Faculty Bibliography

: Melanoma is the most aggressive type of skin cancer in the United States with an increasing incidence. Melanoma lesions often exhibit high immunogenicity, with infiltrating immune cells playing important roles in regression of tumors occurring spontaneously or caused by therapeutic treatment. Computational and experimental methods have been used to estimate the abundance of immune cells in tumors, but their applications are limited by the requirement of large gene sets or multiple antibodies. Although the prognostic role of immune cells has been appreciated, a systematic investigation of their association with clinical factors, genomic features, prognosis and treatment response in melanoma is still lacking. This study, identifies a 25-gene signature based on RNA-seq data from The Cancer Genome Atlas (TCGA)-Skin Cutaneous Melanoma (TCGA-SKCM) dataset. This signature was used to calculate sample-specific Leukocyte Infiltration Scores (LIS) in six independent melanoma microarray datasets and scores were found to vary substantially between different melanoma lesion sites and molecular subtypes. For metastatic melanoma, LIS was prognostic in all datasets with high LIS being associated with good survival. The current approach provided additional prognostic information over established clinical factors, including age, tumor stage, and gender. In addition, LIS was predictive of patient survival in stage III melanoma, and treatment efficacy of tumor-specific antigen vaccine. IMPLICATIONS: This study identifies a 25-gene signature that effectively estimates the level of immune cell infiltration in melanoma, which provides a robust biomarker for predicting patient prognosis.

MicroRNAs are critical post-transcriptional regulators of a majority of genes, of which the FOXO family of transcription factors is no exception. Here, we describe generalizable methods, including 3' UTR reporter assays and western blotting after microRNA manipulation, to test if a candidate miRNA (miR-182) directly targets a candidate (FOXO3) gene product. We also provide guidance on candidate miRNA selection and unbiased miRNA-target identification methods.

Factors influencing melanoma survival include sex, age, clinical stage, lymph node involvement, as well as Breslow thickness, presence of tumor-infiltrating lymphocytes based on histological analysis of primary melanoma, mitotic rate, and ulceration. Identification of genes whose expression in primary tumors is associated with these key tumor/patient characteristics can shed light on molecular mechanisms of melanoma survival. Here, we show results from a gene expression analysis of formalin-fixed paraffin-embedded primary melanomas with extensive clinical annotation. The Cancer Genome Atlas data on primary melanomas were used for validation of nominally significant associations. We identified five genes that were significantly associated with the presence of tumor-infiltrating lymphocytes in the joint analysis after adjustment for multiple testing: IL1R2, PPL, PLA2G3, RASAL1, and SGK2. We also identified two genes significantly associated with melanoma metastasis to the regional lymph nodes (PIK3CG and IL2RA), and two genes significantly associated with sex (KDM5C and KDM6A). We found that LEF1 was significantly associated with Breslow thickness and CCNA2 and UBE2T with mitosis. RAD50 was the gene most significantly associated with survival, with a higher level of expression associated with worse survival.

The T1 parvovirus Minute Virus of Mice (MVM) was used to study the roles that phosphorylation and N-terminal domains (Nt) configuration of capsid subunits may play in icosahedral nuclear viruses assembly. In synchronous MVM infection, capsid subunits newly assembled as two types of cytoplasmic trimeric intermediates (3VP2, and 1VP1:2VP2) harbored a VP1 phosphorylation level fivefold higher than that of VP2, and hidden Nt. Upon nuclear translocation at S phase, VP1-Nt became exposed in the heterotrimer and subsequent subviral assembly intermediates. Empty capsid subunits showed a phosphorylation level restored to VP1:VP2 stoichiometry, and the Nt concealed in their interior. However ssDNA-filled virus maturing at S/G2 lacked VP1 phosphorylation and one major VP2 phosphopeptide, and exposed VP2-Nt. Endosomal VP2-Nt cleavage resulted in VP3 subunits devoid of any phospholabel, implying that incoming viral particles specifically harbor a low phosphorylation status. Phosphorylation provides a mechanistic coupling of parvovirus nuclear assembly to the cell cycle.

Lysyl oxidase-like 3 (LOXL3) is a member of the lysyl oxidase family comprising multifunctional enzymes with depicted roles in extracellular matrix maturation, tumorigenesis, and metastasis. In silico expression analyses followed by experimental validation in a comprehensive cohort of human cell lines revealed a significant upregulation of LOXL3 in human melanoma. We show that LOXL3 silencing impairs cell proliferation and triggers apoptosis in various melanoma cell lines. Further supporting a pro-oncogenic role in melanoma, LOXL3 favors tumor growth in vivo and cooperates with oncogenic BRAF in melanocyte transformation. Upon LOXL3 depletion, melanoma cells display a faulty DNA damage response (DDR), characterized by ATM checkpoint activation and inefficient ATR activation leading to the accumulation of double-strand breaks (DSBs) and aberrant mitosis. Consistent with these findings, LOXL3 binds to proteins involved in the maintenance of genome integrity, in particular BRCA2 and MSH2, whose levels dramatically decrease upon LOXL3 depletion. Moreover, LOXL3 is required for efficient DSB repair in melanoma cells. Our results reveal an unexpected role for LOXL3 in the control of genome stability and melanoma progression, exposing its potential as a novel therapeutic target in malignant melanoma, a very aggressive condition yet in need for more effective treatment options.

Melanoma treatment with the BRAF V600E inhibitor vemurafenib (VMF) provides therapeutic benefits but the common emergence of drug resistance remains a challenge. We generated A375 melanoma cells resistant to VMF with the goal of investigating changes in miRNA expression patterns that might contribute to resistance. Increased expression of miR-204-5p and miR-211-5p occurring in VMF-resistant cells was determined to impact VMF response. Their expression was rapidly affected by VMF treatment through RNA stabilization. Similar effects were elicited by MEK and ERK inhibitors but not AKT or Rac inhibitors. Ectopic expression of both miRNA in drug-naive human melanoma cells was sufficient to confer VMF resistance and more robust tumor growth in vivo. Conversely, silencing their expression in resistant cells inhibited cell growth. Joint overexpression of miR-204-5p and miR-211-5p durably stimulated Ras and MAPK upregulation after VMF exposure. Overall, our findings show how upregulation of miR-204-5p and miR-211-5p following VMF treatment enables the emergence of resistance, with potential implications for mechanism-based strategies to improve VMF responses.

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a transmembrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

A variety of non-coding RNAs have been reported as endogenous sponges for cancer-modulating miRNAs. However, miRNA trapping by transcripts with protein-coding functions is less understood. The mRNA of TYRP1 is now found to sequester the tumour suppressor miR-16 on non-canonical miRNA response elements in melanoma, thereby promoting malignant growth.

Circular RNAs are a novel class of non-coding RNAs with functions that remain poorly characterized in normal and pathological conditions. CDR1as is a non-canonical circRNA observed to act as a sponge for miR-7 in brain tissues. Analysis of RNA-seq data of melanocytes and melanoma cell lines and short-term cultures revealed loss of CDR1as expression as a hallmark of melanoma cells. We confirmed silencing of CDR1as in melanoma cells and tissues by RT-qPCR using divergent primers. Clinically, we observed CDR1as loss associated with metastatic progression and poor patient outcomes in a cohort of fresh-frozen melanoma tissue samples. Depletion of CDR1as in melanoma cell lines enhanced invasion in vitro and lung metastasis in vivo, demonstrating functional significance of CDR1as silencing. Surprisingly, CDR1as depletion had no clear effect on miR-7 activity in melanoma cells, and miR-7 inhibition was insufficient to rescue CDR1as silencing-induced invasion. Moreover, GSEA analyses of proteomic profiling of melanoma cells depleted of CDR1as revealed reductions of proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial function, suggesting CDR1as loss may alter metabolism of melanoma cells. Mining of CLIP-Seq data sets and subsequent RIP-PCR revealed direct interactions of CDR1as with the IGF2BP family of proteins and TAR

Background: Recently, tumor mutation burden (TMB) has been shown to increase the presentation of neoantigens that stimulate immune tumor recognition, resulting in improved immunotherapy (IT) outcomes in melanoma and other cancers. As melanoma is highly immunogenic, here we tested whether TMB associates with immune recognition during tumor progression, hence impacting melanoma overall survival (OS), independently of IT treatment. Methods: We have generated somatic mutation data from 314 IT-naive metastatic melanomas from The Cancer Genome Atlas (TCGA). In the TCGA cohort, TMB has been calculated for 210 genes (200GS) previously established from TMB studies of anti-CTLA4 and anti-PD1/PD-L1 IT. For validation, we have sequenced exonic regions of 20 genes (20GS) with the highest TMB among 200GS in 89 IT-naive metastatic melanomas ascertained at New York University Langone Medical Center. The TMB was defined using total number of somatic, non-synonymous mutations in either 200GS (TCGA discovery) or 20GS (validation), respectively. For discovery and validation cohorts, OS from primary diagnosis of samples with high TMB was compared against low TMB, using thresholds established in previous studies. Results: We found that total TMB predicts better OS (p = 0.03, HR = 2.64) in TCGA melanomas. Restricting the analysis only to the established 200GS, this association became more significant in all patients (p = 0.01, HR = 2.67) as well as in patients without IT (p = 0.01, HR = 2.67). In the validation stage of 89 melanomas without prior IT treatment, a high TMB in a subset of 20GS accurately determined favorable OS (p = 0.02, HR = 2.69) and confirmed TCGA observations from the 200GS. Conclusions: Here we show, for the first time, that in addition to IT, high TMB predicts more favorable OS in patients that never received IT, potentially serving as a novel marker of prognosis of melanoma and likely other immunogenic tumors at early stages. In addition, our study suggests that TMB test can be robust when applied to only a small subset of genes that trigger significantly higher immunogenicity. This may also eventually assist with accurate sub-selection of early stage patients likely to respond to IT regimens