Kerrington R. Molhoek

Synergistic inhibition of human melanoma proliferation by combination treatment with B-Raf inhibitor BAY43-9006 and mTOR inhibitor Rapamycin.

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.

Human Melanoma Cytolysis by Combined Inhibition of Mammalian Target of Rapamycin and Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor-2

Vascular endothelial growth factor (VEGF) plays a vital role in tumor angiogenesis. VEGF is produced by human melanomas, and the VEGF receptor 2 (VEGFR-2) is expressed by most advanced stage melanomas, suggesting the possibility of an autocrine loop. Here, we show that bevacizumab, an anti-VEGF antibody, inhibits proliferation of VEGFR-2(+) melanoma cell lines by an average of 41%; however, it failed to inhibit proliferation of VEGFR-2(neg) melanoma cell lines. The growth inhibitory effect of bevacizumab was eliminated by VEGFR-2 knockdown with small interfering RNA, showing that VEGF autocrine growth in melanoma is mediated through VEGFR-2. However, bevacizumab inhibition of autocrine signals did not completely inhibit cell proliferation nor cause cell death. Cell survival is mediated partially through mammalian target of rapamycin (mTOR), which is inhibited by rapamycin. Combination of bevacizumab with rapamycin caused loss of half of the VEGFR-2(+) melanoma cells, but no reduction in the number of VEGFR-2(neg) melanoma cells. The results show (a) an autocrine growth loop active in VEGFR-2(+) melanoma, (b) a nonangiogenic mechanism for inhibition of melanoma by blocking autocrine VEGFR-2 activation, and (c) a possible therapeutic role for combination of inhibitors of mTOR plus VEGF in selected melanomas.

Comprehensive analysis of receptor tyrosine kinase activation in human melanomas reveals autocrine signaling through IGF-1R.

Melanomas depend on autocrine signals for proliferation and survival; however, no systematic screen of known receptor tyrosine kinases (RTKs) has been performed to identify which autocrine signaling pathways are activated in melanoma. Here, we performed a comprehensive analysis of 42 RTKs in six individual human melanoma tumor specimens as well as 17 melanoma cell lines, some of which were derived from the tumor specimens. We identified five RTKs that were active in almost every one of the melanoma tissue specimens and cell lines, including two previously unreported receptors, insulin-like growth factor receptor 1 (IGF-1R) and macrophage-stimulating protein receptor (MSPR), in addition to three receptors (vascular endothelial growth factor receptor, fibroblast growth factor receptor, and hepatocyte growth factor receptor) known to be autocrine activated in melanoma. We show, by quantitative real time PCR, that all melanoma cell lines expressed genes for the RTK ligands such as HGF, IGF-1, and MSP. Addition of antibodies to either IGF-1 or HGF, but not to MSP, to the culture medium blocked melanoma cell proliferation, and even caused net loss of melanoma cells. Antibody addition deactivated IGF-1R and hepatocyte growth factor receptors, as well as mitogen-activated protein kinase signaling. Thus, IGF-1 is a new growth factor for autocrine driven proliferation of human melanoma in vitro. Our results suggest that IGF-1—IGF-1R autocrine pathway in melanoma is a possible target for therapy in human melanomas.

The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides.

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 (DC) 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 2 clinical trials of melanoma patients. In 1 study (Mel38), patients received 1 injection with an adjuvant mixture alone, composed of incomplete Freunds 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 DCs, and vessels resembling high endothelial venules (HEVs). 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 DCs, 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.

Clinical activity and safety of combination therapy with temsirolimus and bevacizumab for advanced melanoma: a phase II trial (CTEP 7190/Mel47).

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.

Evaluation of molecular markers of mesenchymal phenotype in melanoma.

The epithelial to mesenchymal transition is a developmental process allowing epithelial cells to dedifferentiate into cells displaying mesenchymal phenotypes. The pathological role of epithelial to mesenchymal transition has been implicated in invasion and metastasis for numerous carcinomas, yet limited data exist addressing whether mesenchymal transition (MT) occurs in malignant melanoma cells. Our group developed an in-vitro three-dimensional culture system to address MT in melanoma cells upon transforming growth factor-β/ tumor necrosis factor-α treatment. Loss of E-cadherin is one of the best indicators of MT in epithelial cells. Not surprisingly, E-cadherin was expressed in only three of 12 (25%) melanoma cell lines and all three mesenchymal proteins, N-cadherin, vimentin, and fibronectin, were expressed by seven (58%) melanoma cell lines. However, after cytokine treatment, two or more mesenchymal proteins were elevated in nine (75%) melanoma cell lines. Data support the transforming growth factor-β production by melanoma cells which may induce/support MT. Evaluation of E-cadherin, N-cadherin, and Snail expression in melanoma tissue samples are consistent with an inverse coupling of E-cadherin and N-cadherin expression, however, there are also examples suggesting a more complex control of their expression. These results indicate that malignant melanoma cell lines are susceptible to MT after cytokine treatment and highlight the importance of understanding the effects of cytokines on melanoma to undergo MT.

MicroRNAs induced in melanoma treated with combination targeted therapy of Temsirolimus and Bevacizumab.

BackgroundTargeted 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.MethodsUsing 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 < 0.01 for significance.ResultsmicroRNA 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.ConclusionsTo 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.

VEGFR-2 expression in human melanoma: revised assessment.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that also functions as an autocrine growth factor for VEGF receptor (VEGFR)‐2+ melanomas. In multiple studies, VEGFR‐2 was detected by immunostaining in 78–89% of human melanoma cells, suggesting that most patients with melanoma would benefit from anti‐VEGF therapy. Here, we evaluated 167 human melanoma specimens in a tissue microarray to verify the presence of VEGFR‐2, but found disparities in staining with commercial antibodies A‐3 and 55B11. Antibody A‐3 stained melanoma cells in 79% of specimens, consistent with published results; however, we noted extensive nonspecific staining of other cells such as smooth muscle and histiocytes. In contrast, antibody 55B11 stained melanoma cells in only 7% (95% confidence interval: 3.3–11.5) of specimens. As an internal positive control for VEGFR‐2 detection, vascular endothelial cells were stained with antibody 55B11 in all specimens. We compared VEGFR‐2+ and VEGFR‐2− melanoma cell lines by immunoblotting and immunohistochemistry after small interfering RNA (siRNA) knockdown and transient overexpression of VEGFR‐2 to validate antibody specificity. Immunoblotting revealed that A‐3 primarily cross‐reacted with several proteins in both cell lines and these were unaffected by siRNA knockdown of VEGFR‐2. In contrast, 55B11 staining of VEGFR‐2+ cells was mostly eliminated by siRNA knockdown of VEGFR‐2 and increased in VEGFR‐2− melanoma cell lines following transfection to express ectopic VEGFR‐2. Our results show that relatively few melanoma cells (<10%) express detectable levels of VEGFR‐2, and therefore, the majority of patients with melanoma are unlikely to benefit from antiproliferative effects of anti‐VEGF therapy.

Interface of Signal Transduction Inhibition and Immunotherapy in Melanoma

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.

Abstract 3843: Rational selection of combinatorial therapeutic targets for melanoma identified by synthetic lethal screening with small molecule inhibitors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.