Faculty Bibliography

Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low- versus high-risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state whereas invasiveness genes were linked with Epgn3 high-risk status. We identified ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/β-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the 'cold tumor' phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by β-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).

IMPORTANCE:Therapy for advanced melanoma has transformed during the past decade, but early detection and prognostic assessment of cutaneous melanoma (CM) remain paramount goals. Best practices for screening and use of pigmented lesion evaluation tools and gene expression profile (GEP) testing in CM remain to be defined. OBJECTIVE:To provide consensus recommendations on optimal screening practices and prebiopsy diagnostic, postbiopsy diagnostic, and prognostic assessment of CM. EVIDENCE REVIEW:Case scenarios were interrogated using a modified Delphi consensus method. Melanoma panelists (n = 60) were invited to vote on hypothetical scenarios via an emailed survey (n = 42), which was followed by a consensus conference (n = 51) that reviewed the literature and the rationale for survey answers. Panelists participated in a follow-up survey for final recommendations on the scenarios (n = 45). FINDINGS:The panelists reached consensus (≥70% agreement) in supporting a risk-stratified approach to melanoma screening in clinical settings and public screening events, screening personnel recommendations (self/partner, primary care provider, general dermatologist, and pigmented lesion expert), screening intervals, and acceptable appointment wait times. Participants also reached consensus that visual and dermoscopic examination are sufficient for evaluation and follow-up of melanocytic skin lesions deemed innocuous. The panelists reached consensus on interpreting reflectance confocal microscopy and some but not all results from epidermal tape stripping, but they did not reach consensus on use of certain pigmented lesion evaluation tools, such as electrical impedance spectroscopy. Regarding GEP scores, the panelists reached consensus that a low-risk prognostic GEP score should not outweigh concerning histologic features when selecting patients to undergo sentinel lymph node biopsy but did not reach consensus on imaging recommendations in the setting of a high-risk prognostic GEP score and low-risk histology and/or negative nodal status. CONCLUSIONS AND RELEVANCE:For this consensus statement, panelists reached consensus on aspects of a risk-stratified approach to melanoma screening and follow-up as well as use of visual examination and dermoscopy. These findings support a practical approach to diagnosing and evaluating CM. Panelists did not reach consensus on a clearly defined role for GEP testing in clinical decision-making, citing the need for additional studies to establish the clinical use of existing GEP assays.

Accurate prognostic biomarkers in early-stage melanoma are urgently needed to stratify patients for clinical trials of adjuvant therapy. We applied a previously developed open source deep learning algorithm to detect tumor-infiltrating lymphocytes (TILs) in hematoxylin and eosin (H&E) images of early-stage melanomas. We tested whether automated digital (TIL) analysis (ADTA) improved accuracy of prediction of disease specific survival (DSS) based on current pathology standards. ADTA was applied to a training cohort (n = 80) and a cutoff value was defined based on a Receiver Operating Curve. ADTA was then applied to a validation cohort (n = 145) and the previously determined cutoff value was used to stratify high and low risk patients, as demonstrated by Kaplan-Meier analysis (p ≤ 0.001). Multivariable Cox proportional hazards analysis was performed using ADTA, depth, and ulceration as co-variables and showed that ADTA contributed to DSS prediction (HR: 4.18, CI 1.51-11.58, p = 0.006). ADTA provides an effective and attainable assessment of TILs and should be further evaluated in larger studies for inclusion in staging algorithms.

Mounting evidence supports a role for dysregulated long non-coding RNAs (lncRNA) in the development of many cancers. A recently discovered function of lncRNAs is to act as microRNA (miR) decoys or competing endogenous RNAs, which sequester specific miRs and relieve negative regulation of mRNA expression by miRs. Although a large number of non-coding RNAs are thought to function as competing endogenous RNAs, miR-sequestering lncRNAs involved in nevus to melanoma transformation remain largely unknown. In this study, we applied a bioinformatics approach to a unique dataset of benign melanocytic nevi and primary melanomas of the skin in order to fill this research gap. We modified a previously published miR target prediction algorithm, RNAhybrid, and improved its search efficiency. We reported the presence of many lncRNAs and miRs deregulated when transitioning from a senescence-like state of nevi to melanoma. We provided evidence of a relatively new and understudied mechanism of gene regulation during this process and identified for the first time lncRNAs (n = 122) that may potentially function as miR decoys as well as their target miRs during nevus to melanoma transformation. The knowledge presented here can be employed for developing biomarkers for diagnostic and risk stratification purposes.

FBXW7 is well characterized as a tumor suppressor in many human cancers including melanoma; however, the mechanisms of tumor-suppressive function have not been fully elucidated. We leveraged two distinct RNA sequencing datasets: human melanoma cell lines (n = 10) with control versus silenced FBXW7 and a cohort of human melanoma tumor samples (n = 51) to define the transcriptomic fingerprint regulated by FBXW7. Here, we report that loss of FBXW7 enhances a mitochondrial gene transcriptional program that is dependent on MITF in human melanoma and confers poor patient outcomes. MITF is a lineage-specific master regulator of melanocytes and together with PGC-1alpha is a marker for melanoma subtypes with dependence for mitochondrial oxidative metabolism. We found that inactivation of FBXW7 elevates MITF protein levels in melanoma cells. In vitro studies examining loss of FBXW7 and MITF alone or in combination showed that FBXW7 is an upstream regulator for the MITF/PGC-1 signaling.