The BRAF and MEK Inhibitors Dabrafenib and Trametinib

Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2339

Cancer Therapy: Preclinical Clinical Cancer Research 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 Liu1, Patrick A. Mayes1, Stephen Eastman1, Hong Shi1, Sapna Yadavilli1, Tianqian Zhang1, Jingsong Yang1, Laura Seestaller-Wehr1, Shu-Yun Zhang1,Chris Hopson1, Lyuben Tsvetkov1, Junping Jing2, Shu Zhang3, James Smothers1, and Axel Hoos1

Abstract

Purpose: To assess the immunologic effects of dabrafenib and suppressive factors such as PD-L1, IL1, IL8, NT5E, and VEGFA. PD- trametinib in vitro and to test whether trametinib potentiates or L1 expression in tumor cells was upregulated after acquiring antagonizes the activity of immunomodulatory antibodies in vivo. resistance to BRAF inhibition in vitro. Combinations of trametinib Experimental Design: Immune effects of dabrafenib and tra- with immunomodulators targeting PD-1, PD-L1, or CTLA-4 in a þ þ metinib were evaluated in human CD4 and CD8 T cells from CT26 model were more efficacious than any single agent. The healthy volunteers, a panel of human tumor cell lines, and in vivo combination of trametinib with anti–PD-1 increased tumor-infil- þ using a CT26 mouse model. trating CD8 T cells in CT26 tumors. Concurrent or phased Results: Dabrafenib enhanced pERK expression levels and did sequential treatment, defined as trametinib lead-in followed by þ þ not suppress human CD4 or CD8 T-cell function. Trametinib trametinib plus anti–PD-1 antibody, demonstrated superior effi- reduced pERK levels, and resulted in partial/transient inhibition cacy compared with anti–PD-1 antibody followed by anti–PD-1 of T-cell proliferation/expression of a cytokine and immunomod- plus trametinib. ulatory gene subset, which is context dependent. Trametinib Conclusion: These findings support the potential for synergy effects were partially offset by adding dabrafenib. Dabrafenib between targeted therapies dabrafenib and trametinib and and trametinib in BRAF V600E/K, and trametinib in BRAF immunomodulatory antibodies. Clinical exploration of such wild-type tumor cells induced apoptosis markers, upregulated combination regimens is under way. Clin Cancer Res; 21(7); 1639–51. HLA molecule expression, and downregulated certain immuno- 2015 AACR.

Introduction ticulin (CRT), HSP70 and 90 proteins, HMGB1, and ATP, increased expression of tumor antigens and HLA molecules, and Immunotherapies and targeted therapies have distinctly differ- decreased expression of immunosuppression factors are desirable ent mechanisms of action and have both been shown to be features for potential immune sensitization (3, 4). These effects efficacious in patients with advanced cancers (1, 2). It is expected may allow targeted agents to not only directly inhibit tumor that combinations of both modalities may create synergies with growth, but also further enhance immune response by immuno- increased benefit for patients with cancer. To enable such combi- therapy, through either tumor cell intrinsic or extrinsic immuno- nations, it is critical to determine how targeted therapies affect modulatory mechanisms, thus making the cancer therapy more immune function in the tumor microenvironment and peripheral effective and durable. systems. Immunogenic cell death, characterized by secretion of Inhibition of oncogenic MAPK signaling by dabrafenib, tra- cell damage–associated hallmark molecules consisting of calre- metinib, or the combination of dabrafenib and trametinib has been an effective strategy and approved for the treatment of metastatic melanoma tumors bearing BRAF V600E and V600K 1Immuno-Oncology and Combination DPU, GlaxoSmithKline, mutations (5). The first generation of checkpoint immunomod- 2 Collegeville, Pennsylvania. Molecular Medicine Unit, Oncology R&D, ulatory antibodies targeting either CTLA-4 or PD-L1/PD-1 has GlaxoSmithKline, Collegeville, Pennsylvania. 3Statistical Science, GlaxoSmithKline, Collegeville, Pennsylvania. demonstrated impressive clinical activity resulting in durable responses in subsets of patients with various cancer types Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). (1, 6, 7). BRAF inhibitors, such as vemurafenib, have been shown to increase immune response and efficacy in combination with L. Liu and P.A. Mayes contributed equally to this article. immunomodulators in preclinical models (8, 9). However, MEK Corresponding Author: Axel Hoos, Immuno-Oncology and Combination DPU, inhibitors, including trametinib, have been reported to be immu- GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426. Phone: nosuppressive in vitro, which has limited the in vivo assessment of 610-427-3733; E-mail: [email protected] MEK inhibitor combinations with immunotherapies (10). In this doi: 10.1158/1078-0432.CCR-14-2339 study, we assessed the immunologic effects of dabrafenib and 2015 American Association for Cancer Research. trametinib at clinically relevant exposures on both immune and

www.aacrjournals.org 1639

Liu et al.

protocol. Human whole blood was obtained from GlaxoSmithK- Translational Relevance line's blood donation unit (Upper Providence site, PA) under the Combining MAPK pathway inhibitors, such as dabrafenib Institutional Review Board (IRB) approval. and trametinib, with immunomodulatory antibodies target- In vitro T-cell assays ing CTLA-4 or PD-L1/PD-1 is of high interest in clinical þ þ development. However, comprehensive in vitro/in vivo preclin- Human CD4 and CD8 cells were activated with anti-CD3/ ical translational studies for these agents, especially MEK anti-CD28 antibodies either in bead- or plate-bound forms. inhibitors, such as trametinib, their mechanisms of action Trametinib and dabrafenib were added at same time or sequen- around immunomodulation, and their impact on combina- tially with activation. T-cell proliferation, cytokine secretion, and tion sequences are lacking. In this study, we assessed the apoptosis induction, cell signaling, surface markers, and gene immunologic effects of dabrafenib and trametinib at clinically expression levels were measured. Protocols are described in the relevant exposures on both immune and tumor cells in vitro, Supplementary Methods. fi and tested antitumor ef cacy and/or pharmacodynamic mar- Human and mouse tumor cell assays kers for trametinib alone, and in combination with several The expression levels of immunomodulators, HLA molecules, immunomodulatory antibodies, in a CT26 immunocompe- and tumor-associated antigens from tumor cells were determined tent syngeneic mouse model. Our in vivo data demonstrate the by NanoString, RT-PCR, flow cytometry, and/or Western blot fi – superior ef cacy by the combination of trametinib with anti analyses. Procedures, antibodies, and reagents are described in fi PD-1 antibody concurrently or sequentially phased, when rst the Supplementary Methods. treated with trametinib followed by trametinib plus anti–PD-1 antibody. These findings support clinical exploration of both In vivo evaluation in CT26 murine carcinoma syngeneic mouse trametinib and dabrafenib in combination with specific model immunomodulatory antibodies. Female BALB/C mice (Charles River) received food and water ad libitum and were housed in GlaxoSmithKline in compliance with the recommendations of the Guide for Care and Use of Laboratory Animals. Tumors were established by subcutaneously tumor cells in vitro. The effect on immune cells by dabrafenib was implanting 5 104 CT26 cells in suspension into the right consistent with the literature reports for BRAF inhibitors (10, 11). flank of mice. Tumor weights were calculated using the equation However, we found that the effect of trametinib on immune cells (l w2)/2, where l and w refer to the larger and smaller dimensions was both complex and context dependent. The antiproliferative collected at each measurement. Treatments began at day 11 or effect of trametinib on T cells was partial and transient in vitro. 12 with tumor size 40 to 100 mm3. Tumors were monitored and Furthermore, we tested trametinib alone and in combination with mice were euthanized when an endpoint was reached, defined as several immunomodulatory antibodies in an immunocompetent tumor volume greater than 2,000 mm3, tumor ulceration, or study syngeneic mouse model. Our in vivo data demonstrate that tra- end (21 or 68 days after initial dosing), whichever came first. metinib had minimal inhibitory effects on circulating immune Tumor regressions, median tumor volume, and treatment toler- þ cells, and enhanced efficacy and/or tumor infiltration of CD8 ability were also considered. effective cells. These findings support clinical exploration of both Percentage tumor growth inhibition (% TGI) was defined as the trametinib and dabrafenib in combination with specific immu- difference between the mean tumor volume (MTV) of the desig- nomodulatory antibodies. nated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the designated control ¼ Materials and Methods group:% TGI [1 (MTVdrug-treated/MTVcontrol)] 100. The Kaplan–Meier method was carried out to estimate the survival Cell lines and reagents probability of different treatment groups at a given time. The Human melanoma cell line, A375PF11, was derived from a median time to endpoint (TTE) and its corresponding 95% clonal isolate of the A375 cell line obtained from the American confidence interval (CI) were calculated. Type Culture Collection (ATCC). Human melanoma line YUSIT1 For pharmacodynamic analysis, fresh tumors, lymph nodes, was obtained from Yale Dermatology Cell Culture Facility (12). spleen, serum, and whole blood were collected 4 and 24 hours Human melanoma lines: SK-MEL-24, CHL-1, HMVII, and SK- after last dose on days 7 or 8. Flow cytometric analysis of MEL-2; human non–small cell lung cancer (NSCLC) lines: Calu6, lymphocytes from mouse blood, tumor tissues, lymph nodes, A549, and H358; and mouse colon carcinoma line, CT26, were and spleens, cytokine analysis from serum, and gene expression obtained from the ATCC and cultured in RPMI with 10% fetal and immunohistochemistry (IHC) analyses from tumor tissues bovine serum (FBS) media. All cell lines were characterized by are described in the Supplementary Methods. genotypic and RNA expression analyses using the Affymetrix 500K Statistical analysis of the results was performed by contrast SNP chip and HG-U133Plus2 chip, respectively (Affymetrix, Inc.) analysis following one-way ANOVA, and described in the Sup- for human lines, and using Exome Seq and RNA-Seq (Illumina) plementary Methods. for CT26, and kept in culture for <3 months. 12R5-1, 12R5-3, 16R6-3, 16R5-5, and 16R6-4 were dabrafenib-resistant clones derived from A375PF11 (referred to henceforth as A375; ref. 13). Results þ þ Human CD4 and CD8 T cells were isolated from whole Trametinib, but not dabrafenib, partially and transiently blood using STEMCELL Technologies RosetteSep Human T Cell inhibits T-cell proliferation and cytokine production in vitro þ þ Enrichment Cocktails (STEMCELL Technologies) and Lympho- We used CD4 and CD8 cells isolated from healthy volun- Prep density gradient medium according to the manufacturer's teers to assess whether dabrafenib and trametinib could affect

1640 Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research

Dabrafenib and Trametinib Effect on Immune Function

T-cell proliferation and function in vitro. T cells were activated proliferation, apoptosis, or cytokine production of activated þ þ with anti-CD3/anti-CD28 antibodies coated either on beads or CD4 and CD8 T cells (Fig. 1). Unlike dabrafenib, trametinib þ plates. Trametinib and dabrafenib were added either simulta- alone resulted in partial inhibition of CD4 T-cell proliferation þ neously or sequentially with activation. At clinically relevant after 3 days, but not after 7 days of treatment if CD4 T cells exposures, both dabrafenib and trametinib had little to no were dosed with compound 24 hours before activation (Fig. 1A, effect on na€ve T cells (data not shown). At clinically relevant Drug>Act., top). At 7 days, partial growth inhibition was concentrations, dabrafenib alone did not signiﬁcantly inhibit apparent only at trametinib concentrations greater than 100

Un. Act. D T D+T A C Time = 72 hours Time = 168 hours 2.5 2.5 150,000 25,000

2.0 2.0 20,000 100,000 15,000 1.5 1.5 10,000 1.0 1.0 50,000

Drug>Act. 5,000 0.5 0.5 0 0 0.0 0.0 Drug>Act. Act.>Drug Drug>Act. Act.>Drug γ 111 IFN IL2

111/0. 0.014/0.0010.123/0.0121.111/0.111 0.014/0.0010.123/0.0121. 15,000 8,000 CONTROLS0.002/0.0002 10.000/1.000 CONTROLS0.002/0.0002 10.000/1.000

6,000 10,000

Average cell division 4 4 4,000

3 3 5,000 2,000

2 2 0 0 Drug>Act. Act.>Drug Drug>Act. Act.>Drug Act.>Drug 1 1 TFNα IL8

0 1,500 3,000 0 Concentration (pg/mL)

111/0.111 1,000 2,000 0.014/0.0010.123/0.0121.111/0.111 0.014/0.0010.123/0.0121. CONTROLS0.002/0.0002 10.000/1.000 CONTROLS0.002/0.0002 10.000/1.000 m D+T ( mol/L) 500 1,000

0 0 B Drug>Act. Act.>Drug Drug>Act. Act.>Drug Drug>Act. Act.>Drug IL4 IL10 10,000 300,000 800 6,000

8,000 600 200,000 4,000 6,000 400

(RLU) 4,000 100,000 2,000 200 2,000 Caspase-3/7 GLO 0 0 0 0 TD+TDAct. TD+TDAct. Drug>Act. Act.>Drug Drug>Act. Act.>Drug IL5 IL13

Figure 1. Trametinib transiently inhibits T-cell proliferation and reduces activation-induced apoptosis. A, the proliferation of CD4þ 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 CD4þ T cells following treatment with dabrafenib (370 nmol/L), trametinib (37 nmol/L), or the combination of dabrafenib and trametinib (dabrafenib þ trametinib, 370 nmol/L/37 nmol/L) for 24 hours. C, levels of soluble cytokines in the media of CD4þ 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.

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1641

Liu et al.

þ þ nmol/L, which is above clinical exposure levels. The partial little to no effect on activated CD4 and CD8 T cells. No þ inhibitory effect was not observed if CD4 T cells were activated apoptosis genes, including CASP1, CASP8, BCL2, and TNFSF10 before adding trametinib (Fig. 1A, Act.>Drug, bottom). In (TRAIL), were modulated by trametinib and dabrafenib alone and addition, treatment with trametinib alone (Drug>Act. and in combination. Other immunomodulatory genes associated Act.>Drug) and in combination with dabrafenib (Act.>Drug) with activated T cells [FOXP3, CD274 (PD-L1), TNFRSF4 (OX40), for 24 hours resulted in decreased activation-induced apoptosis ICOS, CTLA4, TNFRSF9 (4-1BB), CD25, and IFNG] were not (AID) measured by caspase-3/7 activity following T-cell acti- affected substantially by dabrafenib and trametinib (< 2-fold) in þ vation (Fig. 1B). This was despite similar levels of proliferation CD4 cells. However, OX40, ICOS, CTLA-4, 4-1BB, and IFNg in T- and dabrafenib/trametinib–treated cells at the time and were partially reduced by trametinib alone and in combination þ concentration tested. The effects of trametinib on cytokine with dabrafenib (2-fold) in CD8 cells. Both dabrafenib and þ þ production were variable depending on the cytokine analyzed, trametinib had little-to-no effect on na€ve CD4 and CD8 T cells resulting in little to no change (IFNg, IL5, and IL10), or partial (data not shown). inhibition of some cytokines (IL2, TNFa, and IL8), while Multicolor flow cytometry confirmed cell-surface expression þ inducing expression of others (IL4) when it was added before changes of CD69, CD25, PD-1, OX40, and CTLA-4 in CD4 and þ (Fig. 1B, Drug>Act.) or simultaneously (data not shown) with CD8 cells (Fig. 2C and data not shown). Dabrafenib effects T-cell activators. Observed cell growth inhibition and cytokine were similar to vehicle control–treated samples. Treatment with þ changes by trametinib were transient and minimized if CD4 T trametinib decreased the expression of CD25, CD69, OX40, þ þ þ cells were activated first (Fig. 1, Act.>Drug). In the setting of and PD-1 in CD4 and CD8 T cells, and CTLA-4 in CD4 trametinib and dabrafenib combination, the effects of trame- T cells only. However, the expression levels of CD69 and OX40 tinib appeared to dominate but were partially offset by dab- in trametinib-treated cells were still well above nonactivated rafenib in some instances. Similar results were also observed in T cells. Combining dabrafenib with trametinib partially offset þ CD8 cells (data not shown). the inhibitory effects seen with trametinib alone (Fig. 2C and Supplementary Tables S3A and S3B). Dabrafenib and trametinib differentially affect the expression levels of pERK and a subset of genes/proteins in human Dabrafenib and trametinib alone and in combination reduce activated T cells in vitro the expression of tumor suppression factors and increase the Cell signaling critical to MAPK–PI3K–mTOR pathways was expression of HLA-class I molecules and tumor antigens in þ þ measured in CD4 and CD8 cells and representative data are BRAF V600–mutant melanoma cell lines shown in Fig. 2A. Dabrafenib alone enhanced pERK expression To determine how MAPK pathway inhibition affects the expres- levels, an observation consistent with previously reported para- sion levels of immunoregulatory genes/proteins in BRAF V600E doxical effects of BRAF inhibitors in BRAF wild-type (WT) cells or V600K–mutant melanoma cells, we first treated A375 mela- (14); with no observed changes in pAKT and pS6 protein levels noma cells with dabrafenib and trametinib either alone or in (Fig. 2A and data not shown). In contrast, trametinib alone combination with the absence and presence of IFNg. As shown reduced pERK levels, but not pAKT and pS6 expression levels, as in Fig. 3A, INFg was able to induce both PD-L1 and HLA-A compared with controls (Fig. 2A and data not shown). expression in A375 cells. Dabrafenib and trametinib (either alone Furthermore, immune gene expression profiling using the or in combination) decreased PD-L1 and increased HLA-A regard- NanoString nCounter gene expression system demonstrated less of IFNg exposure. Interestingly, the inhibition of PD-L1 by þ þ unique gene signatures associated with both CD4 and CD8 dabrafenib, trametinib, and dabrafenib þ trametinib was tran- T-cell activation by anti-CD3/CD28 antibodies (Fig. 2B). We sient in vitro, and did not track with the activation status of identified genes with expression levels changed equal to or greater the pathway over time. In A375 cells treated with dabrafenib, than 3-fold upon activation. Of the 525 genes in the panel, in trametinib, or dabrafenib þ trametinib over a 30-day time course, þ CD4 T cells, 88 genes (17%) were upregulated and 55 genes PD-L1 mRNA levels increased steadily out to day 30 after an initial þ (10%) were downregulated; however, in CD8 T cells, 39 genes reduction through day 8 (Supplementary Fig. S3A). In contrast, (7%) were upregulated and 88 genes (17%) were downregulated. DUSP6 mRNA levels, a reliable surrogate of MAPK activation, Interestingly, more genes were downregulated in CD8 cells upon remained low throughout the 30-day time course (Supplemen- activation, whereas more genes were upregulated in CD4 cells. tary Fig. S3B), indicating an uncoupling of PD-L1 expression and Ontology enrichment indicated activation modulated sets of MAPK activation status in cancer cells chronically exposed to genes with specific functions related to the activity of the T-cell dabrafenib, trametinib, or dabrafenib þ trametinib. In addition, receptors, accompanied by genes encoding cytokines, chemo- we observed that a number of BRAF inhibitor resistant clones kines, as well as genes involved in cell proliferation, transcription, (12R5-1, 12R5-3, 16R6-3, 16R5-5, and 16R6-4; ref. 13), which and growth (Supplementary Tables S2A, S2B, and S2C). When developed from the parental line A375, expressed high levels of trametinib was added simultaneously with T-cell activators, it PD-L1 as determined by Western blot analysis, flow cytometry, partially offset the upregulation of 10 genes, CCL3, CCL4, GZMB, and RT-PCR (Fig. 3A–B and C and data not shown). In A375 IL2, IL3, IL9, IL10, IL17A, IL17F, and IL23R, mostly cytokines and dabrafenib-resistant clones, increased PD-L1 protein expression chemokines. It also enhanced the expression of three upregulated tended to correlate with increased pSTAT levels (Fig. 3C), a cytokines, IL4, IL5, and IL13, while it attenuated the downregula- result which was consistent with a previous report (15). IFNg þ tion of FCER1A, MX1, and RARRES3 genes in CD4 cells. Sim- expression was below the level of detection in all cell samples ilarly, trametinib showed an offset of 11 upregulated genes, CCL3, tested and adding IFNg did not further increase PD-L1 expres- CCL4, GZMB, IFNG, IL2, IL3, LTA, CD82, IL1R1, TNF, and XCL1, sion in 12R5-1–resistant line (data not shown). In the 12R5-1 and three downregulated genes CD244, CXCR4, and SIGIRR in cell line, PD-L1 expression was still partially responsive to þ CD8 cells in response to T-cell activation. Dabrafenib alone had MAPK pathway inhibition as PD-L1 mRNA levels were reduced

1642 Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research

Dabrafenib and Trametinib Effect on Immune Function

A 10 B Unact. CD4 CD8 8 2 h 24 h Un. D+TTDAct. D+TTDAct.Un. 6

pERK/tERK 2 +3.0 (% normalized)

0 T T T V V V D D D D+T D+T D+T

80 Unact. 2 h 60 24 h 40 -3.0 pS6/tS6 20 (% normalized) 0 T T T V V V D D D D+T D+T D+T

C Act. D T D+T CD25 CD69 PD-1 CTLA-4 90 120 45 35 80 40 100 30 70 35

(%) 25 + 60 80 30 50 25 20 Drug>Act. 60

CD25 40 20 15 + 30 40 15 10

CD4 20 10 20 10 5 5 0 0 0 0

0 h 6 h 0 h 6 h 0 h 6 h 0 h 6 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

Baseline Baseline Baseline Baseline

100 120 90 80 90 80 70 80 100 70 60

(%) 70

+ 80 60 60 50 50 50 60 40 Act.>Drug 40 CD25 40 + 30 30 40 30 20

CD4 20 20 20 10 10 10 0 0 0 0

0 h 6 h 0 h 6 h 0 h 6 h 0 h 6 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

Baseline Baseline Baseline Baseline

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 þ 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 þ trametinib, dabrafenib/trametinib ¼ 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 þ trametinib (300 nmol/L/10 nmol/L) were added concurrently with CD3/CD28 activation beads to CD4þ and CD8þ T cells for 24 hours. C, time course of T-cell surface marker expression in CD4þ T cells following treatment with dabrafenib (100 nmol/L), trametinib (10 nmol/L), or dabrafenib þ 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.

by 39%, 34%, and 77%, respectively, in response to trametinib, sure the expression of genes including tumor antigens, dabrafenib, and dabrafenib þ trametinib treatment. We also HLA molecules as well as markers associated with immuno- used a NanoString custom-built codeset (302 genes) to mea- modulation, apoptosis, and MAPK signaling in both A375 and

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1643

Liu et al.

A B

3.0 x 105 PDL-1 48 hours 1,500 Flow cytometry

2.0 x 105 1,000

RT-PCR 1.0 x 10 500

0 MFI ( a -PDL1-APC) 0

A375 DMSO 12R5-1 12R5-3 16R5-2 16R5-3 INFg+MEKi BRAFi+MEKiINFg-3 ng/mL INFg+BRAFi MEKi-10 nmol/L BRAF-300 nmol/L INFg+BRAFi+Meki C Western blot

6 9.0 x 10 HLA-A PDL1 pSTAT3 6.0 x 106 (Y705)

6 STAT3

RT-PCR 3.0 x 10 Actin 0

DMSO A375 BRAFi+MEKi INFg+MEKiINFg+BRAFi 12R5-1 12R5-3 16R6-3 16R5-5 16R6-4 MEKi-10 nmol/L INFg-3 ng/mL BRAF-300 nmol/L INFg+BRAFi+Meki D Tumor antigens NY-ESO-1, BAGE, TRP1, gp100

HLA class I and II +4.0 HLA-A,B,C,E, HLA-DMA,DPA, DRA, DRB3

Immunomodulation CD40, CD68, CD70, CD83, GBP1, ICOSLG, IL15, IRF1, OX40L, SPP1, STAT1, STAT3, TOX, B7-H3, PDCD2

Immunosuppression PD-L1, IL1A, IL8, NT5E, VEGFA -4.0 Apoptosis/tumor suppressors BIM1, DPP4, PIK3IP1, RARRES3,TP53IP

MAPK pathway Ki67, AREG, CDKN3, DUSP4/6, ETV4/5, SPRY4 TIMP1,TRIB2

DMSO T D D+T

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 IFNg 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).

SK-MEL-24 BRAF V600E–mutant cells. As shown in Fig. 3C and OX40L, SPP1, STAT1/3, TOX, B7-H3, PDCD2,andpro-apopto- Supplementary Table S4A for A375 cells, MAPK inhibition by sis/tumor suppression genes including BCL2L11 (BIM1), DPP4, dabrafenib and trametinib upregulated tumor antigens NY- PIK3IP1, RARRES3,andTP53IP, and downregulated a subset of ESO-1, BAGE, TRP1, gp100, HLA-class I and class II molecules, immunosuppressive factors such as PD-L1, VEGFA, IL1A,and immunomodulation factors such as CD40, ICOSLG, IL15, IRF1, NT5E (CD73). Similar results were seen in SK-MEL-24 cells

1644 Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research

Dabrafenib and Trametinib Effect on Immune Function

Table 1. In vitro effects on human tumor cell lines PD-L1 T

baseline (IC50, Cell lines (RT-PCR) nmol/L) CTG Gene expression changes by T at 10 nmol/L, 48 h of treatmenta Ct Levels PD-L1 MAPK Apoptosis HLA-I HLA-II IL8 IL1A, TGFA, (DUSP4/6) (PIK3IP1, (A, B, C) (DMA, DPA, IL1B, EREG, TP53INP1, DRA) VEGFA, AREG BCL2L11) NTE5 BRAF-mutant melanoma YUSITc 28 Low 1 — # ND — ##ND ND 12R5-1c 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.

(Table 1 and Supplementary Table S4B). In addition, down- sion level was monitored as a measure of MAPK pathway inhi- regulation of PD-L1 and upregulation of HLA-I and II mole- bition by compound treatment. Trametinib induced an upregula- cules were confirmed in A375 and SK-MEL-24 and observed in tion of apoptosis markers, PIK3IP1, TP53INP1, BCL2L11 12R5-1, but not in YUSIT BRAF V600K–mutant cells, by RT- (BIM1), and HLA-I and/or II expression in fiveoutofsixcell PCR and/or flow cytometry analyses (Table 1 and data not lines tested except in the CHL-1. Whereas it decreased IL8 levels shown). Furthermore, out of these four tested lines, tumor in two out of three melanoma lines (HMVII and SK-MEL-2) and antigen NY-ESO-1 mRNA was increased 2-fold in A375 cells reduced the expression of NTE5 (CD73) and/or VEGFA in all measured by NanoString, but not by Taqman, and at the three lung lines. It also reduced TGFA, EREG.andAREG genes protein level by Western blot analysis; whereas MART1 upre- in two out of three lung lines (H358 and Calu6). Baseline level gulation by MAPK inhibition was observed at mRNA level (6- of IL6 was extremely low in all lines except in CHL1. RT-PCR, fold) in YUSIT and both mRNA (37–93-fold) and protein levels but not NanoString, detected some level of IL6 induction by in SK-MEL-24 cells. trametinib in two out of the six lines (A549 and Calu6). All cell lines except CHL-1 showed a reduction in DUSP4/6 levels by Trametinib increases apoptosis markers and the expression of compound treatment, indicating that upregulation of markers HLA-molecules in non–BRAF-mutant tumor cells involved in apoptosis, HLA-I/II, and downregulation a subset To evaluate the immunomodulatory effects of trametinib of immunosuppression IL8, NTE5, VEGFA may be associated on non–BRAF-mutant tumor cells, we monitored the same key with MAPK pathway inhibition by trametinib. In addition, markers with the NanoString custom-built codeset in three BRAF trametinib dose dependently increased MART1, GP100, TRP- WT melanoma lines, HMVII and SK-MEL-2 (both have a NRAS 1,andTYR (3–10-fold at 3–10 nmol/L) tumor antigen gene mutation and are sensitive to trametinib), CHL1 (resistant expression in HMVII BRAF WT melanoma line, but not in the to trametinib), and three KRAS-mutant NSCLC lines, Calu6, rest of the lines tested. H358, and A549 (sensitive to trametinib). The representative genes, PD-L1, DUSP4, DUSP6, HLA-I (A, B, C), HLA-II (DMA, Combinations of trametinib with immunomodulatory DPA, DRA), IL6, and IL8 were confirmed with RT-PCR. In addi- antibodies targeting PD-1, PD-L1, or CTLA-4 in CT26 murine tion, surface protein expression levels of PD-L1 and HLA-I mole- syngeneic tumor models are superior to single agents cules were evaluated by flow cytometry analysis. The data are The in vivo antitumor effect of trametinib was evaluated in the shown in Supplementary Table S4B (NanoString) and summa- murine immunocompetent BALB/C syngeneic CT26 tumor mod- rized in Table 1. Baseline level of PD-L1 is low in most of the lines, el. CT26 mouse colorectal tumor cells harbor the homozygous except H358 with moderate expression. Trametinib displayed KRAS G12D mutation and MAPK1 and MET amplifications (16). mixed effects on PD-L1 expression with upregulation in Calu-6 In vitro, trametinib caused dose-dependent inhibition of cell and A549 (only detected by RT-PCR), downregulation in H358 proliferation with a mean IC50 value of 20 nmol/L and blocked NSCLC line, and no change in three out of three melanoma lines MAPK signaling measured by pERK (Fig. 4A). In vivo, as shown tested. IFNg increased the expression levels of PD-L1 and HLA-I in in Fig. 4B, 18 days of daily trametinib monotherapy at 1 mg/kg all tested cell lines (data not shown). DUSP4 and DUSP6 expres- resulted in moderate antitumor activity with 61% TGI.

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1645

Liu et al.

A B 2,500 Rat-I gG2b 100 α-PD-1

) α-PD-L1

3 2,000 α-CTLA4 75 T IC = 20 ± 16 nmol/L 50 1,500 T+α-PD-1 T+α-PD-L1 50 1030 nmol/L 1,000 T+α-CTLA4 pErk Cell proliferation 25 Tumor volume (mm (% vehicle control) 500 * P < 0.05 Erk 0 0 0 0.1 1 10 100 0 7 14 21 Trametinib (nmol/L) Days of treatment

C D 100 2,000 80 ) 3 1,500 60

1,000 40 Survival (%) * * P < 0.05 500 20 Tumor volume (mm

0 0 0 7 14 21 0 14 28 42 56 70 Days of treatment Days of treatment Untreated PD-1-2nd PD-1-1st+MEKi-2nd Untreated PD-1-2nd PD-1-1st+MEKi-2nd Veh+IgG2a MEKi-1st MEKi-1st+PD-1-1st Veh+IgG2a MEKi-1st MEKi-1st+PD-1-1st PD-1-1st MEKi-2nd MEKi-1st+PD-1-2nd PD-1-1st MEKi-2nd MEKi-1st+PD-1-2nd

MEKi-1st PD-1-2nd PD-1-1st MEKi-2nd End of study 11- 1 8 86 )syad(

CT26 cell 68 Days of treatment implant 61 Days of treatment

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 ¼ 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, a-mouse PD-1 (RMP1-14 clone, rat IgG2a), a-mouse PD-L1 (10F.9G2 clone, rat IgG2b), or a-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-1st)and a-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 a-PD-1 (PD-1-2nd). Mice (n ¼ 10 per group) were treated with trametinib at 1 mg/kg orally one daily, or with antibodies rat-IgG2a or a-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.

Anti-mouse PD-1, PD-L1, and CTLA-4 antibodies dosed alone CTLA4 antibodies demonstrated much more profound activity showed minimal to low efficacy with 2%, 18%, and 32% TGI with 80%, 81%, and 84% TGI, respectively. No overt toxicity, as respectively. However, concurrent combinations beginning with defined by weight loss, unkempt appearance, mortality and the first dose of trametinib with anti-mouse PD-1, PD-L1, or behavior, was observed in any of the groups during the course

1646 Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research

Dabrafenib and Trametinib Effect on Immune Function

A Untreated IgG2a MEK a-PD-1 MEK/a-PD-1 5 5 5 5 5 10 10 10 10 10 17.7% 20.6% 30.5% 18.7% 21.7% 4 4 4 4 4 10 10 10 10 10 35.9% 33.2% 25.5% 24% 58.2% 3 3 3 3 3 10 10 10 10 10 2 2 2 2 2 10 10 10 10 10 CD4 FITC-A

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 CD8 APC-Cv7-A 5 5 5 40.4% %7.14 5 %1.43 %1.14 5 %6.63 10 10 10 10 10 4 4 4 4 4 10 10 10 10 10 3 3 3 3 3 10 10 10 10 10 2 2 2 2 2 10 10 10 10 10 CD25 APC-A

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 Foxp3 PE-A Lymphocytes % in tumor B C 16 α-PD-1 Untreated Untreated IgG2a α-PD-1 +MEK MEK IgG2a 12 T α-PD-1 pERK 8 T+α-PD-1 * 4 tERK * *

Total cell from tumor (%) 0 Lymphocytes CD3+ CD4+ CD8+ D

Untreated

IgG2a

α-PD-1

α-PD-1 +MEK

MEK C3 Cfd Il2ra Mif * Ccl5 Ccl8 Ccl3 Cd36 Ccl11 Cxcl9 Cxcl3 Cxcl1 Tnf ** Cd7 * Cd4 * Il1rn * Il1r2 * Gzmb Il33 ** Il1b ** Icosl * Sell ** Ccr7 * C1s ** Trem1 Cd274 Cxcl11 Batf3 * Ccl2 ** Nfil3 ** Ccl4 ** Ccl6 ** Ccl9 ** Ccl7 ** Cxcl10 Cxcl12 Csf1r * Plau ** Sele ** Cd74 * Cx3cr1 Il1rl1 ** Ccl22 * C1ra ** Il12a ** Msr1 ** Cxcr3 * Tgfbi ** Itga6 ** II12b ** Fcgrt ** Tnfaip6 Plaur ** Cd22 ** Cd14 ** Cd44 ** Cd209g Ccl12 ** Ptgs2 ** Tgfb3 ** Cxcr2 ** Tnfrsf9 * Ptpn22 * Ltb4r1 ** Pdgfrb ** Cd79b ** Cd163 ** H2-Eb1 * II11ra1 ** II11ra1 Il18rap ** Clec4e ** H2Eaps * Cdkn1a ** S100a9 ** S100a8 ** Mapk11 ** Mapk11 Ceacam1 *

| || ||| -2.50 2.50

Figure 5. þ þ 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 þ þ þ þ 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 ¼ 3; , P < 0.05 vs. untreated and IgG2aþvehicle 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; a-PD-1, anti–PD-1 antibody treatment; MEK, trametinib treatment.

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1647

Liu et al.

þ þ of treatment. The above data indicate that concurrent combina- CD69 , and PD-1 cells from spleens and lymph nodes (data tions of trametinib with immunomodulatory antibodies targeting not shown). Of note, these immune surface markers and circu- PD-1, PD-L1, and CTLA-4 potentiate antitumor activity as com- lating cytokines from peripheral blood samples were variable pared with the single agents at their tolerated doses. from mouse to mouse, and marginally (< 2-fold) or not signif- Next, we explored whether the combination of trametinib icantly affected by any treatment in the study (data not shown). with the anti–PD-1 antibody in different lead-in sequences IHC analysis revealed that treatment with trametinib alone and in would affect in vivo efficacy. As shown in Fig. 4C and D and combination with anti–PD-1 antibody led to 70% to 75% inhi- Supplementary Fig. S1 (tumor growth curves from individual bition of pERK in the tumor (Fig. 5C and Supplementary Tables mice), three combination regimens were evaluated: trametinib S5A and S5B), demonstrating effective MAPK signaling inhibition and anti–PD-1 antibody given concurrently starting in the first by trametinib in the CT26 tumor model in immunocompetent week (MEK-1stþPD-1-1st); trametinib given in the first week as mice. single agent followed by adding anti–PD-1 antibody in the second In addition, tumor gene expression was profiled using a Nano- week with continued trametinib dosing (MEK-1stþPD-1-2nd); String-based immunology panel and follow-up with quantitative and anti–PD-1 antibody given in the first week as single agent RT-PCR (qRT-PCR). Among the 561 mouse genes profiled, 77 followed by adding trametinib in the second week with continued showed a 1.5-fold change by trametinib, anti–PD-1, or the anti–PD-1 dosing (PD-1-1stþMEK-2nd). All three combination combination of the two in comparison with untreated and regimens showed inhibition of tumor growth more effectively non-specific IgG2a controls (Supplementary Table S6). A heat- than their single-agent controls during the initial 2 to 3 weeks of map was generated by gene clustering of these 77 genes (Fig. 5D). treatment (Fig. 4C). However, only two of three combination The group I genes are those upregulated by trametinib alone and/ treatment groups, concurrent MEK-1stþPD-1-1st and trametinib or trametinib in combination with anti–PD-1 but not by anti–PD- lead-in followed by trametinib þ PD-1 (MEK-1stþPD-1-2nd) 1 alone. Interestingly, three MHC class II genes, H2-Ea-ps, CD74, produced profound delay in median tumor growth with TTE of and H2-Eb1, along with CD4 and IL12b were upregulated most 39 and 49 days, two out of 10 and four out of 10 68-day survivors, significantly by the combination treatment. Group II includes 12 respectively, at the end of the study, and differed significantly from genes mainly associated with T-cell IFNg inducible cytotoxic untreated and vehicle/isotype controls by log-rank survival anal- factor (GZMB), immunoregulators (CD274-PD-L1 and IL2RA- ysis (P < 0.05; Fig. 4D). Conversely, all PD-1-1stþMEK-2nd CD25), chemokines (CXCL9, CXCL11, CXCL10, CXCL12, showed minimal benefit in long-term survival. Finally, it is worth CCL5-RENTES, and CCL8-MCP-1), and other immune factors noting that both PD-1-1st monotherapy and PD-1-1stþMEK-2nd (CFD, CD36, and C3). Although not statistically significant, treated groups had one out of 10 68-day survivors although tumors from 2 out of 3 mice treated with anti–PD-1 antibody majority of the groups showed no response. All treatments were alone showed unique and noticeable upregulation of all of these well tolerated. genes, implying a gene signature of anti–PD-1 effect on tumors. Group III has 42 genes that were downregulated by trametinib Trametinib alone or in combination with anti–PD-1 antibody alone and in combination with anti–PD-1. These genes includes in vivo reduced immunosuppression factors, increased inflammation factors (e.g., SELE-E-selectin, PTGS2-COX-2, MIF, þ HLA-class II genes and lead to increased intratumoral CD4 and TNF, and PLAUR), chemokines (CCL2, CCL3, CCL4, CCL6, CCL7, þ CD8 T cells CCL9, CCL11, CCL12, CXCL1, CXCL3, and CXCR2), pro-inflam- Given that trametinib can potentiate the efficacy of immuno- matory IL1 family cytokines mostly associated with immunosup- modulators in vivo, we investigated potential immunomodulato- pression (IL33, IL1Rl1, IL1RN, IL1B, IL1R2, and IL18RAP), tumor ry mechanisms of trametinib in combination with an anti–PD-1 metastasis factors (S100A8 and S100a9, PLAU, and ITGA6), blocking antibody in lymphocyte tissues and CT26 tumors in vivo. markers associated with immunosuppression (TGFB1), and mar- We collected whole blood, tumors, spleens, and lymph nodes kers for monocytes (CD163 and CD14), macrophage and den- after 7 days of treatment with trametinib and anti–PD-1 antibody dritic cells (MSR1 and CD209G) and myeloid cells (TREM1). A alone, or in concurrent combination. In tumors, trametinib alone few selected genes were further evaluated using qRT-PCR that has and in combination with anti–PD-1 antibody, but not anti–PD-1 more sensitive detection and showed consistent results with þ antibody alone, significantly increased CD4 T cells by 4.3- and NanoString. As illustrated in Supplementary Fig. S2, anti–PD-1 3.4-fold, respectively, compared with untreated and nonspecific antibody increased IFNG and GZMB by itself, and PD-L1, ICOS, IgG2a controls (P < 0.05) measured by flow cytometry (Fig. 5A and CTLA-4 with and without trametinib. Only the combination and B and Supplementary Table S5). Only the combination of trametinib and anti–PD-1 increased CD4, OX40, and PD-1. of trametinib and anti–PD-1 antibody significantly increased trametinib alone and in combination with anti–PD-1 showed þ CD8 T cells by 4.7-fold vs. untreated (P < 0.05) and 3.6-fold reduction of CCL2 (consistent with NanoString data), DUSP6 þ vs. non-specific IgG2a control (P ¼ 0.05; Fig. 5B). CD69 cells (correlated with pERK IHC) and IL6 (only detected by RT-PCR; þ from CD4 population were reduced by 53% from trametinib Supplementary Fig. S2). þ þ treatment (P < 0.05 vs. untreated). CD69 cells from CD8 All data generated demonstrated that trametinib in concurrent population were reduced by 57% from the combination of combination with anti–PD-1 downregulated immunosuppres- trametinib with anti–PD-1 antibody (P < 0.05 vs. untreated). sion factors, upregulated HLA molecules, and increased immune þ Other immune cell subtypes, such as Treg, measured by Foxp3 response in tumors with little or no immune phenotypical/func- þ þ þ þ þ /CD25 from CD4 population, PD-1 , PD-L1 , OX40 , and tional changes from circulating cell populations and immune þ þ þ ICOS from both CD4 and CD8 cells were changed less than 2- organs in vivo. All of these effects contribute to and support the fold or not statistically significant (P > 0.05) by any treatment rationale that trametinib in combination with immunomodula- (Supplementary Table S5). There were no significant alterations in tory antibodies targeting PD-1, PD-L1, and CTLA-4 is a more þ þ þ the numbers and expression levels of CD3 , CD4 , CD25 , effective antitumor therapeutic approach.

1648 Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research

Dabrafenib and Trametinib Effect on Immune Function

Discussion tinib, and the combination partially downregulated PD-L1 Although the mechanisms of action for BRAF and MEK inhi- expression in a subset, but not all cell lines tested, an observation bitors, such as dabrafenib and trametinib, regarding tumor- similar to data from recent publications (15, 22, 23). Interestingly, growth inhibition are well studied, their impact on immune cells we also observed that trametinib could induce PD-L1 expression and the tumor microenvironment is less understood. Preclinical in two of the NSCLC lines with low baseline level of PD-L1 and limited clinical findings have suggested that immunomod- expression. Our data in A375 cells chronically exposed to dabra- ulatory effects in the tumor microenvironment and on circulating fenib, trametinib, or dabrafenib þ trametinib, suggests that PD-L1 immune cells by a BRAF inhibitor alone and the combination of expression may be regulated via a compensatory pathway in a BRAF inhibitor with a MEK inhibitor is context-dependent response to sustained inhibition of MAPK signaling. Although (8, 17–20). On the other hand, concerns have been raised regard- the mechanisms of PD-L1 regulation appear to be multifactorial, ing the potential immunosuppressive activity of MEK inhibitors MAPK signaling and feedback regulation by MAPK inhibition due to their immunosuppressive activity in vitro (10). However, likely contribute, in part, to the expression levels of PD-L1 in the studies have been limited in scope and lack in vivo validation in tumors. However, PD-L1 expression level could be affected by mouse models. Here, we present data showing that the immu- multiple pathways including the MAPK pathway, and the change nomodulatory effects of trametinib on activated T cells were of PD-L1 expression is accompanied by multiple other genetic and multifaceted and context dependent. Changes that could be morphologic changes that collectively contribute to tumor growth considered immunosuppressive were only observed in vitro, were and metastasis (24). As a result, PD-L1 may serve as a marker, but transient, and did not translate in vivo using an immunocompe- may not be the driver determining patient response to BRAF tent mouse tumor model. The differences between data from this inhibitor treatment. Future studies should further investigate the study and those of prior reports may be explained by a number of compensatory pathways responsible for PD-L1 regulation in the factors, including: (i) the in vitro experiments done by others had a context of MAPK inhibition. As reported, during the initial treat- short period of time exposure to MEK inhibitors without mon- ment with either BRAF inhibitor alone or BRAF þ MEK inhibitor þ itoring longer-term effects, therefore only the transient suppres- in patients BRAF-mutant melanoma, both PD-L1 and CD8 T-cell þ sive activity by MAPK inhibition was captured; (ii) MEK inhibitors infiltrates were increased in treated tumors (18). However, CD8 transiently block the MAPK signaling during the initial T-cell T-cell infiltrates declined when these patients progressed from the activation, thus delay the kinetics of T-cell activation and cytokine treatment. It was also noted that PD-L1 expression increased secretion in vitro; (iii) the circulating T cells collected in vivo are when patients became refractory to BRAF or BRAF þ MEK inhib- mostly in a na€ve state, reflected in this study by the low number of itor treatment. The evidence of PD-L1 modulation data from activated T cells from blood and immune organs with and without both preclinical and clinical studies indicates that upregulation anti–PD-1 antibody treatment in mice and much less active than T of PD-L1 may be a marker of acquired resistance to BRAF, MEK, or cells activated by anti-CD3/CD28 in vitro. Our preclinical data are BRAFþMEK inhibition, and may provide rationale for PD-L1 complementary to the recent report showing that both dabrafenib inhibitor use in BRAF-resistant patients. and trametinib alone or the combination increased tumor infil- In this study, we also demonstrated that inhibition of MAPK tration of lymphocytes, and enhanced the antitumor effect with by trametinib, dabrafenib, or the combination of trametinib adoptive T-cell immunotherapy in the syngeneic murine model and dabrafenib in BRAF-mutant melanoma and trametinib in of BRAF V600E–mutant melanoma (21). Our studies indicate BRAF WT melanoma and NSCLC lines increased the expression þ that trametinib alone increases tumor infiltrating CD4 lympho- of apoptosis markers and HLA-class I and/or II molecules and þ cytes (TIL), does not negatively affect the prevalence of CD8 TILs decreased a subset of immunosuppression factors in vitro.The þ while significantly increasing CD8 TILs when combined with increase of apoptosis markers PIK3IP1, TP53INP1, and anti–PD-1 antibody. Most importantly, combining trametinib BCL2L11 (BIM1) may sensitize tumor cell killing by cytotoxic with anti–PD-1 either concurrently or in phased sequence, with T cells. Tumor cells secrete IL1, IL8, and VEGFA soluble factors trametinib administered first followed by trametinib plus anti– to induce immunosuppression (25, 26). NTE5 (CD73) gene is PD-1, resulted in much more effective and durable antitumor highly expressed in many human solid tumors. It encodes an activity than both single agents in the KRAS-mutant CT26 colo- ectoenzyme generating extracellular adenosine that induces rectal tumor syngeneic mouse model. Interestingly, phased potent immunosuppressive effectsandimpairedanti-tumor sequence of anti–PD-1 antibody dosed first, followed by anti– T-cell responses (27, 28). EGFR ligands, such as TGFA, EREG, PD-1 antibody plus trametinib showed minimal benefit in long- and AREG, can activate EGFR, promote tumor metastasis, and term survival compared with single-agent treatment. The obser- enhance regulatory T cell-suppressive function via the EGFR vation suggests that blockage of oncogenic MAPK signaling by (29). HLA-class I molecules are often downregulated or lost in trametinib is critical and effective to prime and synergize tumors tumor cells and play a key role in immune escape (30–32). A in response to immunotherapy through the induction of apo- study by Carreteto and colleagues (33) showed that higher HLA ptosis markers, upregulation of HLA molecules, and reduction class I gene expression was observed in regressing but not in of immunosuppression factors from tumors. Our data support progressing metastases from microdissected tumor regions, the rationale to evaluate the sequencing of these agents, includ- supporting the idea that the nature of HLA class I alterations ing giving the targeted therapy as a lead-in to an immunomod- in tumor cells may contribute to antitumor effects of thera- ulatory antibody or concurrently in clinical trial design to peutic interventions. Our observation of MAPK inhibition maximize antitumor efficacy and with the hope of minimizing induced apoptosis, increase of MHC class I and/or II along side effects. with the decrease of immunosuppression factors in most of the Our data show that PD-L1 was not only induced by IFN-g in all human tumor cell lines, and in CT26 tumors when combined cell lines tested, but was also upregulated in cell lines with with anti–PD-1 antibody in vivo is intriguing. These effects may acquired resistance to dabrafenib. Conversely, dabrafenib, trame- potentially increase immune response when MAPK pathway

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1649

Liu et al.

inhibitors combine with immunotherapy and may lead to Analysis and interpretation of data (e.g., statistical analysis, biostatistics, therapeutic synergy. computational analysis): L. Liu, P.A. Mayes, S. Eastman, H. Shi, S. Yadavilli, These data taken together further expand the understanding of T. Zhang, J. Yang, S.-Y. Zhang, C. Hopson, L. Tsvetkov, J. Jing, S. Zhang, A. Hoos Writing, review, and/or revision of the manuscript: L. Liu, P.A. Mayes, BRAF and MEK inhibitor effects on the immune system and S. Eastman, H. Shi, T. Zhang, J. Yang, L. Seestaller-Wehr, S.-Y. Zhang, C. Hopson, provide scientific evidence to support the investigation of the J. Jing, J. Smothers, A. Hoos combination of trametinib, with or without dabrafenib, with Administrative, technical, or material support (i.e., reporting or organizing immunomodulators in the clinic. Clinical studies are under way data, constructing databases): L. Liu to test the combination of these important agents in patients with Study supervision: L. Liu, P.A. Mayes, J. Yang, A. Hoos metastatic melanoma. Acknowledgments Disclosure of Potential Conflicts of Interest The authors thank Amber Anderson, Vivian Zhang, Bao Hoang, Yaobin Liu, Meixia Bi, David Kilian (GlaxoSmithKline), Xiaoyu Pan, Drew M Pardoll (Johns A. Hoos and J. Jing have ownership interests (including patents) in Hopkins University, Baltimore, MD), and Lisa Dauffenbach (Mosaic Labora- GlaxoSmithKline. No potential conflicts of interest were disclosed by the tories) for their technical and consultation assistance. other authors.

Authors' Contributions Grant Support ﬁ Conception and design: L. Liu, P.A. Mayes, S. Eastman, H. Shi, J. Yang, This study was nancially supported by GlaxoSmithKline. J. Smothers, A. Hoos The costs of publication of this article were defrayed in part by the payment of Development of methodology: L. Liu, P.A. Mayes, S. Eastman, H. Shi, page charges. This article must therefore be hereby marked advertisement in S. Yadavilli, T. Zhang, L. Seestaller-Wehr accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Liu, S. Eastman, H. Shi, S. Yadavilli, L. Seestaller- Received September 19, 2014; revised December 4, 2014; accepted December Wehr, S.-Y. Zhang, L. Tsvetkov, A. Hoos 23, 2014; published OnlineFirst January 15, 2015.

References 1. Mackiewicz-Wysocka M, Zolnierek J, Wysocki PJ. New therapeutic options rafenib, mediated by NRAS or MEK mutations. Mol Cancer Ther in systemic treatment of advanced cutaneous melanoma. Expert Opin 2012;11:909–20. Investig Drugs 2013;22:181–90. 14. Callahan MK, Masters G, Pratilas CA, Ariyan C, Katz J, Kitano S, et al. 2. Pardoll DM. The blockade of immune checkpoints in cancer immuno- Paradoxical activation of T cells via augmented ERK signaling mediated by therapy. Nat Rev Cancer 2012;12:252–64. a RAF inhibitor. Cancer Immunol Res 2014;2:70–9. 3. Dudek AM, Garg AD, Krysko DV, De Ruysscher D, Agostinis P. Inducers of 15. Jiang X, Zhou J, Giobbie-Hurder A, Wargo J, Hodi FS. The activation of immunogenic cancer cell death. Cytokine Growth Factor Rev 2013;24: MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 319–33. expression that is reversible by MEK and PI3K inhibition. Clin Cancer Res 4. Yaguchi T, Sumimoto H, Kudo-Saito C, Tsukamoto N, Ueda R, Iwata- 2013;19:598–609. Kajihara T, et al. The mechanisms of cancer immunoescape and develop- 16. Castle JC, Loewer M, Boegel S, de Graaf J, Bender C, Tadmor AD, et al. ment of overcoming strategies. Int J Hematol 2011;93:294–300. Immunomic, genomic and transcriptomic characterization of CT26 colo- 5. Menzies AM, Long GV. Dabrafenib and trametinib, alone and in combi- rectal carcinoma. BMC Genomics 2014;15:190. nation for BRAF-mutant metastatic melanoma. Clin Cancer Res 2014;20: 17. Cooper ZA, Frederick DT, Juneja VR, Sullivan RJ, Lawrence DP, Piris A, et al. 2035–43. BRAF inhibition is associated with increased clonality in tumor-infiltrating 6. Forde PM, Reiss KA, Zeidan AM, Brahmer JR. What lies within: novel lymphocytes. Oncoimmunology 2013;2:e26615. strategies in immunotherapy for non-small cell lung cancer. Oncologist 18. Frederick DT, Piris A, Cogdill AP, Cooper ZA, Lezcano C, Ferrone CR, et al. 2013;18:1203–13. BRAF inhibition is associated with enhanced melanoma antigen expres- 7. Merelli B, Massi D, Cattaneo L, Mandala M. Targeting the PD1/PD-L1 axis sion and a more favorable tumor microenvironment in patients with in melanoma: biological rationale, clinical challenges and opportunities. metastatic melanoma. Clin Cancer Res 2013;19:1225–31. Crit Rev Oncol 2014;89:140–65. 19. Knight DA, Ngiow SF, Li M, Parmenter T, Mok S, Cass A, et al. Host 8. Hong DS, Vence L, Falchook G, Radvanyi LG, Liu C, Goodman V, et al. immunity contributes to the anti-melanoma activity of BRAF inhibitors. BRAF(V600) inhibitor GSK2118436 targeted inhibition of mutant BRAF in J Clin Invest 2013;123:1371–81. cancer patients does not impair overall immune competency. Clin Cancer 20. Wilmott JS, Long GV, Howle JR, Haydu LE, Sharma RN, Thompson JF, et al. Res 2012;18:2326–35. Selective BRAF inhibitors induce marked T-cell infiltration into human 9. Koya RC, Mok S, Otte N, Blacketor KJ, Comin-Anduix B, Tumeh PC, et al. metastatic melanoma. Clin Cancer Res 2012;18:1386–94. BRAF inhibitor vemurafenib improves the antitumor activity of adoptive 21. Hu-Lieskovan S, Mok S, Faja LR, Goedert L, Comin-Anduix B, Koya RC, cell immunotherapy. Cancer Res 2012;72:3928–37. et al. Combinatorial effect of dabrafenib, trametinib, and adoptive cell 10. Vella LJ, Pasam A, Dimopoulos N, Andrews M, Knights A, Puaux AL, et al. transfer (ACT) in an immune-competent murine model of BRAFV600E MEK inhibition, alone or in combination with BRAF inhibition, affects mutant melanoma. J Clin Oncol 2014;32:2512. multiple functions of isolated normal human lymphocytes and dendritic 22. Atefi M, Avramis E, Lassen A, Wong DJ, Robert L, Foulad D, et al. Effects of cells. Cancer Immunol Res 2014;2:351–60. MAPK and PI3K pathways on PD-L1 expression in melanoma. Clin Cancer 11. Boni A, Cogdill AP, Dang P, Udayakumar D, Njauw CN, Sloss CM, et al. Res 2014;20:3446–57. Selective BRAFV600E inhibition enhances T-cell recognition of mela- 23. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of noma without affecting lymphocyte function. Cancer Res 2010; PD-L1 on tumor cells in the escape from host immune system and tumor 70:5213–9. immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 2002; 12. Halaban R, Zhang W, Bacchiocchi A, Cheng E, Parisi F, Ariyan S, et al. 99:12293–7. PLX4032, a selective BRAFV600E kinase inhibitor, activates the ERK 24. Massi D, Brusa D, Merelli B, Ciano M, Audrito V, Serra S, et al. PD-L1 marks pathway and enhances cell migration and proliferation of BRAFWT mel- a subset of melanomas with a shorter overall survival and distinct genetic anoma cells. Pigment Cell Melanoma Res 2010;23:190–200. and morphological characteristics. Ann Oncol 2014;25:2433–42. 13. Greger JG, Eastman SD, Zhang V, Bleam MR, Hughes AM, Smitheman 25. Liu C, Peng W, Xu C, Lou Y, Zhang M, Wargo JA, et al. BRAF inhibition KN, et al. Combinations of BRAF, MEK, and PI3K/mTOR inhibitors increases tumor infiltration by T cells and enhances the antitumor activity overcome acquired resistance to the BRAF inhibitor GSK2118436 dab- of adoptive immunotherapy in mice. Clin Cancer Res 2013;19:393–403.

1650 Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research

Dabrafenib and Trametinib Effect on Immune Function

26. Khalili JS, Liu S, Rodriguez-Cruz TG, Whittington M, Wardell S, Liu C, et al. 30. Garrido C, Paco L, Romero I, Berruguilla E, Stefansky J, Collado A, et al. Oncogenic BRAF(V600E) promotes stromal cell-mediated immunosup- MHC class I molecules act as tumor suppressor genes regulating the cell pression via induction of interleukin-1 in melanoma. Clin Cancer Res cycle gene expression, invasion and intrinsic tumorigenicity of melanoma 2012;18:5329–40. cells. Carcinogenesis 2012;33:687–93. 27. Jin D, Fan J, Wang L, Thompson LF, Liu A, Daniel BJ, et al. CD73 31. Garcia-Lora A, Algarra I, Garrido F. MHC class I antigens, immune sur- on tumor cells impairs antitumor T-cell responses: a novel mech- veillance, and tumor immune escape. J Cell Physiol 2003;195:346–55. anism of tumor-induced immune suppression. Cancer Res 2010; 32. Carretero R, Romero JM, Ruiz-Cabello F, Maleno I, Rodriguez F, Camacho 70:2245–55. FM, et al. Analysis of HLA class I expression in progressing and regressing 28. Muller-Haegele S, Muller L, Whiteside TL. Immunoregulatory activity of metastatic melanoma lesions after immunotherapy. Immunogenetics adenosine and its role in human cancer progression. Expert Rev Clin 2008;60:439–47. Immunol 2014:10:1–18. 33. Wilmott JS, Haydu LE, Menzies AM, Lum T, Hyman J, Thompson JF, et al. 29. Zaiss DM, van Loosdregt J, Gorlani A, Bekker CP, Grone€ A, Sibilia M, et al. Dynamics of chemokine, cytokine, and growth factor serum levels in BRAF- Amphiregulin enhances regulatory T cell-suppressive function via the mutant melanoma patients during BRAF inhibitor treatment. J Immunol epidermal growth factor receptor. Immunity 2013;38:275–84. 2014;192:2505–13.

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1651

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, et al.

Clin Cancer Res 2015;21:1639-1651. Published OnlineFirst January 14, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-14-2339

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2015/01/15/1078-0432.CCR-14-2339.DC1

Cited articles This article cites 33 articles, 17 of which you can access for free at: http://clincancerres.aacrjournals.org/content/21/7/1639.full#ref-list-1

Citing articles This article has been cited by 40 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/21/7/1639.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/21/7/1639. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.