Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAF(V600E) melanoma

Abstract

Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA4 antibody ipilimumab was terminated early because of substantial liver toxicities. MEK [MAPK (mitogen-activated protein kinase) kinase] inhibitors can potentiate the MAPK inhibition in BRAF mutant cells while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild-type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAF(V600E)-driven melanoma, SM1, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors, and improved in vivo cytotoxicity. Single-agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and major histocompatibility complex (MHC) expression and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested the combination of dabrafenib, trametinib, and anti-PD1 therapy in SM1 tumors, and observed superior antitumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAF(V600E) mutant metastatic melanoma.

Fig. 1. Enhanced in vivo antitumor activity with pmel-1 adoptive cell transfer (ACT) plus dabrafenib (D) and/or trametinib (T)

(A) Western blot analysis of MAPK pathway. SM1 cells were treated with serial dilutions of D, T, or D+T for 1 and 24 hours. L: low dose (D 0.1μM/T 0.005μM). M: medium dose (D 5μM/T 0.25μM). H: high dose (D 20μM/T 1μM). (B) In vivo tumour growth curves with 4 mice in each group (mean+/−SD). SM1 bearing C57BL/6 mice were treated with D 30mg/kg, T 0.15mg/kg, or the combination via oral gavage daily, started when tumours were 3-5mm. (C) Schema of pmel-1 ACT model. C57BL/6 mice had myeloid-depleting total body irradiation (TBI) followed by bone marrow transplant (BMT) and SM1 tumour injections. When tumours reached 3mm, three million gp100_25-33 peptide activated pmel-1 splenocytes (pmel-1 transgenic mice carrying T cell receptor specific for melanoma antigen gp100) were injected. Wild type C57 BL/6 mouse splenocytes activated by CD3 and CD28 were mock ACT controls. Both ACT were followed by high dose IL-2 for 3 days. Daily oral gavage of vehicle control (V), D 30mg/kg, T 0.6mg/kg, or the combination were started on the day of ACT. (D) In vivo SM1 tumour growth curves with 3-4 mice in each group (mean+/−SD), after D, T and ACT treatments. P < 0.0001 by unpaired t test on day 30, pmel-1 ACT+D+T vs pmel-1 ACT +T, or vs mock ACT +D+T.

Fig. 2. Increased tumor infiltrating lymphocytes (TILs) with pmel-1 ACT plus dabrafenib and/or trametinib in SM1 tumors

(A) Quantification of TILs. Splenocytes and TILs harvested on day 5 after ACT were counted and analysed by flow cytometry for Thy1.1/CD3/CD8 staining (3 mice in each group, mean+/−SD). Percentage of effectors (CD3+CD8+ or CD3+Thy1.1+) were shown to be statistically significantly changed by unpaired t test in several subgroups (CD3+CD8+ TILs: p=0.049 pmel D vs pmel V, p=0.02 pmel T vs pmel V, p=0.004 pmel D+T vs pmel V, p=0.035 pmel D+T vs pmel T; CD3+Thy1.1+: p=0.03 pmel D vs pmel V, p=0.02 pmel T vs pmel V, p=0.006 pmel D+T vs pmel V, p=0.047 pmel D+T vs pmel T). (B) Representative flow data of percentage of CD3+Thy1.1+ TILs is shown. (C) In vivo bioluminescent imaging (BLI) of adoptively transferred lymphocytes. Pmel-1 transgenic T cells were transduced with a retrovirus-firefly luciferase and used for ACT. Representative figure on day 5 depict 4 replicate mice per group. (D) Quantification of BLI of serial images with region of interest (ROI) analysis at the site of tumours (counts/pixel) obtained through day 18 post-ACT of luciferase expressing pmel-1 T-cells (4 mice per group, mean+/−SD). On Day 5, p=0.0009 pmel D vs pmel V, p<0.0001 pmel T vs pmel V, p<0.0001 pmel D+T vs pmel V, p=0.01 pmel T or pmel D+T vs pmel D (unpaired t test, n=4).

Fig. 3. Dabrafenib, trametinib or combination impairs effector T cell function in vitro but not in vivo

(A) In vitro study of cytokine-producing function of effector cells. Gp100_25-33-activated pmel-1 mouse splenocytes were treated at serial dilutions of D, T, or D+T for 72 hours. L: low dose (D 0.1μM/T 0.005μM). M: medium dose (D 5μM/T 0.25μM). H: high dose (D 20μM/T 1μM). Cells were analysed by FACS for CD3/CD8/IFN-γ staining. Bar graph of percentage of IFN-γ expressing CD3+ CD8+ cells are shown (mean+/−SD). p=0.002 DM or DH vs DL, p=0.045 DH vs DM, p=0.003 TM or TH vs TL, p=0.0002 D+T M or D+T H vs D+T L (unpaired t test, n=3). (B) In vivo effect on cytokine production upon antigen re-stimulation. SM1 tumour-bearing C57BL/6 mice received pmel-1 ACT with or without D and T. On day 5 post-ACT, spleens and TILs were isolated for intracellular IFN-γ staining analysed by FACS after 5-hour ex vivo exposure to the gp100_25-33 peptide. Percentage of IFN-γ expressing CD3+Thy1.1 cells in the spleen and tumor was normalized to Pmel + V (mean+/−SD). (C) Gating strategy and representative flow data is shown. (D) Schema of the in vivo cytotoxic T cell assay. C57BL/6 mice received ACT of 5 × 10⁴ pmel-1 splenocytes and daily D, T, D+T or vehicle via oral gavage. On day 5, mice received an intravenous challenge with CFSE-labelled target cells (splenocytes pulsed with gp100_25-33 peptide or control OVA peptide). Gp100_25-33 pulsed targets were pulsed with 10 times more concentrated of CFSE than OVA pulsed cells. Ten hours later, splenocytes were harvested and analysed by FACS. (E) Bar graph representation of the in vivo cytotoxicity study result (mean+/−SD). p=0.01 pmel V vs mock V (34% down, unpaired t test, n=3). (F). Representative flow data is shown.

Fig. 4. Dabrafenib and trametinib changed the cellular components of the tumour microenvironment

On day 5 post-ACT, spleens and tumours were isolated and stained with fluorescent-labelled antibodies, analysed by FACS with 3 mice in each group (mean+/−SD). (A) MO-MDSC (CD11b+Ly6C^Hi Ly6G^Lo) presented as percentage of CD11b+ cells. * p=0.06 pmel D vs pmel V in spleen, p=0.009 pmel V vs Mock V in tumor (unpaired t test, n=3). (B) PMNMDSC (CD11b+Ly6C^LowLy6G^Hi) presented as percentage of CD11b+ cells. * p=0.002 mock D+T vs mock V in tumor (unpaired t test, n=3). (C) Analysis of macrophages (F4/80+CD11b+). * p=0.04 pmel D vs pmel V, p=0.002 pmel D+T vs pmel V, both in tumors (unpaired t test, n=3). (D) Analysis of T regulatory cells (Tregs, CD4+CD25+FOXp3+). * P=0.002 pmel D vs pmel V in tumors (unpaired t test, n=3). (E) Gating strategy and representative FACS plots in tumours.

Fig. 5. Microarray analysis of tumors treated by dabrafenib, trametinib, or combination of dabrafenib and trametinib combined with pmel-1 ACT or mock ACT

On day 5 post-ACT, tumours were isolated and snap frozen immediately (two to three mice in each group). RNA isolation was done after all samples were collected. (A) Principal component analysis of gene expression profile of the tested samples. (B) Clustering of immune-related genes with ANOVA filter p<0.05. Gene names in individual clusters are listed in supplemental tables 1-3. (C) Clustering of chemokines and their receptors. (D) Clustering of MDAs and MHC class I and II molecules.

Fig. 6. Up-regulation of PD-L1 and triple combination of dabrafenib, trametinib with PD-1 blockade is superior in antitumor effect against SM1

(A) Heat map representation of CD8, granzyme B, IFNγ, PD-1, PD-L1 gene expression from microarray data (PD-L1: p=0.01 mock D+T vs mock V, p=0.004 pmel T vs pmel V, p=0.004 pmel D+T vs pmel V, p=0.03 pmel T vs pmel D+T, unpaired t test, n=3). (B) Percentage of PD-L1-expressing cells in the spleen and tumours 5 days after ACT and drug treatments started, 3 mice in each group (mean+/−SD). P=0.006 mock D+T vs mock V, p=0.04 pmel D vs pmel V, p=0.007 pmel T vs pmel V, p=0.001 pmel D+T vs pmel V. (C) Expression of PD-L1 on SM1 after 18 hours of IFNγ stimulation at different concentrations. B16 cells served as positive control. (D) In vivo SM1 tumour growth curves after D, T and anti-PD1 treatments, 4 mice in each group (mean+/−SD). SM1 tumour bearing C57 BL/6 mice received anti-PD1 (Merck DX400) 200μg via intraperitoneal injection every 4 days, started when tumours reaches 3-5mm. Daily oral gavage of vehicle control (V), D 30mg/kg, T 0.6mg/kg, or the combination were started on the same day as anti-PD1.