Faculty Bibliography

T cells play a critical role in host defense against viruses, intra- and extracellular microbes, and tumors. Because foreign antigen is presented amongst a vast majority of self-antigens, T cells have evolved the unique ability to discriminate "self" from "non-self" with high sensitivity and selectivity, enabling the elimination of foreign pathogens while largely avoiding self-reactivity. However, tissue-specific autoimmunity and tolerance to or eradication of cancer does not fit neatly into the self/non-self paradigm because the T cell responses in these situations are not directed to an exogenous pathogen, but rather most often to non-mutated self-proteins. Therefore, an important question is how the immune system establishes suitable thresholds that allow positively selected T cells to interact with selfligands in the periphery without causing overt activation. One hypothesis to explain how a T cell distinguishes among different types of self-ligands is the kinetic proof-reading theory, which relates signaling efficacy to the lifetime of the TCR (T cell receptor)-pMHC (peptide-major histocompatibility complex) interaction. More recently, T cell maturation associated signaling feedback pathways have also been hypothesized to play a role in T cell discrimination of between self-ligands. We are taking a variety of biophysical and cellular imaging approaches to determine how specific thresholds for T cell recognition of self-antigens are set. Our recent results [1] indicate that antitumor activity and autoimmunity are coupled and have a similar kinetic threshold; reducing autoimmunity cannot be accomplished without sacrificing efficacy of tumor killing. Therefore, an "optimal TCR affinity range" that leads to optimal tumor regression and minimal autoimmunity is elusive and treatment strategies focusing on increasing TCR affinities to a supraphysiological level has most likely little therapeutic benefit. Therefore, other approaches are needed to improve the balance between anti-tumor responses and autoimmunity. Our strategy to overcome this issue includes novel methods for careful biophysical engineering of tumor-specific TCRs to carefully balance tumorreactivity and autoimmunity. Furthermore, our recent preliminary data show that TCR-proximal signaling differs significantly between effector memory and central memory T cells due to differential constitutive activity and localization of signaling molecules. Understanding how activation signaling contributes to differences in memory T cell subset sensitivity may provide insight into how T cells can be manipulated to achieve optimal anti-tumor sensitivity. This could lead to adjuvants that target and enhance antigenspecific T cell anti-tumor efficacy. Together may lead to development of cancer immunotherapy approaches with improved outcomes

Introduction: The immunoregulatory protein T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) contributes towards T cell exhaustion in several chronic diseases, including melanoma [1]. NK cells from the latter were shown to be functionally impaired/exhausted, as they failed to proliferate, produce cytokines or kill target cells. In addition they down regulated activating receptors (NKG2D and NKp46) and upregulated inhibitory receptors (KIRB1, KIRNKAT2 and Tim-3). Notably Tim-3 blockade reversed this exhausted phenotype, implicating this molecule as a major checkpoint inhibitor in advanced melanoma [2]. To further evaluate NK cell phenotype and function as a consequence of progressive melanoma, we monitored NK cells from a large cohort of patients with stage I-IV melanoma tested the association between NK cell phenotype and clinicopathological variables associated with melanoma prognosis. Expression of MICA (NKG2D ligand) and HMGB1 (Tim-3 ligand) in the plasma/sera of our main cohort was also monitored in an independent validation cohort. Methods: NK cells were purified from the peripheral blood of melanoma patients. They were evaluated for the expression of activating and inhibitory receptors. Cytotoxicity was measured by Lamp-1 expression. IFN-gamma production was measured after 4h stimulation with rhIL-12. Proliferation was quantified by CFSE after 6 days in the presence of rhIL- 2. MICA and HMGB1 expression on patients' plasma/sera was measured by ELISA. Event-time distributions were estimated with the use of the Kaplan-Meier method. Two tailed t-test unpaired was used to compare samples from different stages. Results: NK cells gradually develop a phenotypic and functional profile consistent with progressive exhaustion, from stage I to stage IV characterized by: 1) up-regulation of inhibitory receptors (Tim-3, KIRB1 and KIRNKAT2); 2) down-regulation of activating receptors (NKG2D and NKp46); 3) loss of IFN-gamma production, proliferation and cytotoxicity. Interestingly, the expression of Tim-3 is higher, while the cytotoxicy and IFN-gamma production is reduced in patients with melanomas thicker than 1mm. Moreover, higher expression of Tim-3 and KIRB1, and a lower cytotoxic ability and T-bet expression is associated with local or distant metastases. Higher expression of MICA in the plasma was associated with worse prognosis, as was validated in an independent cohort (Figure 1). Conclusions: These data demonstrate that NK cells become progressively exhausted in the context of melanoma progression and that Tim-3 blockade possibly earlier in disease may have some benefit on innate immune function. Finally, our data suggest that soluble MICA is a potential prognostic marker which may contribute to the NK cell exhaustion through its interactions with NKG2D

To better identify melanoma patients who are, at the time of primary melanoma diagnosis, at high risk of developing brain metastases, primary melanoma characteristics were examined as risk factors for brain metastasis development. In a study of two patient cohorts, clinicopathological characteristics prospectively collected at primary cutaneous melanoma diagnosis for patients with/without brain metastasis were assessed in univariate and multivariate analyses using data from two prospectively collected databases: the Melanoma Cooperative Group (MCG) (1972-1982) and the Interdisciplinary Melanoma Cooperative Group (IMCG) (2002-2009). Candidate risk factors were evaluated in association with time to brain metastasis using either the log-rank test or Cox proportional hazards regression analysis with/without considering competing risks. Out of 2341 total patients included in the study, 222 (9.5%) developed brain metastases (median follow-up: 98 months). The median time to brain metastases was 30.5 months and the median survival time after brain metastases was 4 months. Increased hazard ratios (HRs) for brain metastasis were found among thicker (logarithmic value in mm) (MCG: HR=1.97, P<0.0001; IMCG: HR=1.31, P=0.018), ulcerated (MCG: HR=1.93, P=0.01; IMCG: HR=3.14, P<0.0001), and advanced-stage (MCG: HR=2.08, P=0.008; IMCG: HR=2.56, P=0.0002) primary melanomas on the basis of multivariate Cox regression analysis assuming the presence of competing risks. Primary cutaneous melanoma thickness, ulceration, and stage were identified and validated as risk factors associated with time to melanoma brain metastasis.

Previous reports have demonstrated a role for hedgehog signaling in melanoma progression, prompting us to explore the therapeutic benefit of targeting this pathway in melanoma. We profiled a panel of human melanoma cell lines and control melanocytes for altered expression of hedgehog pathway members and determined the consequences of both genetic and pharmacological inhibition of the hedgehog pathway activator Smoothened (SMO) in melanoma, both in vitro and in vivo. We also examined the relationship between altered expression of hedgehog pathway mediators and survival in a well-characterized cohort of metastatic melanoma patients with prospectively collected follow up information. Studies revealed that over 40% of the melanoma cell lines examined harbored significantly elevated levels of the hedgehog pathway mediators SMO, GLI2, and PTCH1 compared to melanocytes (p < 0.05). SMO inhibition using siRNA and the small molecule inhibitor, NVP-LDE-225, suppressed melanoma growth in vitro, particularly in those cell lines with moderate SMO and GLI2 expression. NVP-LDE-225 also induced apoptosis in vitro and inhibited melanoma growth in a xenograft model. Gene expression data also revealed evidence of compensatory up-regulation of two other developmental pathways, Notch and WNT, in response to hedgehog pathway inhibition. Pharmacological and genetic SMO inhibition also downregulated genes involved in human embryonic stem cell pluripotency. Finally, increased SMO expression and decreased expression of the hedgehog pathway repressor GLI3 correlated with shorter post recurrence survival in metastatic melanoma patients. Our data demonstrate that hedgehog pathway inhibition might be a promising targeted therapy in appropriately selected metastatic melanoma patients.

BACKGROUND: Steadily high melanoma mortality rates urge for the availability of novel biomarkers with a more personalized ability to predict melanoma clinical outcomes. Germline risk variants are promising candidates for this purpose; however, their prognostic potential in melanoma has never been systematically tested. METHODS: We examined the effect of 108 melanoma susceptibility single nucleotide polymorphisms (SNPs), associated in recent GWAS with melanoma and melanoma-related phenotypes, on recurrence-free survival (RFS) and overall survival (OS), in 891 prospectively accrued melanoma patients. Cox proportional hazards models (Cox PH) were used to test the associations between 108 melanoma risk SNPs and RFS and OS adjusted by age at diagnosis, gender, tumor stage, histological subtype and other primary tumor characteristics. RESULTS: We identified significant associations for rs7538876 (RCC2) with RFS (HR = 1.48, 95% CI = 1.20-1.83, p = 0.0005) and rs9960018 (DLGAP1) with both RFS and OS (HR = 1.43, 95% CI = 1.07-1.91, p = 0.01, HR = 1.52, 95% CI = 1.09-2.12, p = 0.01, respectively) using multivariable Cox PH models. In addition, we developed a logistic regression model that incorporates rs7538876, rs9960018, primary tumor histological type and stage at diagnosis that has an improved discriminatory ability to classify 3-year recurrence (AUC = 82%) compared to histological type and stage alone (AUC = 78%). CONCLUSIONS: We identified associations between melanoma risk variants and melanoma outcomes. The significant associations observed for rs7538876 and rs9960018 suggest a biological implication of these loci in melanoma progression. The observed predictive patterns of associated variants with clinical end-points suggest for the first time the potential for utilization of genetic risk markers in melanoma prognostication.

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor-treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma. Cancer Res; 73(20); 6264-76. (c)2013 AACR.