 week old Braf^V600E/+/Pten^−/− mice bearing on average 10 mm² melanomas were placed on mock (red line), PLX4720 (blue line), GSK212 (green line), or PLX4720+GSK212 combo treatment (gray line), and tumor size was subsequently followed in two dimensions over time (mm²). The indicated treatments were started either directly (A) or after 53 days of single PLX4720 treatment (B). Three mice were omitted from combo treatment at day 92 (purple line) and two mice continued PLX4720 treatment (orange line). Abbreviations: MEK, mitogen-activated protein kinase kinase; MEKi, MEK inhibitor; BRAFi, BRAF inhibitor; GSK212, GSK1120212.

Figure 2

MEKi-associated skin toxicity can be reduced by addition of BRSFi. Notes: The occurrence of skin toxicity was evaluated in a Kaplan–Meier analysis for the cohort of mice. The arrow indicates the moment at which treatments were switched for the mice. Representative macroscopic examples of skin toxicity observed after 58 days of GSK212 treatment (C). Representative hematoxylin and eosin (H&E) stain of skin from a single GSK212 treated mouse and inflamed skin from a combination PLX4720+GSK212 treated mouse after 26 and 172 days of treatment, respectively (D). Abbreviations: MEK, mitogen-activated protein kinase kinase; MEKi, MEK inhibitor; BRAFi, BRAF inhibitor; GSK212, GSK1120212.

Figure 3

MEKi dose reduction does not achieve reduced skin toxicity without impairing tumor control. Notes: 4–10 week old Braf^V600E/+/Pten^−/− mice bearing on average 10 mm² melanomas were placed on 0.01 (gray line), 0.05 (orange line), 0.1 (blue line), 0.5 (purple line), or 0.75 (green line) mg/kg GSK212 drug treatment. subsequently, the tumor outgrowth was followed over time and compared to mock-treated mice (red line) (A). For all cohorts of mice, the occurrence of skin toxicity was evaluated using a Kaplan–Meier analysis (B). Abbreviations: MEK, mitogen-activated protein kinase kinase; MEKi, MEK inhibitor; BRAFi, BRAF inhibitor; GSK212, GSK1120212.

Figure 4

Skin pERK levels do not correlate with reduced MEKi-induced skin toxicity upon synchronous BRAFi. Notes: Littermate mice of the inducible melanoma model were mock, PLX4720, GSK212, or PLX4720+GSK212 combination treated. Immunohistochemistry was used to detect pERK1/2 (Thr202/Tyr204) in skin samples from treated mice (A). Representative Western blot for pERK1/2 (Thr202/Tyr204) (green bands) and β-actin (red bands) in skin from mice treated for 2 weeks (B). Relative quantification of Western blot analysis of skin from multiple (7–8) individual mice. Expression values are corrected for equal loading and normalized against mock treatment (C). Abbreviations: MEK, mitogen-activated protein kinase kinase; MEKi, MEK inhibitor; BRAFi, BRAF inhibitor; GSK212, GSK1120212.