Immunologic Effects of An Oral BRAF Inhibitor in a BRAF Wild-Type Murine Model

Metastatic melanoma represents a clinical challenge as aggressive therapy often results in unacceptable toxicity and less intensive therapy results in suboptimal clinical outcomes. PLX-4032 is an orally available small molecule which targets constitutively activated BRAFV600E in melanoma cells. BRAF is an important regulator of cell growth, proliferation and migration. We examined the effect of PLX-4032 on the immune system in two BRAF wild type murine systems: the first to determine the effect of PLX-4032 on cytokine production and the second to determine any functional immune effect of PLX-4032 in an insulinoma model.

Effect of PLX-4032 on cytokine production: B10.D2 BRAF WT mice were injected with CD8 lymphocytes expressing a TCR specific for HA_518-526 peptide (clone 1) and CD4 lymphocytes expressing a TCR specific for HA_110-119 peptide (SFE) on Day 0 and concurrently simulated with appropriate peptide, incomplete Freund's adjuvant, and poly IC. Mice received PLX-4032 200mg/kg or placebo daily for 5 days. On day 6, splenic lymphocytes were harvested and flow cytometry performed to determine CD4 and CD8 IL2, IFN gamma, and TNF alpha production. Effect of PLX-4032 on functional immune response: B10.D2 BRAF WT rat insulin promoter (RIP)-Tag2-hemagglutinin mice bearing insulinoma were injected with clone 1 and SFE lymphocytes on Day 0 and received concurrent stimulation as above. Mice received PLX-4032 200mg/kg or placebo daily for 30 days and serum glucose levels were monitored weekly.

Effect of PLX-4032 on cytokine production: We noted no significant difference in the absolute number of splenic lymphocytes recovered in the two groups. There was no significant difference in the percent of CD8+ clone 1 cells producing TNF alpha, IL2 or IFN gamma. There was similarly no significant difference in the percent of CD4+/SFE lymphocytes recovered between the treatment and control groups, nor was there a significant difference in the percent of cells producing IFN gamma. Effect of PLX-4032 on functional immune response: Treatment with PLX-4032 resulted in no significant inhibition of the immune response of transferred lymphocytes against insulinoma. Mice in both groups developed significantly elevated serum glucose levels at the same time and remained diabetic for a similar duration.

Our data demonstrate that PLX-4032 does not appear to exert a quantitative or functional effect on the immune system in BRAF WT mice. Melanoma therapies often include medications which augment patient immune function or specifically target immune regulation; these data suggest that PLX-4032 does not inhibit normal immune function and therefore may have a role in combination with immunologically directed therapy, possibly resulting in synergistic anti-tumor effect by targeting multiple pathways involved in tumor survival.

No relevant conflicts of interest to declare.