Research ArticleCancer

Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma

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Science Translational Medicine  18 Mar 2015:
Vol. 7, Issue 279, pp. 279ra41
DOI: 10.1126/scitranslmed.aaa4691
  • Fig. 1. Enhanced in vivo antitumor activity with pmel-1 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 (0.1 μM)/T (0.005 μM)]. M, medium dose [D (5 μM)/T (0.25 μM)]; H, high dose [D (20 μM)/T (1 μM)]. (B) In vivo tumor growth curves with four mice in each group (mean ± SD). SM1-bearing C57BL/6 mice were treated with D (30 mg/kg), T (0.15 mg/kg), or the combination via daily oral gavage, starting when tumors were 3 to 5 mm. (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 tumor injections. When tumors reached 3 mm, 3 million gp10025-33 peptide–activated pmel-1 splenocytes (pmel-1 transgenic mice carrying TCR specific for melanoma antigen gp100) were injected. Wild-type C57BL/6 mouse splenocytes activated by CD3 and CD28 were mock ACT controls. Both ACTs were followed by high-dose IL-2 for 3 days. Daily oral gavage of vehicle control (V), D (30 mg/kg), T (0.6 mg/kg), or the combination was started on the day of ACT. i.v., intravenously; s.c., subcutaneously; i.p., intraperitoneally. (D) In vivo SM1 tumor growth curves with three to four 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 versus pmel-1 ACT + T, or versus 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 analyzed by flow cytometry for Thy1.1/CD3/CD8 staining (three mice in each group, mean ± SD). Percentage of effectors (CD3+CD8+ or CD3+Thy1.1+) was shown to be statistically significantly changed by unpaired t test in several subgroups (CD3+CD8+ TILs: P = 0.049, pmel D versus pmel V; P = 0.02, pmel T versus pmel V; P = 0.004, pmel D + T versus pmel V; P = 0.035, pmel D + T versus pmel T; CD3+Thy1.1+: P = 0.03, pmel D versus pmel V; P = 0.02, pmel T versus pmel V; P = 0.006, pmel D + T versus pmel V; P = 0.047, pmel D + T versus pmel T). (B) Representative flow data of percentage of CD3+Thy1.1+ TILs are shown. (C) In vivo bioluminescence 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 depicts four replicate mice per group. (D) Quantification of BLI of serial images with region of interest (ROI) analysis at the site of tumors (counts per pixel) obtained through day 18 after ACT of luciferase-expressing pmel-1 T cells (four mice per group, mean ± SD). On day 5, P = 0.0009, pmel D versus pmel V; P < 0.0001, pmel T versus pmel V; P < 0.0001, pmel D + T versus pmel V; P = 0.01, pmel T or pmel D + T versus pmel D (unpaired t test, n = 4).

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

    (A) In vitro study of cytokine-producing function of effector cells. Gp10025-33–activated pmel-1 mouse splenocytes were treated at serial dilutions of D, T, or D + T for 72 hours. L, low dose [D (0.1 μM)/T (0.005 μM)]; M, medium dose [D (5 μM)/T (0.25 μM)]; H, high dose [D (20 μM)/T (1 μM)]. Cells were analyzed by fluorescence-activated cell sorting (FACS) for CD3/CD8/IFN-γ staining. Bar graphs of percentage of IFN-γ expressing CD3+CD8+ cells are shown (mean ± SD). P = 0.002, D M or D H versus D L; P = 0.045, D H versus D M; P = 0.003, T M or T H versus T L; P = 0.0002, D + T M or D + T H versus D + T L (unpaired t test, n = 3). (B) In vivo effect on cytokine production upon antigen restimulation. SM1 tumor–bearing C57BL/6 mice received pmel-1 ACT with or without D and T. On day 5 after ACT, spleens and TILs were isolated for intracellular IFN-γ staining analyzed by FACS after 5-hour ex vivo exposure to the gp10025-33 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 are 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 (carboxyfluorescein diacetate succinimidyl ester)–labeled target cells (splenocytes pulsed with gp10025-33 peptide or control OVA peptide). Gp10025-33–pulsed targets were pulsed with 10 times more concentration of CFSE- than OVA-pulsed cells. Ten hours later, splenocytes were harvested and analyzed by FACS. (E) Bar graph representation of the in vivo cytotoxicity study result (mean ± SD). P = 0.01, pmel V versus mock V (34% down, unpaired t test, n = 3). (F). Representative flow data are shown.

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

    On day 5 after ACT, spleens and tumors were isolated and stained with fluorescent-labeled antibodies and then analyzed by FACS, with three mice in each group (mean ± SD). (A) MO-MDSC (CD11b+Ly6CHiLy6GLo) presented as percentage of CD11b+ cells. *P = 0.06, pmel D versus pmel V in spleen; P = 0.009, pmel V versus mock V in tumor (unpaired t test, n = 3). (B) PMN-MDSC (CD11b+Ly6CLowLy6GHi) presented as percentage of CD11b+ cells. *P = 0.002, mock D + T versus mock V in tumor (unpaired t test, n = 3). (C) Analysis of macrophages (F4/80+CD11b+). *P = 0.04, pmel D versus pmel V; P = 0.002, pmel D + T versus pmel V, both in tumors (unpaired t test, n = 3). (D) Analysis of Tregs (CD4+CD25+FOXp3+). *P = 0.002, pmel D versus pmel V in tumors (unpaired t test, n = 3). (E) Gating strategy and representative FACS plots in tumors.

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

    On day 5 after ACT, tumors were isolated and snap-frozen immediately (two to three mice in each group). RNA isolation was done after all samples were collected. (A) PCA of gene expression profile of the tested samples. (B) Clustering of immune-related genes with analysis of variance (ANOVA) filter, P < 0.05. Gene names in individual clusters are listed in tables S1 to S3. (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, and PD1 blockade is superior in antitumor effect against SM1.

    (A) Heat map representation of CD8, granzyme B, IFN-γ, PD1, and PD-L1 gene expression from microarray data (PD-L1: P = 0.01, mock D + T versus mock V; P = 0.004, pmel T versus pmel V; P = 0.004, pmel D + T versus pmel V; P = 0.03, pmel T versus pmel D + T; unpaired t test, n = 3). (B) Percentage of PD-L1–expressing cells in the spleen and tumors 5 days after ACT and drug treatments started; three mice in each group (mean ± SD). P = 0.006, mock D + T versus mock V; P = 0.04, pmel D versus pmel V; P = 0.007, pmel T versus pmel V; P = 0.001, pmel D + T versus pmel V. (C) Expression of PD-L1 on SM1 after 18 hours of stimulation with IFN-γ at different concentrations. B16 cells served as a positive control. (D) In vivo SM1 tumor growth curves after D, T, and anti-PD1 treatments; four mice in each group (mean ± SD). SM1 tumor–bearing C57BL/6 mice received anti-PD1 (Merck DX400; 200 μg) via intraperitoneal injection every 4 days, starting when tumors reached 3 to 5 mm. Daily oral gavage of vehicle control (V), D (30 mg/kg), T (0.6 mg/kg), or the combination was started on the same day as anti-PD1.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/279/279ra41/DC1

    Fig. S1. In vitro study of effects of dabrafenib and trametinib on effector T cell and gating strategies.

    Fig. S2. Microarray analysis quality control, all gene clustering, and expression of interested genes.

    Fig. S3. Source data of Western blots in Fig. 1A.

    Table S1. Immune A genes.

    Table S2. Immune B genes.

    Table S3. Immune C genes.

    Table S4. Source data of tumor growth curves in Figs. 1B, 1D, and 6D.

  • Supplementary Material for:

    Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma

    Siwen Hu-Lieskovan, Stephen Mok, Blanca Homet Moreno, Jennifer Tsoi, Lidia Robert, Lucas Goedert, Elaine M. Pinheiro, Richard C. Koya, Thomas G. Graeber, Begoña Comin-Anduix, Antoni Ribas*

    *Corresponding author. E-mail: aribas{at}mednet.ucla.edu

    Published 18 March 2015, Sci. Transl. Med. 7, 279ra41 (2015)
    DOI: 10.1126/scitranslmed.aaa4691

    This PDF file includes:

    • Fig. S1. In vitro study of effects of dabrafenib and trametinib on effector T cell and gating strategies.
    • Fig. S2. Microarray analysis quality control, all gene clustering, and expression of interested genes.
    • Fig. S3. Source data of Western blots in Fig. 1A.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Immune A genes.
    • Table S2 (Microsoft Excel format). Immune B genes.
    • Table S3 (Microsoft Excel format). Immune C genes.
    • Table S4 (Microsoft Excel format). Source data of tumor growth curves in Figs. 1B, 1D, and 6D.

    [Download Tables S1 to S4]

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