Faculty Bibliography

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.

The prognostic value of mitotic rate in melanoma is increasingly recognized, particularly in thin melanoma in which the presence or absence of a single mitosis/mm can change staging from T1a to T1b. Still, accurate mitotic rate calculation (mitoses/mm) on hematoxylin and eosin (H&E)-stained sections can be challenging. Antimonoclonal mitotic protein-2 (MPM-2) and antiphosphohistone-H3 (PHH3) are 2 antibodies reported to be more mitosis-specific than other markers of proliferation such as Ki-67. We used light microscopy and computer-assisted image analysis software to quantify MPM-2 and PHH3 staining in melanoma. We then compared mitotic rates by each method with conventional H&E-based mitotic rate for correlation with clinical outcomes. Our study included primary tissues from 190 nonconsecutive cutaneous melanoma patients who were prospectively enrolled at New York University Langone Medical Center with information on age, gender, and primary tumor characteristics. The mitotic rate was quantified manually by light microscopy of corresponding H&E-stained, MPM-2-stained, and PHH3-stained sections. Computer-assisted image analysis was then used to quantify immunolabeled mitoses on the previously examined PHH3 and MPM-2 slides. We then analyzed the association between mitotic rate and both progression-free and melanoma-specific survival. Univariate analysis of PHH3 found significant correlation between increased PHH3 mitotic rate and decreased progression-free survival (P=0.04). Computer-assisted image analysis enhanced the correlation of PHH3 mitotic rate with progression-free survival (P=0.02). Regardless of the detection method, neither MPM-2 nor PHH3 offered significant advantage over conventional H&E determination of mitotic rate.

Background: Manipulation of key epigenetic regulators in melanoma proliferation is emerging as a new therapeutic strategy. Bromodomain-containing proteins such as the extraterminal domain (BET) family are components of transcription factor complexes and determinants of epigenetic memory. We investigated the expression of BRD4, a BET family member in melanoma cell lines and tissues, and the effects of its inhibition with the small molecule compounds MS436 and MS417 in in vitro and in vivo models of melanoma. Methods: BRD2 and BRD4 expression were analyzed by immunohistochemistry. We tested the effects of pharmacological or RNAi-mediated inhibition of BRD4 in melanoma cells using crystal violet-based assays for proliferation/colony formation and flow-cytometry for cell cycle analysis. The molecular effects of BRD4 suppression were examined using RNA sequencing, Real-Time quantitative PCR and western blots for p27, p21, MYC, ERK1 and SKP2. In the in vivo xenograft experiments NOD/SCID/IL2R-/-mice were injected with melanoma cells and treated with MS417. Statistical significance was determined by unpaired t-test (GraphPad). Results: BRD4 was found significantly upregulated in primary and metastatic melanoma tissues compared to melanocytes and nevi (p<0.001). 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. Rapidly after BET displacement, key cell cycle genes (SKP2, ERK1 and c-MYC) were downregulated concomitantly with the accumulation of CDK inhibitors (p21, p27), followed by melanoma cell cycle arrest. BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status. Conclusions: Our results demonstrate for the first time a role for BRD4 in melanoma maintenance and support the role of BET proteins as novel targets in melanoma. Further investigation in the clinical setting is warranted

Background: Estimating the risk of recurrence in patients with melanoma is extremely challenging. Standard of care is AJCC staging system, but the accuracy and robustness of this method is still under development. We conducted a proof of concept study exploring the use of machine-learned Bayesian Belief Networks (ml-BBNs) using a miRNA profiled cohort of melanoma patients with extended follow up to create Bayesian Biological Systems Models (BBSMs). We sought to determine if ml-BBNs could describe the biological system and if we could use the model to identify new cases with higher risk of recurrence. Methods: Our study cohort consisted of 89 patients (42 of which recurred) with a median follow up time of 118 months, that were examined for 869 miRNAs. Prior to modeling we segmented the data into training data (72 cases/80%) and testing data (17 cases/20%) at random. We recursively trained ml-BBNs on the training set, using all miRNAs. We used the directed graph structure of the ml-BBNs to identify miRNAs that consistently had more connectivity and goodness of fit as determined by Bayesian Information Criteria (BIC) scoring. MiRNAs that were in the top 50 BIC-scoring nodes across all models were selected for use to train the recurrence BBSMs. To compensate for a small number, bootstrapping was used to increase the sample to 100 records. We then compared our test set cases to our recurrence model, and used a similarity scoring algorithm to evaluate the similarity of values in each test instance to our biological models. For comparison we also trained an ml-BBN using clinical data from the same cohort. We then evaluated the scores against known recurrence outcome using Receiver Operating Characteristic (ROC) curve analysis. Results: BIC-scoring analysis selected 35 miRNAs for use in BBSM modeling. Area Under the Curve (AUC) for detection of recurrence is 0.76 in the training set and 0.62 in the testing set, while the clinical data yielded an AUC of 0.5 in this cohort. Conclusions: Our data suggest !

Background: PP6C binds to regulatory units toaffect a number of important pathways including cell proliferation and DNA repair. Recently two independent groups reported for the first time the presence of somatic mutations in the PPP6C gene in ~10% of short term cultures and limited number of human melanoma tissues. However, the clinical or biological relevance of PPP6C mutations in melanoma patients is unknown. Our objectives were to examine the clinical relevance of PPP6C mutations in a well characterized cohort of melanoma specimens linked to extensive, prospectively-collected clinical information and to explore the functional consequence of different categories of mutations. Methods: Sanger dideoxy sequencing was performed on PCR-amplified DNA from macro-dissected FFPE tumors. Associations between PPP6C mutations and baseline characteristics, recurrence, survival, and BRAF/ NRAS mutational status were examined. The impact of mutations on binding PP6C regulatory units was assessed as well as the effect on additional downstream pathways. Results: 308 primary melanoma patients (118 Stage I, 92 Stage II, and 98 Stage III) were examined (median follow up: 5.3 years). 50 PPP6C mutations in 33 patients (10.7%) were identified with 11 tumors harboring more than one mutation. One mutation (R301C) was identified in 6 patients. PPP6C mutations occurred with similar frequencies across stages and showed no association with BRAF or NRAS mutations.Mutations were categorized into 3 groups: Mutations resulting in premature stop codon (n=9), those occurring in the active site (n=16) and others (n=8). 8/9 (89%) patients with stop mutations recurred and developed visceral metastases. Functional studies revealed that PPP6C mutants also behaved differently; some PPP6C mutations led to decreased binding to regulatory subunits, others, including the R301C mutation did not. Conclusions: Our data suggest that PPP6C mutation is an early event in melanoma progression and independent of BRAF or NRAS mutations. Data also!

Background: Although patient age at diagnosis is not currently included in guidelines for treatment of primary melanoma, several lines of evidence suggest that patient age is an important, yet understudied, factor when considering treatment options. Here, we attempt to address the limited knowledge of the impact of age on primary melanoma treatment. Methods: In a prospectively enrolled and followed-up cohort of melanoma patients at NYU, we used logistic regression models to evaluate the association between patient age at diagnosis, tumor baseline characteristics, including BRAF and NRAS mutation status, and likelihood of receiving and responding to adjuvant therapy. We examined adjuvant therapy effectiveness using recurrence and melanoma-specific survival as endpoints. Results: 444 primary melanoma patients were included in the study (median follow-up: 6.3 years; age range: 19-95 years). Age was categorized into three groups spanning the range of age at presentation: younger (19-45 years; 24%), middle (46-70 years; 50%), and older (71-95 years; 26%). Older patients were significantly more likely to have advanced stage, nodular subtype (P < 0.01, both variables), and BRAF wildtype tumors (P = 0.04). Controlling for these factors as well as gender, older patients experienced a higher risk of recurrence (HR older vs. younger 3.34, 95% CI 1.53-7.25; P < 0.01). Of the 128/444 (29%) patients who were eligible for adjuvant treatment (clinical stage IIB), only 67/128 (52%) received treatment. Using a propensity score that accounts for stage at presentation, patients in the middle age group were more likely to receive adjuvant therapy than those in the older group (OR 2.61, 95% CI 1.12-6.08; P = 0.03). In addition, a trend suggesting benefit from adjuvant therapy (defined as longer melanoma-specific survival) was observed only in the middle age group (P = 0.07). Conclusions: Our data suggest that older melanoma patients, despite having a significantly worse prognosis, are less likely to receive and bene!

Background: Patients with metastatic melanoma are eligible for BRAF inhibitor therapy if the BRAF V600E mutation can be identified in their tumor specimen. Patients lacking an available specimen for genotyping are unable to receive inhibitor therapy. We developed two mutation-specific genotyping platforms and tested their ability to detect BRAF and NRAS mutations in archived plasma and tumor samples to determine the potential utility of blood-based tumor genotyping in melanoma. Methods: We analyzed a group of 96 patients with stage III or IV melanoma, prospectively enrolled and followed in the NYU Melanoma Biorepository program. Each patient had a plasma sample and one or more tumor samples available for analysis. We used a combination of allele-specific PCR (Taqman) and SNaPshot assays to identify BRAF V600 and NRAS Q61 mutations in the tumor and plasma samples. Results: Among the 96 patients, 51 had stage III disease at the time of analysis; 45 had stage IV disease. Seventy-two patients had 2 or more tumor samples available for analysis, for a total of 204 tumors analyzed. In total, 52/96 (54%) patients had one or more BRAF or NRAS mutant tumors, including one patient with separate BRAF and NRAS mutant tumors (BRAF, n=35 (36%); NRAS, n=18 (19%)). We successfully amplified plasma DNA from 39/52 (75%) patients with tumor-associated mutations. Among those patients with amplifiable plasma DNA we detected mutations in 7 (18%) patients including 3 BRAF V600E, one V600K, 2 NRAS Q61K and one Q61L. Plasma-based mutations matched tumor-associated mutations in all 7 patients. All 7 patients had active disease at the time of blood draw. There were 32 patients with tumor-associated mutations in which a mutation could not be detected in the plasma. Only 15 of those 32 (47%) had active disease at the time of blood draw. There were no mutations detected in the plasma of the 44 patients whose tumors lacked BRAF or NRAS mutations. Conclusions: These data suggest that plasma-based detection of BRAF and NRAS mut!