Chengwen Liu

BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor Microenvironment in Patients with Metastatic Melanoma

Purpose: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. Experimental Design: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines. Results: Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition. Conclusions: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma. Clin Cancer Res; 19(5); 1225–31. ©2013 AACR.

Loss of PTEN promotes resistance to T cell–mediated immunotherapy

UNLABELLED T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. SIGNIFICANCE This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.

Persistent antigen at vaccination sites induces tumor-specific CD8+ T cell sequestration, dysfunction and deletion

To understand why cancer vaccine–induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freunds adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8+ T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

Plasmacytoid dendritic cells induce NK cell–dependent, tumor antigen–specific T cell cross-priming and tumor regression in mice

A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-gamma production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8(+) T cells.

BRAF Inhibition Increases Tumor Infiltration by T cells and Enhances the Antitumor Activity of Adoptive Immunotherapy in Mice

Purpose: Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors. Experimental Design: BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2Db to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen–independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples. Results: We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model. Conclusion: These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell–based immunotherapy for the treatment of patients with melanoma. Clin Cancer Res; 19(2); 393–403. ©2012 AACR.

PD-1 BLOCKADE ENHANCES T CELL MIGRATION TO TUMORS BY ELEVATING IFN-γ INDUCIBLE CHEMOKINES

Adoptive cell transfer (ACT) is considered a promising modality for cancer treatment, but despite ongoing improvements, many patients do not experience clinical benefits. The tumor microenvironment is an important limiting factor in immunotherapy that has not been addressed fully in ACT treatments. In this study, we report that upregualtion of the immunosuppressive receptor programmed cell death-1 (PD-1) expressed on transferred T cells at the tumor site, in a murine model of ACT, compared with its expression on transferred T cells present in the peripheral blood and spleen. As PD-1 can attenuate T-cell-mediated antitumor responses, we tested whether its blockade with an anti-PD-1 antibody could enhance the antitumor activity of ACT in this model. Cotreatment with both agents increased the number of transferred T cells at the tumor site and also enhanced tumor regressions, compared with treatments with either agent alone. While anti-PD-1 did not reduce the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells present in tumor-bearing mice, we found that it increased expression of IFN-γ and CXCL10 at the tumor site. Bone marrow-transplant experiments using IFN-γR-/- mice implicated IFN-γ as a crucial nexus for controlling PD-1-mediated tumor infiltration by T cells. Taken together, our results imply that blocking the PD-1 pathway can increase IFN-γ at the tumor site, thereby increasing chemokine-dependent trafficking of immune cells into malignant disease sites.

ONCOGENIC BRAF(V600E) PROMOTES STROMAL CELL-MEDIATED IMMUNOSUPPRESSION VIA INDUCTION OF INTERLEUKIN-1 IN MELANOMA

Purpose: In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF). Experimental Design: Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression. Results: Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1α) and IL-1β in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient–derived TAFs with IL-1α/β significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs. Conclusions: This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions. Clin Cancer Res; 18(19); 5329–40. ©2012 AACR.

BRAF(V600) Inhibitor GSK2118436 Targeted Inhibition of Mutant BRAF in Cancer Patients Does Not Impair Overall Immune Competency

Purpose: An intact immune system likely contributes to the outcome of treatment and may be important for clearance of drug-resistant tumor cells and for prevention of recurrence. Although pharmacologic inhibition of BRAF(V600E) in melanoma patients, which is linked to immune suppression, results in an initial response rate, these responses are typically of limited duration. Combining immunotherapeutic drugs with kinase-targeted agents is an attractive strategy to increase clinical efficacy. Evidence suggesting that mitogen-activated protein kinase pathway activation in tumor cells contributes to immune suppression suggests that the two approaches may be synergistic, provided that BRAF(V600E) inhibitors are nontoxic to immune cells. Methods: To assess effects of mutant BRAF inhibition on systemic immunity, we studied 13 patients with tumors carrying a BRAF mutation who underwent treatment with GSK2118436, a V600 mutant BRAF-specific inhibitor. We carried out peripheral blood immunomonitoring before and following one or two 28-day cycles of treatment. Results: GSK2118436 treatment had no detectable impact on most immune parameters tested, including serum cytokine levels, peripheral blood cell counts, leukocyte subset frequencies, and memory CD4+ and CD8+ T-cell recall responses. A slight increase in serum TNF-α over the course of treatment was observed. In addition, three of the four human leukocyte antigen-A2–positive patients experienced a modest increase in circulating tumor antigen–specific CD8+ T cells following BRAF(V600) inhibitor therapy. Conclusions: GSK2118436 treatment results in no detectable negative impact on existing systemic immunity or the de novo generation of tumor-specific T cells. These findings suggest that future trials combining specific BRAF(V600E) inhibition with immunotherapy should not impair immune response. Clin Cancer Res; 18(8); 2326–35. ©2012 AACR.

Transduction of Tumor-Specific T Cells with CXCR2 Chemokine Receptor Improves Migration to Tumor and Antitumor Immune Responses

Purpose: One of the most important rate-limiting steps in adoptive cell transfer (ACT) is the inefficient migration of T cells to tumors. Because melanomas specifically express the chemokines CXCL1 and CXCL8 that are known to facilitate the CXCR2-dependent migration by monocytes, our aim is to evaluate whether introduction of the CXCR2 gene into tumor-specific T cells could further improve the effectiveness of ACT by enhancing T-cell migration to tumor. Experimental Design: In this study, we used transgenic pmel-1 T cells, which recognize gp100 in the context of H-2Db, that were transduced with luciferase gene to monitor the migration of transferred T cells in vivo. To visualize luciferase-expressing T cells within a tumor, a nonpigmented tumor is required. Therefore, we used the MC38 tumor model, which naturally expresses CXCL1. Results: Mice bearing MC38/gp100 tumor cells treated with CXCR2/luciferase-transduced pmel-1 T cells showed enhanced tumor regression and survival compared with mice receiving control luciferase-transduced pmel-1 T cells. We also observed preferential accumulation of CXCR2-expressing pmel-1 T cells in the tumor sites of these mice using bioluminescence imaging. A similar enhancement in tumor regression and survival was observed when CXCR2-transduced pmel-1 T cells were transferred into mice bearing CXCL1-transduced B16 tumors compared with mice treated with control pmel-1 T cells. Conclusions: These results implicate that the introduction of the CXCR2 gene into tumor-specific T cells can enhance their localization to tumors and improve antitumor immune responses. This strategy may ultimately enable personalization of cancer therapies based on chemokine expression by tumors. Clin Cancer Res; 16(22); 5458–68. ©2010 AACR.

IFN-α Enhances Peptide Vaccine-Induced CD8+ T Cell Numbers, Effector Function, and Antitumor Activity

Type I IFNs, including IFN-α, enhance Ag presentation and promote the expansion, survival, and effector function of CD8+ CTL during viral infection. Because these are ideal characteristics for a vaccine adjuvant, we examined the efficacy and mechanism of exogenous IFN-α as an adjuvant for antimelanoma peptide vaccination. We studied the expansion of pmel-1 transgenic CD8+ T cells specific for the gp100 melanocyte differentiation Ag after vaccination of mice with gp10025–33 peptide in IFA. IFN-α synergized with peptide vaccination in a dose-dependent manner by boosting relative and absolute numbers of gp100-specific T cells that suppressed B16 melanoma growth. IFN-α dramatically increased the accumulation of gp100-specific, IFN-γ-secreting, CD8+ T cells in the tumor through reduced apoptosis and enhanced proliferation of Ag-specific CD8+ T cells. IFN-α treatment also greatly increased the long-term maintenance of pmel-1 CD8+ T cells with an effector memory phenotype, a process that required expression of IFN-α receptor on the T cells and IL-15 in the host. These results demonstrate the efficacy of IFN-α as an adjuvant for peptide vaccination, give insight into its mechanism of action, and provide a rationale for clinical trials in which vaccination is combined with standard-of-care IFN-α therapy for melanoma.