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Purpose: A CTEP-sponsored phase II trial was conducted to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma. Experimental Design: Patients with unresectable stage III to IV melanoma were treated intravenously with temsirolimus 25 mg weekly and bevacizumab 10 mg every 2 weeks. Adverse events were recorded using CTCAE v3.0. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors and overall survival was recorded. Correlative studies measured protein kinases and histology of tumor biopsies and immune function in peripheral blood. Results: Seventeen patients were treated. Most patients tolerated treatment well, but 2 had grade 4 lymphopenia and 1 developed reversible grade 2 leukoencephalopathy. Best clinical response was partial response (PR) in 3 patients [17.7%, 90% confidence interval (CI) 5, 0–39.6], stable disease at 8 weeks (SD) in 9 patients, progressive disease (PD) in 4 patients, and not evaluable in 1 patient. Maximal response duration for PR was 35 months. Ten evaluable patients had BRAFWT tumors, among whom 3 had PRs, 5 had SD, and 2 had PD. Correlative studies of tumor biopsies revealed decreased phospho-S6K (d2 and d23 vs. d1, P < 0.001), and decreased mitotic rate (Ki67+) among melanoma cells by d23 (P = 0.007). Effects on immune functions were mixed, with decreased alloreactive T-cell responses and decreased circulating CD4+FoxP3+ cells. Conclusion: These data provide preliminary evidence for clinical activity of combination therapy with temsirolimus and bevacizumab, which may be greater in patients with BRAFwt melanoma. Mixed effects on immunologic function also support combination with immune therapies. Clin Cancer Res; 19(13); 3611–20. ©2013 AACR.

Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in two clinical trials of melanoma patients. In one study (Mel38), patients received one injection with an adjuvant mixture alone, comprised of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature dendritic cells (DC) and vessels resembling high endothelial venules (HEV). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DC, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4+ and CD8+ T lymphocytes, as well as FoxP3+CD4+ lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination.

Food and Drug Administration-approved treatment for metastatic melanoma, including interferon alpha and interleukin-2, offer a modest benefit. Immunotherapy, although has not enjoyed high overall response rates, is capable of providing durable responses in a subset of patients. In recent years, new molecular-targeted therapies have become available and offer promise of clinical benefit, although low durability of response. It is not yet clear how best to integrate these 2 novel modalities that target the immune response to melanoma (immune therapy) or that target molecular signaling pathways in the melanoma cells (targeted therapy). Many signal transduction pathways are important in both tumor cell and T-cell proliferation and survival, which generate risk in combining targeted therapy and immunotherapy. This review focuses on the role of targeted therapy and immunotherapy in melanoma, and discusses how to combine the 2 modalities rationally for increased duration and response.

Background Targeted therapies directed at commonly overexpressed pathways in melanoma have clinical activity in numerous trials. Little is known about how these therapies influence microRNA (miRNA) expression, particularly with combination regimens. Knowledge of miRNAs altered with treatment may contribute to understanding mechanisms of therapeutic effects, as well as mechanisms of tumor escape from therapy. We analyzed miRNA expression in metastatic melanoma tissue samples treated with a novel combination regimen of Temsirolimus and Bevacizumab. Given the preliminary clinical activity observed with this combination regimen, we hypothesized that we would see significant changes in miRNA expression with combination treatment. Methods Using microarray analysis we analyzed miRNA expression levels in melanoma samples from a Cancer Therapy Evaluation Program-sponsored phase II trial of combination Temsirolimus and Bevacizumab in advanced melanoma, which elicited clinical benefit in a subset of patients. Pre-treatment and post-treatment miRNA levels were compared using paired t-tests between sample groups (patients), using a p-value Results microRNA expression remained unchanged with Temsirolimus alone; however, expression of 15 microRNAs was significantly upregulated (1.4 to 2.5-fold) with combination treatment, compared to pre-treatment levels. Interestingly, twelve of these fifteen miRNAs possess tumor suppressor capabilities. We identified 15 putative oncogenes as potential targets of the 12 tumor suppressor miRNAs, based on published experimental evidence. For 15 of 25 miRNA-target mRNA pairings, changes in gene expression from pre-treatment to post-combination treatment samples were inversely correlated with changes in miRNA expression, supporting a functional effect of those miRNA changes. Clustering analyses based on selected miRNAs suggest preliminary signatures characteristic of clinical response to combination treatment and of tumor BRAF mutational status. Conclusions To our knowledge, this is the first study analyzing miRNA expression in pre-treatment and post-treatment human metastatic melanoma tissue samples. This preliminary investigation suggests miRNAs that may be involved in the mechanism of action of combination Temsirolimus and Bevacizumab in metastatic melanoma, possibly through inhibition of oncogenic pathways, and provides the preliminary basis for further functional studies of these miRNAs.

BackgroundTargeted inhibition of protein kinases is now acknowledged as an effective approach for cancer therapy. However, targeted therapies probably have limited success because cancer cells have alternate pathways for survival and proliferation thereby avoiding inhibition. We tested the hypothesis that combination of targeted agents would be more effective than single agents in arresting melanoma cell proliferation.MethodsWe evaluated whether BAY43-9006, an inhibitor of the B-Raf kinase, and rapamycin, an inhibitor of the mTOR kinase, would inhibit serum-stimulated proliferation of human melanoma cell lines, either alone or in combination. Proliferation was measured by quantitating melanoma cell numbers with a luciferase for ATP. Phosphorylation of proteins downstream of targeted kinase(s) was assayed by immunoblots. Statistical significance was determined with the Student-T test. Isobologram analysis was performed to distinguish additive versus synergistic effects of combinations of drugs.ResultsSerum-stimulated proliferation of multiple human melanoma cell lines was inhibited by BAY43-9006 and by rapamycin. Melanoma cells containing the B-Raf mutation V599E were more sensitive than cells with wild-type B-raf to 10 nM doses of both BAY43-9006 and rapamycin. Regardless of B-Raf mutational status, the combination of low dose rapamycin and BAY43-9006 synergistically inhibited melanoma cell proliferation. As expected, rapamycin inhibited the phosphorylation of mTOR substrates, p70S6K and 4EBP1, and BAY43-9006 inhibited phosphorylation of ERK, which is dependent on B-Raf activity. We also observed unexpected rapamycin inhibition of the phosphorylation of ERK, as well as BAY43-9006 inhibition of the phosphorylation of mTOR substrates, p70S6K and 4EBP1.ConclusionThere was synergistic inhibition of melanoma cell proliferation by the combination of rapamycin and BAY 43-9006, and unexpected inhibition of two signaling pathways by agents thought to target only one of those pathways. These results indicate that combinations of inhibitors of mTOR and of the B-raf signaling pathways may be more effective as a treatment for melanoma than use of either agent alone.

8530 Background: A CTEP-sponsored phase II trial was performed to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma. Correlative studies assessed mTOR signaling in tumor biopsies and evidence of induced immunologic dysfunction systemically. Methods: 17 patients with stage III or IV melanoma were enrolled and treated with temsirolimus (25 mg IV weekly) and bevacizumab (10 mg/kg IV every 14d, starting d.8) for up to 13 months. Clinical response was determined by RECIST criteria. Adverse events were assessed (CTCAE v3.0). Blood was collected d.1, 2, and 23 (to assess immune function), and tumor biopsies were obtained (to assess protein kinase activity and melanoma cell proliferation). Results: Treatment-related grade 3 or 4 adverse events occurred in 5 and 1 patients, respectively; 1 patient developed reversible leukoencephalopathy. In 16 patients evaluable for clinical response, best overall response was a partial response (PR) in 3 patients (19%), stable disease at 8 weeks (SD) in 9 patients (56%), and progressive disease in 4 patients. Thus, disease control rate (DCR = PR + SD) was 75%. Ten of the patients had BRAF wild-type (BRAFwt) melanomas: these accounted for the 3 PRs (30%), and a DCR of 100%. Maximal response duration has exceeded 3 years for a BRAFwt patient. mTOR signaling was inhibited in melanoma metastases, based on decreased phospho-S6 kinase after 24h temsirolimus. Ki67+ melanoma cells in tumor biopsies decreased significantly by day 23 (p = 0.007, F-test), most notably in clinical responders. There was no significant alteration of T cell and NK function with combination treatment, by ELIspot and cytotoxicity assays. Conclusions: Combination therapy with temsirolimus and bevacizumab is well-tolerated in patients with advanced melanoma and has intriguing clinical activity. The most notable responses were in patients with BRAFwt tumors, a population with no accepted effective targeted therapy. Decreases in Ki67+ melanoma cells may be associated with clinical response. The lack of immunologic dysfunction supports future combination with immune therapies.

Cellular signals are not necessarily transmitted via linear pathways, but through a dynamic interconnected network. Effective pharmacologic inhibition of cellular signaling in malignant cells will thus require identifying the key nodes, and intervening at more than one site within the network. To systematically identify functional interactions between signaling proteins within the cell signaling network we have screened nine melanoma cell lines of diverse genetic backgrounds for sensitivity to combinations of drugs targeting the major signaling pathways altered in cancer. Seven drugs representative of standard targets were designated as “primary drugs”. These seven primary targeted therapies were screened against each other and an additional 67 signal transduction inhibitors. The screen was performed robotically with a Biomek NX workstation in a 96-well format. Cell viability was measured with alamarBlue on a microplate reader. Various concentrations of the secondary drug were tested against a partial inhibitory concentration of the primary drug. Twelve percent of a total of 4672 drug dose and cell combinations tested displayed superadditivity in vitro. Nearly 10% of those hits demonstrated greater than 50% superadditivity according to first principles analysis; the actual growth inhibition of the combination was more than 50% greater than the sum of the growth inhibition produced by each drug alone. One intriguing drug combination showing superadditivity in most cell lines tested was sorafenib plus diclofenac. The degree of inhibition and concentration in which superadditivity was observed was cell line dependent, and was independent of known mutational status. The MEK inhibitor, PD325901, was able to substitute qualitatively for sorafenib indicating that Raf may be the major target for sorafenib when used in combination with diclofenac. Consistent with this, sorafenib inhibited MEK and ERK activity at doses that achieved superadditivity with diclofenac. However, the combination of PD325901 with diclofenac was less robust at inhibiting growth than sorafenib plus diclofenac suggesting that the alternate sorafenib targets may play a role in the observed superadditivity. Celecoxib, a COX2 inhibitor, or ibuprofen, a preferential COX1 inhibitor, substituted for diclofenac indicating that diclofenac was acting as a COX inhibitor, although the inhibition was considerably less robust, suggesting a role for a balanced inhibition of COX1 and COX2, or a role for off-target effects. Collectively, these results indicate an unexpected functional interaction between Raf and COX signaling in melanoma. Since both sorafenib and diclofenac are in clinical use, the therapeutic potential of these observations is striking and is being further explored in pre-clinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3843.