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PDF file - 190K, Flow cytometric analysis of H2-Db expression by SM1 with or without exposure to vemurafenib for different periods

PDF file - 77K, 18FFAC PET imaging of adoptively transferred splenocytes with or without vemurafenib

PDF file - 41K, Effects of vemurafenib on the cytotoxic effects of pmel-1 adoptive cell transfer (ACT) at a high initial dose of cells

PDF file - 117K, Analysis of melanoma-like genome-wide DNA copy number alterations in the SM1 cell line

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PDF file - 109K, Effects of vemurafenib on the systemic distribution of adoptively transferred pmel-1 splenocytes

Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma. Cancer Res; 72(16); 3928–37. ©2012 AACR.

Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

Adoptive T cell transfer (ACT) based immunotherapy for melanoma can induce remarkable and highly durable tumor responses, which may last many years. Vemurafenib (Vmf) is a potent inhibitor of BRAF mutated at V600E with response rates of up to 80% in patients with metastatic melanoma. However, drug resistance develops in most of patients leading to response durations of only several months. We previously showed that the combined approach of BRAF inhibition with adoptive cell immunotherapy leads to an improved outcome in vivo. Here we show that Vmf also directly and independently affects T lymphocytes, resulting in favorable anti-tumoral phenotypic changes. We had created a transplantable murine melanoma cell-line driven by V600E BRAF oncogene (SM1) derived from a spontaneously arising melanoma in transgenic mice harboring the V600E BRAF mutation under the control of tyrosinase promoter. SM1 cells stably expressing the ovalbumin (OVA) model antigen were implanted in C57BL6 mice. Daily i.p. Vmf combined with ACT of OVA-specific TCR transgenic cells generated by retroviral transduction demonstrated superior tumor control of the combined treatment in comparison to each treatment alone. We also confirmed better outcomes with this combination in the pmel-1 model, which is based on the ACT of TCR transgenic cells against the endogenously expressed and relevant melanoma antigen, gp100. We then cultured primary T cells in the presence of Vmf with a broad range of concentration (0.1 to up to 100 uM). There was no evidence of cytotoxicity, but interestingly, T cells differentiated into a phenotype resembling T central memory (CM) cells (CD44+, CD62L+) in a dose dependent manner as assessed by flow-cytometry. CM T cells were shown to induce superior anti-tumoral responses in comparison to ACT of Effector T cells in murine models. As expected for CM T cells, further analysis of 24 h collection supernatants from in vitro cognate peptide stimulated T cells showed dose-dependent lower interferon-gamma secretion with Vmf as analyzed by ELISA. Pmel-1 T cells were then analyzed by Immunoblotting for phosphorylation status (activation) of protein kinases at 1, 5, 15, 30 min and at 24h after Vmf treatment (concentrations from 1 to 15 uM). Vmf induced increased levels of pERK and pMEK. Co-immunoprecipitation studies and kinase assays further demonstrated a role of C-Raf in this paradoxical activation of the MAPK pathway in T cells induced by Vmf. Furthermore, in vivo studies in C57BL6 mice treated with Vmf daily or vehicle control for 3 weeks demonstrated skewing of CD3+ cells towards a phenotype resembling central memory T cells (CD44+, CD62L+, LY-6C+). Taken all our data together, we provide further support for the rationale of combining BRAF targeted therapy and adoptive T cell immunotherapy and the testing of such combinations in patients with V600E BRAF mutant metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3510. doi:1538-7445.AM2012-3510

Vemurafenib blocks activated BRAF with V600E driver mutation and induces unprecedented high response rates in patients with metastatic melanoma. However, most patients have response durations limited to only several months. Conversely, immunotherapy based on adoptive transfer of T cells has induced low frequency, but highly durable tumor responses. We previously reported (Clin Cancer Res. 16(24), 2010) that T lymphocytes exposed to high concentrations of vemurafenib had preserved viability and function, providing a compelling rationale for a combined targeted drug/immunotherapy approach. We first established an implantable murine melanoma cell line driven by the V600E BRAF oncogene (SM1) from a spontaneously arising melanoma in transgenic mice harboring the V600E BRAF mutation under the control of tyrosinase promoter. SM1 cells exposed to vemurafenib had partial in vitro sensitivity (IC50 of 14 uM) resulting from inhibition of MAPK pathway signaling (as demonstrated by Immunoblotting), while murine lymphocytes were spared. In vitro assays indicated that SM1 cells undergo apoptosis and cell cycle arrest at G1 phase in the presence of vemurafenib. Mice implanted with SM1 tumors responded significantly with daily i.p. injection of vemurafenib, confirming its efficacy in vivo. We then tested the combination of vemurafenib and immunotherapy in vivo using two models of adoptive cell transfer (ACT) therapy. OT-1 T cell receptor-expressing lymphocytes targeting ovalbumin (OVA) present in SM1-OVA tumors or pmel-1 lymphocytes targeting the melanoma-associated-antigen gp100 (endogenously expressed by SM1) combined with daily i.p. injections of vemurafenib resulted in superior antitumor responses compared to either therapy alone. We then quantified the adoptively transferred T cells in spleen and tumor biopsies by tissue immunostaining. There were no significant differences in numbers of T cells infiltrating the tumors with or without vemurafenib. Further analysis with two different molecular imaging-based in vivo T cell tracking (1-Luciferin bioluminescence and 2-Positron Emission Tomography with dFAC tracer) confirmed that vemurafenib did not significantly alter the expansion, distribution or tumor accumulation of the adoptively transferred T cells. Also, vemurafenib did not alter SM1 antigen presentation as demonstrated by lack of significant differences in gp100 expression and MHC-I levels in SM1, as well as, in vivo cytotoxic activity of adoptively transferred cells against their cognate antigen. We then performed immunophenotypic analysis of transferred T cells. Vemurafenib skewed these cells towards a phenotype resembling central memory T cells, a favorable characteristic for superior and prolonged tumor control. Further functional analysis of tumor infiltrating lymphocytes indicated increased interferon-gamma secretion as assayed by intracellular staining and FACS. In conclusion, our data derived from two independent models combining BRAF targeted therapy and immunotherapy indicated favorable changes in T cell function/immunophenotype and support the testing of such combination in patients with BRAFV600 mutant metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A65.

PLX4032 is a potent inhibitor of V600E BRAF with response rates of up to 80% in patients with metastatic melanoma. However, drug resistance develops in most of patients leading to response durations of only several months. Immunotherapy for melanoma has induced low frequency, but highly durable tumor responses, which may last years. Therefore, the concept of a combined approach of BRAF inhibition and immunotherapy is very attractive. We have established a novel animal model which allows the testing of this concept in fully immunocompetent mice. An implantable murine melanoma cell line driven by the V600E BRAF oncogene (SM1) was established from a spontaneously arising murine melanoma in transgenic mice backcrossed to C57BL/6 and harboring the V600E BRAF mutation under the control of tyrosinase promoter. The in vitro IC50 of PLX4032 against SM1 was 14μM, which indicates partial resistance to the drug and therefore useful as a model for combined therapy. In vitro assays indicated that SM1 cells undergo apoptosis and cell cycle arrest at G1 phase in the presence of the PLX4032. Mice implanted with tumors responded significantly with daily i.p. injection of PLX4032, confirming its efficacy in vivo, though there was no complete eradication of the tumor. Cultured primary murine T lymphocytes exposed to PLX4032 at different time points (24, 48 and 72h) with a broad range of concentration (0.1 to up to 100 μM) showed no evidence of cytotoxicity. In vivo real-time T cell tracking by luciferase bioluminescence imaging, as well as PET/CT, confirmed that PLX4032 did not adversarially affect the viability, engraftment or tumor infiltration of T cells in mice. We then tested the combination of PLX4032 and immunotherapy in vivo using two models of adoptive cell transfer (ACT) therapy. SM1 cells stably expressing the chicken ovalbumin (OVA) model antigen were implanted in C57BL6 mice. Daily i.p. PLX4032 combined with ACT of OVA-specific TCR transgenic cells (OTI) generated by retroviral transduction demonstrated superior effects of the combined treatment (tumor size at day 14 after ACT plus PLX4032: 114.9±23.6 mm2, PLX4032: 203.2±6.2, OTI ACT: 192.5±6.2, and vehicle control 241.3±3.1, p In conclusion, our data derived from two independent models combining BRAF targeted therapy and immunotherapy support the testing of such combinations in patients with V600E BRAF mutant metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2011-656