A B S T R A C T PurposeNivolumab, a human immunoglobulin G 4 -blocking antibody against the T-cell programmed death-1 checkpoint protein, has activity against metastatic melanoma. Its safety, clinical efficacy, and correlative biomarkers were assessed with or without a peptide vaccine in ipilimumab-refractory and -naive melanoma. Patients and MethodsIn this phase I study, 90 patients with unresectable stage III or IV melanoma who were ipilimumab naive and had experienced progression after at least one prior therapy (cohorts 1 to 3, 34 patients) or experienced progression after prior ipilimumab (cohorts 4 to 6, 56 patients) received nivolumab at 1, 3, or 10 mg/kg every 2 weeks for 24 weeks, then every 12 weeks for up to 2 years, with or without a multipeptide vaccine. ResultsNivolumab with vaccine was well tolerated and safe at all doses. The RECIST 1.1 response rate for both ipilimumab-refractory and -naive patients was 25%. Median duration of response was not reached at a median of 8.1 months of follow-up. High pretreatment NY-ESO-1 and MART-1-specific CD8 ϩ T cells were associated with progression of disease. At week 12, increased peripheral-blood T regulatory cells and decreased antigen-specific T cells were associated with progression. PD-L1 tumor staining was associated with responses to nivolumab, but negative staining did not rule out a response. Patients who experienced progression after nivolumab could respond to ipilimumab. ConclusionIn patients with ipilimumab-refractory or -naive melanoma, nivolumab at 3 mg/kg with or without peptide vaccine was well tolerated and induced responses lasting up to 140 weeks. Responses to nivolumab in ipilimumab-refractory patients or to ipilimumab in nivolumab-refractory patients support combination or sequencing of nivolumab and ipilimumab.

Expression of PD-1 ligands by tumors and interaction with PD-1 expressing T cells in the tumor microenvironment can result in tolerance. Therapies targeting this co-inhibitory axis have proven clinically successful in the treatment of metastatic melanoma, non-small cell lung cancer and other malignancies. Therapeutic agents targeting the epigenetic regulatory family of histone deacetylases (HDACs) have shown clinical success in the treatment of some hematologic malignancies. Beyond direct tumor cell cytotoxicity, HDAC inhibitors have also been shown to alter the immunogenicity and enhance anti-tumor immune responses. Here we show that class I histone deacetylase inhibitors upregulated the expression of PD-L1 and, to a lesser degree, PD-L2 in melanomas. Evaluation of human and murine cell lines and patient tumors treated with a variety of HDAC inhibitors in vitro displayed upregulation of these ligands. This upregulation was robust and durable, with enhanced expression lasting past 96 hours. These results were validated in vivo in a B16F10 syngeneic murine model. Mechanistically, HDAC inhibitor treatment resulted in rapid upregulation of histone acetylation of the PD-L1 gene leading to enhanced and durable gene expression. The efficacy of combining HDAC inhibition with PD-1 blockade for treatment of melanoma was also explored in a murine B16F10 model. Mice receiving combination therapy had a slower tumor progression and increased survival compared to control and single agent treatments. These results highlight the ability of epigenetic modifiers to augment immunotherapies, providing a rationale for combining HDAC inhibitors with PD-1 blockade.

Calcium signaling mediated by STIM1 and Orai1 activates Src to promote invadopodium assembly while simultaneously promoting MT1-MMP recycling to the plasma membrane to promote ECM degradation.

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PURPOSE Effective treatment options are limited for patients with advanced (metastatic or unresectable) melanoma who progress after immune checkpoint inhibitors and targeted therapies. Adoptive cell therapy using tumor-infiltrating lymphocytes has demonstrated efficacy in advanced melanoma. Lifileucel is an autologous, centrally manufactured tumor-infiltrating lymphocyte product. METHODS We conducted a phase II open-label, single-arm, multicenter study in patients with advanced melanoma who had been previously treated with checkpoint inhibitor(s) and BRAF ± MEK targeted agents. Lifileucel was produced from harvested tumor specimens in central Good Manufacturing Practice facilities using a streamlined 22-day process. Patients received a nonmyeloablative lymphodepletion regimen, a single infusion of lifileucel, and up to six doses of high-dose interleukin-2. The primary end point was investigator-assessed objective response rate (ORR) per RECIST, version 1.1. RESULTS Sixty-six patients received a mean of 3.3 prior therapies (anti–programmed death 1 [PD-1] or programmed death ligand 1 [PD-L1]: 100%; anticytotoxic T-lymphocyte-associated protein-4: 80%; BRAF ± MEK inhibitor: 23%). The ORR was 36% (95% CI, 25 to 49), with two complete responses and 22 partial responses. Disease control rate was 80% (95% CI, 69 to 89). Median duration of response was not reached after 18.7-month median study follow-up (range, 0.2-34.1 months). In the primary refractory to anti–PD-1 or PD-L1 therapy subset, the ORR and disease control rate were 41% (95% CI, 26 to 57) and 81% (95% CI, 66 to 91), respectively. Safety profile was consistent with known adverse events associated with nonmyeloablative lymphodepletion and interleukin-2. CONCLUSION Lifileucel demonstrated durable responses and addresses a major unmet need in patients with metastatic melanoma with limited treatment options after approved therapy, including the primary refractory to anti–PD-1 or PD-L1 therapy subset.

Purpose The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study demonstrates the potential therapeutic utility of the HSP90 inhibitor (XL888) in 6 different models of vemurafenib resistance. Experimental design The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was demonstrated in vitro and in vivo. A novel mass spectrometry-based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts and melanoma biopsies. Mechanistic studies were performed to determine the mechanism of XL888-induced apoptosis. Results XL888 potently inhibited cell growth, induced apoptosis and prevented the growth of vemurafenib resistant melanoma cell lines in 3D cell culture, long-term colony formation assays and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1 and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM expression and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual MEK/PI3K inhibition. Conclusions HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy.

The median survival for metastatic melanoma is in the realm of 8–16 months and there are few therapies that offer significant improvement in overall survival. One of the recent advances in cancer treatment focuses on epigenetic modifiers to alter the survivability and immunogenicity of cancer cells. Our group and others have previously demonstrated that pan-HDAC inhibitors induce apoptosis, cell cycle arrest and changes in the immunogenicity of melanoma cells. Here we interrogated specific HDACs which may be responsible for this effect. We found that both genetic abrogation and pharmacologic inhibition of HDAC6 decreases in vitro proliferation and induces G1 arrest of melanoma cell lines without inducing apoptosis. Moreover, targeting this molecule led to an important upregulation in the expression of tumor associated antigens and MHC class I, suggesting a potential improvement in the immunogenicity of these cells. Of note, this anti-melanoma activity was operative regardless of mutational status of the cells. These effects translated into a pronounced delay of in vivo melanoma tumor growth which was, at least in part, dependent on intact immunity as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Given our findings, we provide the initial rationale for the further development of selective HDAC6 inhibitors as potential therapeutic anti-melanoma agents.

Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to target a variety of other proteins unrelated to the chromatin environment. In this context, our present work demonstrates that the pharmacological or genetic abrogation of HDAC6 in primary melanoma samples and cell lines, down-regulates the expression of PD-L1, an important co-stimulatory molecule expressed in cancer cells, which activates the inhibitory regulatory pathway PD-1 in T-cells. Our data suggests that this novel mechanism of PD-L1 regulation is mainly mediated by the influence of HDAC6 over the recruitment and activation of STAT3. Additionally, we observed that selective HDAC6 inhibitors impairs tumor growth and reduce the in vivo expression of several inhibitory checkpoint molecules and other regulatory pathways involved in immunosurveillance. Most importantly, these results provide a key pre-clinical rationale and justification to further study isotype selective HDAC6 inhibitors as potential immuno-modulatory agents in cancer. ª 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. IntroductionIn spite of recent progress made in understanding the pathobiology, genetics, and immunology of melanoma, the outcome for patients with advanced-stage disease remains poor with a median survival ranging from 8 to 16 months and an overall survival (OS) at 5 years of less than 10% (Nikolaou and Stratigos, 2014