The BRAF and MEK Inhibitors Dabrafenib and Trametinib: Effects on Immune Function and in Combination with Immunomodulatory Antibodies Targeting PD-1, PD-L1, and CTLA-4

Li Liu; Patrick A. Mayes; Stephen Eastman; Hong Shi; Sapna Yadavilli; Tianqian Zhang; Jingsong Yang; Laura Seestaller-Wehr; Shu-Yun Zhang; Chris Hopson; Lyuben Tsvetkov; Junping Jing; Shu Zhang; James Smothers; Axel Hoos

doi:10.1158/1078-0432.CCR-14-2339

DOI: 10.1158/1078-0432.CCR-14-2339 Published April 2015

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Figure 1.
Trametinib transiently inhibits T-cell proliferation and reduces activation-induced apoptosis. A, the proliferation of CD4⁺ T cells as measured by CFSE following treatment with increasing concentrations of dabrafenib and trametinib at the indicated time points following addition of compounds. B, caspase-3/7 activity in CD4⁺ T cells following treatment with dabrafenib (370 nmol/L), trametinib (37 nmol/L), or the combination of dabrafenib and trametinib (dabrafenib + trametinib, 370 nmol/L/37 nmol/L) for 24 hours. C, levels of soluble cytokines in the media of CD4⁺ T cells after treatment with dabrafenib (370 nmol/L), trametinib (37 nmol/L), or the combination of dabrafenib and trametinib (370 nmol/L/37 nmol/L) for 72 hours. Drug>Act. signifies the addition of drug 24 hours before addition of CD3/CD28 activation beads. Act.>Drug signifies the addition of drug 24 hours after activation with CD3/CD28 activation beads. Average of six individual donors are shown.
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Figure 2.
Dabrafenib and trametinib differentially changed pERK and expression levels of a subset of genes/proteins, however, showed no/minimal impact on pS6 in human activated T cells in vitro. A, p-ERK and p-S6 levels were measured by MSD in CD4⁺ T cells treated with dabrafenib (300 nmol/L), trametinib (10 nmol/L), or the combination of dabrafenib and trametinib (dabrafenib + trametinib, dabrafenib/trametinib = 300 nmol/L/10 nmol/L) in the absence (Unact.) and presence of anti-CD3/CD28 activation bead for 2 and 24 hours. B, heatmap from representative genes. NanoString nCounter GX Human Immunology v2 Kit was used. Dabrafenib (300 nmol/L), trametinib (10 nmol/L), or dabrafenib + trametinib (300 nmol/L/10 nmol/L) were added concurrently with CD3/CD28 activation beads to CD4⁺ and CD8⁺ T cells for 24 hours. C, time course of T-cell surface marker expression in CD4⁺ T cells following treatment with dabrafenib (100 nmol/L), trametinib (10 nmol/L), or dabrafenib + trametinib (100 nmol/L/10 nmol/L). Drug>Act. signifies the addition of drug 16 hours before activation; Act.>Drug signifies the addition of drug 16 hours after activation. Un, nonactivated T cells; Act, activated T cells.
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Figure 3.
Immunomodulation by dabrafenib and trametinib in A375 BRAF-mutant melanoma cells. A, RT-PCR quantification of PD-L1 and HLA-A in A375 cells treated with trametinib/dabrafenib with and without IFNγ for 48 hours. B, PD-L1 protein expression from flow cytometry and (C) Western blot analyses in A375 parental and dabrafenib acquired resistant cell lines. D, differential gene expression from A375 cells treated with trametinib (10 nmol/L), dabrafenib (300 nmol/L), and the combination of trametinib and dabrafenib (10 nmol/L/300 nmol/L) for 48 hours (P < 0.05; ≥ 2-fold change).
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Figure 4.
Antitumor activity by trametinib alone and/or in combination with immunomodulators targeting PD-1, PD-L1, or CTLA4 in the CT26 murine syngeneic model. A, in vitro cell growth and pERK inhibition by trametinib in CT26 cells. B, in vivo antitumor growth efficacy. Treatments began at day 12 after cell implant. Mice (n = 10 per group) were treated with vehicle (0.5% HPMC, 0.2% Tween-80, pH 7.0) or trametinib at 1 mg/kg orally once daily for 21 days, or with antibodies rat-IgG2a, α-mouse PD-1 (RMP1-14 clone, rat IgG2a), α-mouse PD-L1 (10F.9G2 clone, rat IgG2b), or α-mouse CTLA-4 (9D9 clone, mouse IgG2b) at 10 mg/kg, i.p. twice weekly for 3 weeks. C, tumor growth inhibition by treatment after initial 3 weeks of treatment. D, Kaplan–Meier survival curves of different treatment groups. Treatments began on day 11 after tumor cell implantation, indicated as day 1 of drug treatment for trametinib (MEK-1^st) and α-PD-1 (PD-1-1st), or on day 18 after tumor cell implantation, indicated as day 8 of drug treatment for trametinib (MEK-2nd) and α-PD-1 (PD-1-2nd). Mice (n = 10 per group) were treated with trametinib at 1 mg/kg orally one daily, or with antibodies rat-IgG2a or α-mouse PD-1 (RMP1-14 clone) at 10 mg/kg i.p. twice weekly until tumors reached the endpoint of 2,000 mm³ or by study end.
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Figure 5.
Trametinib alone and/or in combination with anti–PD-1 increased intratumoral CD4⁺ and CD8⁺ T cells in CT26 murine model. A, representative figures of flow cytometry gating and quantification for CD4⁺ and CD8⁺ (top), and Treg (CD25⁺/FoxP3⁺; bottom) in tumors from each treatment group. B, flow cytometry quantification of tumor-infiltrating immune cells (mean ± SEM; n = 3; *, P < 0.05 vs. untreated and IgG2a+vehicle controls). C, representative pERK and total ERK IHC staining tumor sections from each treatment group. D, heatmap generated by clustering of 77 genes with ≥1.5-fold of tumor gene expression changes by any treatment group. Un; untreated; IgG2a, nonspecific isotype control for anti–PD-1; α-PD-1, anti–PD-1 antibody treatment; MEK, trametinib treatment.

Table 1.

In vitro effects on human tumor cell lines

Cell lines	PD-L1 baseline (RT-PCR)		T (IC₅₀, nmol/L) CTG	Gene expression changes by T at 10 nmol/L, 48 h of treatment^a
	Ct	Levels		PD-L1	MAPK (DUSP4/6)	Apoptosis (PIK3IP1, TP53INP1, BCL2L11)	HLA-I (A, B, C)	HLA-II (DMA, DPA, DRA)	IL8	IL1A, IL1B, VEGFA, NTE5	TGFA, EREG, AREG
BRAF-mutant melanoma
YUSIT^c	28	Low	1	^—	^↓	ND	^—	^↓	^↓	ND	ND
12R5-1^c	22	High	366	^↓	^↓	ND	^—	^↑	^↓	ND	ND
A375	24	Moderate	2	^↓	^↓	^↑	^↑	^↑	^↓	^↓	^↓
SK-MEL-24	30	Very low	10	^—	^↓	^↑	^↑	^↑	^↓	^↓	^↓
BRAF WT melanoma
SK-MEL-2	26	Low	4	^—	^↓	^↑	^↑	^↑	^↓	^—	^—
HMVII	29	Low	2	^—	^↓	^↑	^↑	^↑	^↓	^—	^—
CHL-1	31	Very low	>500	^—	^—	^—	^—	^—	^—	^—	^—
KRAS-mutant NSCLC
H358	23	Moderate	29	^↓	^↓	^↑	^—	^—^b	^—	^↓	^↓
Calu6	29	Low	21	^↑	^↓	^↑	^↑	^—^b	^—	^↓	^↓
A549	29	Low	26	^↑	^↓	^↑	^↑	^—^b	^—	^↓	^—

NOTE: Gene expression levels were evaluated by NanoString and/or RT-PCR.

Abbreviations: CTG, CellTiter-Glo assay for cell growth after 3 days of T treatment; ND, not determined.

↵^aCells were treated with trametinib (10 nmol/L) alone in all lines or in combination with dabrafenib (300 nmol/L) in 12R5-1 line for 48 hours.
↵^bData from RT-PCR only.
↵^cExpression increase by trametinib treatment was only detected by RT-PCR, but not NanoString due to below detection range.
↵↓, designated decrease with ≤0.5-fold of control.
↵↑, designated increase with ≥2-fold of control.
↵— designated no change, with less than 2-fold of changes vs. control or below the detection range.

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Supplementary Data

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Supplementary Data - Supplementary Materials and Methods
Supplementary figure 1 - Supplementary figure 1. Tumor growth kinetics in individual mice treated with trametinib (T) in combination with the immunomodulator-targeting PD1 in the CT26 murine syngeneic model.
Supplementary figure 2 - Supplementary figure 2. Tumor gene expression changes measured by RT-PCR from mice treated with trametinib (T) in combination with the immunomodulator-targeting PD1 in the CT26 murine syngeneic model.
Supplementary tables S1-S6 - Supplementary tables S1-S6. Tumor gene expression data