CD4 Phenotypes Are Associated with Reduced Expansion of Tumor-Infiltrating Lymphocytes in Melanoma Patients Treated with Adoptive Cell Therapy.

Tumor-infiltrating lymphocyte (TIL) adoptive cell therapy is effective in treating malignant melanoma, but its success relies on the adequate ex vivo expansion of TIL. To assess correlates of TIL expansion, CD4+ and CD8+ TIL were analyzed by RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing of acetylated histone 3. Patients were grouped into "TIL high" and "TIL low" based on division at the median number of TIL infused. Greater numbers of TIL infused correlated with longer overall survival, and increased frequencies of CD4+ cells infused were negatively correlated with the number of TIL infused. RNA-seq analysis of CD4+ TIL showed increases in Th2/Th17/regulatory T cell-related transcripts and pathways in the TIL-low group. Analysis of a public single-cell RNA-seq dataset validated findings that increased frequencies of CD4+ cells were negatively correlated with the number of TIL infused. TIL-low patients had significantly increased frequencies of CD4+ cells expressing ETS2 and OSM and trended toward increased expression of TNFRSF18.

A Population of Tumor-Infiltrating CD4+ T Cells Co-Expressing CD38 and CD39 Is Associated with Checkpoint Inhibitor Resistance.

PURPOSE: We previously showed that elevated frequencies of peripheral blood CD3+CD4+CD127-GARP-CD38+CD39+ T cells were associated with checkpoint immunotherapy resistance in patients with metastatic melanoma. In the present study, we sought to further investigate this population of ectoenzyme-expressing T cells (Teee). EXPERIMENTAL DESIGN: Teee derived from the peripheral blood of patients with metastatic melanoma were evaluated by bulk RNA-sequencing (RNA-seq) and flow cytometry. The presence of Teee in the tumor microenvironment was assessed using publically available single-cell RNA-seq datasets of melanoma, lung, and bladder cancers along with multispectral immunofluorescent imaging of melanoma patient formalin-fixed, paraffin-embedded specimens. Suppressive function of Teee was determined by an in vitro autologous suppression assay. RESULTS: Teee had phenotypes associated with proliferation, apoptosis, exhaustion, and high expression of inhibitory molecules. Cells with a Teee gene signature were present in tumors of patients with melanoma, lung, and bladder cancers. CD4+ T cells co-expressing CD38 and CD39 in the tumor microenvironment were preferentially associated with Ki67- CD8+ T cells. Co-culture of patient Teee with autologous T cells resulted in decreased proliferation of target T cells. High baseline intratumoral frequencies of Teee were associated with checkpoint immunotherapy resistance and poor overall survival in patients with metastatic melanoma. CONCLUSIONS: These results demonstrate that a novel population of CD4+ T cells co-expressing CD38 and CD39 is found both in the peripheral blood and tumor of patients with melanoma and is associated with checkpoint immunotherapy resistance.

Phase II clinical and immune correlate study of adjuvant nivolumab plus ipilimumab for high-risk resected melanoma.

BACKGROUND: Adjuvant therapy for high-risk resected melanoma with programmed cell-death 1 blockade results in a median relapse-free survival (RFS) of 5 years. The addition of low dose ipilimumab (IPI) to a regimen of adjuvant nivolumab (NIVO) in CheckMate-915 did not result in increased RFS. A pilot phase II adjuvant study of either standard dose or low dose IPI with NIVO was conducted at two centers to evaluate RFS with correlative biomarker studies. METHODS: Patients with resected stages IIIB/IIIC/IV melanoma received either IPI 3 mg/kg and NIVO 1 mg/kg (cohort 4) or IPI 1 mg/kg and NIVO 3 mg/kg (cohorts 5 and 6) induction therapy every 3 weeks for 12 weeks, followed by maintenance NIVO. In an amalgamated subset of patients across cohorts, peripheral T cells at baseline and on-treatment were assessed by flow cytometry and RNA sequencing for exploratory biomarkers. RESULTS: High rates of grade 3-4 adverse events precluded completion of induction therapy in 50%, 35% and 7% of the patients in cohorts 4, 5 and 6, respectively. At a median of 63.9 months of follow-up, 16/56 patients (29%) relapsed. For all patients, at 5 years, RFS was 71% (95% CI: 60 to 84), and overall survival was 94% (95% CI: 88 to 100). Expansion of CD3+CD4+CD38+CD127-GARP- T cells, an on-treatment increase in CD39 expression in CD8+ T cells, and T-cell expression of phosphorylated signal-transducer-and-activator-of-transcription (STAT)2 and STAT5 were associated with relapse. CONCLUSIONS: Adjuvant IPI/NIVO at the induction doses used resulted in promising relapse-free and overall survival, although with a high rate of grade 3-4 adverse events. Biomarker analyses highlight an association of ectoenzyme-expressing T cells and STAT signaling pathways with relapse, warranting future validation. TRIAL REGISTRATION NUMBER: NCT01176474 and NCT02970981.

Circulating CD8+ mucosal-associated invariant T cells correlate with improved treatment responses and overall survival in anti-PD-1-treated melanoma patients.

OBJECTIVES: While much of the research concerning factors associated with responses to immune checkpoint inhibitors (ICIs) has focussed on the contributions of conventional peptide-specific T cells, the role of unconventional T cells, such as mucosal-associated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are an abundant population of innate-like T cells expressing a semi-invariant T-cell receptor restricted to the MHC class I-like molecule, MR1, presenting vitamin B metabolites derived from bacteria. We sought to characterise MAIT cells in melanoma patients and determined their association with treatment responses and clinical outcomes. METHODS: In this prospective clinical study, we analysed the frequency and functional profile of circulating and tumor-infiltrating MAIT cells in human melanoma patients. Using flow cytometry, we compared these across metastatic sites and between ICI responders vs. non-responders as well as healthy donors. RESULTS: We identified tumor-infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients compared to healthy donors. However, circulating MAIT cell frequencies are restored by ICI treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival. CONCLUSION: Our results suggest that MAIT cells may be a potential predictive marker of responses to immunotherapies and provide rationale for testing MAIT cell-directed therapies in combination with current and next-generation ICIs.

Expression Differences in BCL2 Family Members between Uveal and Cutaneous Melanomas Account for Varying Sensitivity to BH3 Mimetics.

Uveal melanoma (UM) is a subtype of melanoma. Although they share a melanocytic origin with cutaneous melanoma (CM), patients with UM have few treatment options. BCL2 homologous 3 mimetics are small-molecule drugs that mimic proapoptotic BCL2 family members. We compared BCL2 family member expression between UM and CM using immunoblot and The Cancer Genome Atlas transcriptomic analysis. UM has a unique signature of low BFL1 and high PUMA proteins compared with CM and 30 other cancer types, making them an attractive candidate for BCL2 homologous 3 protein mimetics. We tested the efficacy of a BCL2 inhibitor and MCL1 inhibitor (MCL1i) in UM, with viability assays, live-cell imaging, sphere assays, and mouse xenograft models. UM had a higher sensitivity to MCL1i than CM. Overexpression of BFL1 or knockdown of PUMA made the UM more resistant to MCL1i. In contrast, MAPK/extracellular signal‒regulated kinase inhibitor treatment in CM made them more sensitive to MCL1i. However, MCL1i-alone treatment was not very effective to reduce the UM initiating cells; to overcome this, we employed a combination of MCL1i with BCL2 inhibitor that synergistically inhibited UM initiating cell's capacity to expand. Overall, we identify a distinct expression profile of BCL2 family members for UM that makes them susceptible to BCL2 homologous 3 mimetics.

PhenoComb: a discovery tool to assess complex phenotypes in high-dimensional single-cell datasets.

Motivation: High-dimensional cytometry assays can simultaneously measure dozens of markers, enabling the investigation of complex phenotypes. However, as manual gating relies on previous biological knowledge, few marker combinations are often assessed. This results in complex phenotypes with the potential for biological relevance being overlooked. Here, we present PhenoComb, an R package that allows agnostic exploration of phenotypes by assessing all combinations of markers. PhenoComb uses signal intensity thresholds to assign markers to discrete states (e.g. negative, low, high) and then counts the number of cells per sample from all possible marker combinations in a memory-safe manner. Time and disk space are the only constraints on the number of markers evaluated. PhenoComb also provides several approaches to perform statistical comparisons, evaluate the relevance of phenotypes and assess the independence of identified phenotypes. PhenoComb allows users to guide analysis by adjusting several function arguments, such as identifying parent populations of interest, filtering of low-frequency populations and defining a maximum complexity of phenotypes to evaluate. We have designed PhenoComb to be compatible with a local computer or server-based use. Results: In testing of PhenoComb's performance on synthetic datasets, computation on 16 markers was completed in the scale of minutes and up to 26 markers in hours. We applied PhenoComb to two publicly available datasets: an HIV flow cytometry dataset (12 markers and 421 samples) and the COVIDome CyTOF dataset (40 markers and 99 samples). In the HIV dataset, PhenoComb identified immune phenotypes associated with HIV seroconversion, including those highlighted in the original publication. In the COVID dataset, we identified several immune phenotypes with altered frequencies in infected individuals relative to healthy individuals. Collectively, PhenoComb represents a powerful discovery tool for agnostically assessing high-dimensional single-cell data. Availability and implementation: The PhenoComb R package can be downloaded from https://github.com/SciOmicsLab/PhenoComb. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

A Novel Regimen for Treating Melanoma: MCL1 Inhibitors and Azacitidine.

Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials.

Melanoma Metastases to the Adrenal Gland Are Highly Resistant to Immune Checkpoint Inhibitors.

BACKGROUND: Adrenal gland metastases (AGMs) are common in advanced-stage melanoma, occurring in up to 50% of patients. The introduction of immune checkpoint inhibitors (ICIs) has markedly altered the outcome of patients with melanoma. However, despite significant successes, anecdotal evidence has suggested that treatment responses in AGMs are significantly lower than in other metastatic sites. We sought to investigate whether having an AGM is associated with altered outcomes and whether ICI responses are dampened in the adrenal glands. PATIENTS AND METHODS: We retrospectively compared ICI responses and overall survival (OS) in 68 patients with melanoma who were diagnosed with an AGM and a control group of 100 patients without AGMs at a single institution. Response was determined using RECIST 1.1. OS was calculated from time of ICI initiation, anti-PD-1 initiation, initial melanoma diagnosis, and stage IV disease diagnosis. Tumor-infiltrating immune cells were characterized in 9 resected AGMs using immunohistochemical analysis. RESULTS: Response rates of AGMs were significantly lower compared with other metastatic sites in patients with AGMs (16% vs 22%) and compared with those without AGMs (55%). Patients with AGMs also had significantly lower median OS compared with those without AGMs (3.1 years vs not reached, respectively). We further observed that despite this, AGMs exhibited high levels of tumor-infiltrating immune cells. CONCLUSIONS: In this cohort of patients with melanoma, those diagnosed with an AGM had lower ICI response rates and OS. These results suggest that tissue-specific microenvironments of AGMs present unique challenges that may require novel, adrenal gland-directed therapies or surgical resection.

Targeting CDK4/6 Represents a Therapeutic Vulnerability in Acquired BRAF/MEK Inhibitor-Resistant Melanoma.

There is a clear need to identify targetable drivers of resistance and potential biomarkers for salvage therapy for patients with melanoma refractory to the combination of BRAF and MEK inhibition. In this study, we performed whole-exome sequencing on BRAF-V600E-mutant melanoma patient tumors refractory to the combination of BRAF/MEK inhibition and identified acquired oncogenic mutations in NRAS and loss of the tumor suppressor gene CDKN2A We hypothesized the acquired resistance mechanisms to BRAF/MEK inhibition were reactivation of the MAPK pathway and activation of the cell-cycle pathway, which can both be targeted pharmacologically with the combination of a MEK inhibitor (trametinib) and a CDK4/6 inhibitor (palbociclib). In vivo, we found that combination of CDK4/6 and MEK inhibition significantly decreased tumor growth in two BRAF/MEK inhibitor-resistant patient-derived xenograft models. In vitro, we observed that the combination of CDK4/6 and MEK inhibition resulted in synergy and significantly reduced cellular growth, promoted cell-cycle arrest, and effectively inhibited downstream signaling of MAPK and cell-cycle pathways in BRAF inhibitor-resistant cell lines. Knockdown of CDKN2A in BRAF inhibitor-resistant cells increased sensitivity to CDK4/6 inhibition alone and in combination with MEK inhibition. A key implication of our study is that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors, and loss of CDKN2A may represent a biomarker of response to the combination. Inhibition of the cell-cycle and MAPK pathway represents a promising strategy for patients with metastatic melanoma who are refractory to BRAF/MEK inhibitor therapy.

Clinical and molecular features of subungual melanomas are site-specific and distinct from acral melanomas.

Subungual melanomas (SUM) arise beneath the nails of the hands and feet, and account for 0.7-3.5% of all malignant melanomas. Most studies include SUM in the category of acral melanoma, but understanding the specific features of SUM is critical for improving patient care. In this study, we performed a site-specific comparison of the clinical and molecular features between 54 cases of SUM and 78 cases of nonsubungual acral melanoma. Compared to patients with acral melanoma, patients with SUM were younger at diagnosis, had a higher prevalence of primary melanomas on the hand, and had more frequent reports of previous trauma at the tumor site. SUM was deeper than acral melanoma at diagnosis, which correlated with an increased frequency of metastases. Analysis of common melanoma driver genes revealed KIT and KRAS mutations were predominantly found in SUM, whereas BRAF and NRAS mutations occurred almost exclusively in acral melanoma. We also discovered molecular differences in the cell cycle pathway, where CDK4/CCND1 amplifications were more frequent in SUM and CDKN2A/B loss occurred mostly in acral melanoma, and in the PI3K/mTOR pathway, where RICTOR amplification and TSC1 K587R mutations were exclusively in SUM and PTEN loss and AKT1 mutations were exclusively in acral melanoma. Comparison of hand versus foot tumors revealed more frequent ulceration of SUM foot tumors, which correlated with more distal metastases and poorer overall survival. In summary, we find SUM are both clinically and molecularly distinct from acral melanoma, and our data suggest KIT, CDK4/6, and mTOR inhibitors may be particularly relevant and effective treatments for patients with SUM.

Whole-genome sequencing of acral melanoma reveals genomic complexity and diversity.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.

Simultaneously Inhibiting BCL2 and MCL1 Is a Therapeutic Option for Patients with Advanced Melanoma.

There is an urgent need to develop treatments for patients with melanoma who are refractory to or ineligible for immune checkpoint blockade, including patients who lack BRAF-V600E/K mutations. This is often the case in patients diagnosed with rare melanoma subtypes such as mucosal and acral melanoma. Here, we analyzed data from the cutaneous melanoma The Cancer Genome Atlas Network (TCGA) transcriptomic and proteomic databases for differential expression of apoptosis molecules between melanomas with or without BRAF hotspot mutations. Our data indicated higher B-cell CLL/lymphoma 2 (BCL2) expression in melanoma without BRAF hotspot mutations, suggesting that BH3 mimetics, such as ABT-199 (venetoclax, a small molecule against BCL2), may be a potential therapeutic option for these patients. We explored the efficacy of combining two BH3 mimetics, ABT-199 and a myeloid cell leukemia sequence 1 (MCL1) inhibitor (S63845 or S64315/MIK665) in cutaneous, mucosal and acral melanomas, in vitro and in vivo. Our data indicate this combination induced cell death in a broad range of melanoma cell lines, including melanoma initiating cell populations, and was more potent in melanoma cells without BRAF-V600E/K mutations. Our knockdown/knockout experiments suggest that several pro-apoptotic BCL2 family members, BCL2-like 11 (apoptosis facilitator) (BIM), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) or BID, play a role in the combination-induced effects. Overall, our study supports the rationale for combining an MCL1 inhibitor with a BCL2 inhibitor as a therapeutic option in patients with advanced melanoma.

Pre-Treatment Mutational and Transcriptomic Landscape of Responding Metastatic Melanoma Patients to Anti-PD1 Immunotherapy.

Immunotherapy, such as anti-PD1, has improved the survival of patients with metastatic melanoma. However, predicting which patients will respond to immunotherapy remains a significant knowledge gap. In this study we analyzed pre-immunotherapy treated tumors from 52 patients with metastatic melanoma and monitored their response based on RECIST 1.1 criteria. The responders group contained 21 patients that had a complete or partial response, while the 31 non-responders had stable or progressive disease. Whole exome sequencing (WES) was used to identify biomarkers of anti-PD1 response from somatic mutations between the two groups. Variants in codons G34 and G41 in NFKBIE, a negative regulator of NFkB, were found exclusively in the responders. Mutations in NKBIE-related genes were also enriched in the responder group compared to the non-responders. Patients that harbored NFKBIE-related gene mutations also had a higher mutational burden, decreased tumor volume with treatment, and increased progression-free survival. RNA sequencing on a subset of tumor samples identified that CD83 was highly expressed in our responder group. Additionally, Gene Set Enrichment Analysis showed that the TNFalpha signaling via NFkB pathway was one of the top pathways with differential expression in responders vs. non-responders. In vitro NFkB activity assays indicated that the G34E variant caused loss-of-function of NFKBIE, and resulted in activation of NFkB signaling. Flow cytometry assays indicated that G34E variant was associated with upregulation of CD83 in human melanoma cell lines. These results suggest that NFkB activation and signaling in tumor cells contributes to a favorable anti-PD1 treatment response, and clinical screening to include aberrations in NFkB-related genes should be considered.

MCL1 inhibitors S63845/MIK665 plus Navitoclax synergistically kill difficult-to-treat melanoma cells.

Current treatment for patients with metastatic melanoma include molecular-targeted therapies and immune checkpoint inhibitors. However, a subset of melanomas are difficult-to-treat. These melanomas include those without the genetic markers for targeted therapy, non-responsive to immunotherapy, and those who have relapsed or exhausted their therapeutic options. Therefore, it is necessary to understand and explore other biological processes that may provide new therapeutic approaches. One of most appealing is targeting the apoptotic/anti-apoptotic system that is effective against leukemia. We used genetic knockdown and pharmacologic approaches of BH3 mimetics to target anti-apoptotic BCL2 family members and identified MCL1 and BCLXL as crucial pro-survival members in melanoma. We then examined the effects of combining BH3 mimetics to target MCL1 and BCLXL in vitro and in vivo. These include clinical-trial-ready compounds such as ABT-263 (Navitoclax) and S63845/S64315 (MIK655). We used cell lines derived from patients with difficult-to-treat melanomas. In vitro, the combined inhibition of MCL1 and BCLXL resulted in significantly effective cell killing compared to single-agent treatment (p < 0.05) in multiple assays, including sphere assays. The combination-induced cell death was independent of BIM, and NOXA. Recapitulated in our mouse xenograft model, the combination inhibited tumor growth, reduced sphere-forming capacity (p < 0.01 and 0.05, respectively), and had tolerable toxicity (p > 0.40). Taken together, this study suggests that dual targeting of MCL1 and BCLXL should be considered as a treatment option for difficult-to-treat melanoma patients.

BH3 mimetics induce apoptosis independent of DRP-1 in melanoma.

Despite the recent advancement in treating melanoma, options are still limited for patients without BRAF mutations or in relapse from current treatments. BH3 mimetics against members of the BCL-2 family have gained excitement with the recent success in hematological malignancies. However, single drug BH3 mimetic therapy in melanoma has limited effectiveness due to escape by the anti-apoptotic protein MCL-1 and/or survival of melanoma-initiating cells (MICs). We tested the efficacy of the BH3 mimetic combination of A-1210477 (an MCL-1 inhibitor) and ABT-263 (a BCL-2/BCL-XL/BCL-W inhibitor) in killing melanoma, especially MICs. We also sought to better define Dynamin-Related Protein 1 (DRP-1)'s role in melanoma; DRP-1 is known to interact with members of the BCL-2 family and is a possible therapeutic target for melanoma treatment. We used multiple assays (cell viability, apoptosis, bright field, immunoblot, and sphere formation), as well as the CRISPR/Cas9 genome-editing techniques. For clinical relevance, we employed patient samples of different mutation status, including some relapsed from current treatments such as anti-PD-1 immunotherapy. We found the BH3 mimetic combination kill both the MICs and non-MICs (bulk of melanoma) in all cell lines and patient samples irrespective of the mutation status or relapsed state (p < 0.05). Unexpectedly, the major pro-apoptotic proteins, NOXA and BIM, are not necessary for the combination-induced cell death. Furthermore, the combination impedes the activation of DRP-1, and inhibition of DRP-1 further enhances apoptosis (p < 0.05). DRP-1 effects in melanoma differ from those seen in other cancer cells. These results provide new insights into BCL-2 family's regulation of the apoptotic pathway in melanoma, and suggest that inhibiting the major anti-apoptotic proteins is sufficient to induce cell death even without involvement from major pro-apoptotic proteins. Importantly, our study also indicates that DRP-1 inhibition is a promising adjuvant for BH3 mimetics in melanoma treatment.

IMPACT web portal: oncology database integrating molecular profiles with actionable therapeutics.

BACKGROUND: With the advancement of next generation sequencing technology, researchers are now able to identify important variants and structural changes in DNA and RNA in cancer patient samples. With this information, we can now correlate specific variants and/or structural changes with actionable therapeutics known to inhibit these variants. We introduce the creation of the IMPACT Web Portal, a new online resource that connects molecular profiles of tumors to approved drugs, investigational therapeutics and pharmacogenetics associated drugs. RESULTS: IMPACT Web Portal contains a total of 776 drugs connected to 1326 target genes and 435 target variants, fusion, and copy number alterations. The online IMPACT Web Portal allows users to search for various genetic alterations and connects them to three levels of actionable therapeutics. The results are categorized into 3 levels: Level 1 contains approved drugs separated into two groups; Level 1A contains approved drugs with variant specific information while Level 1B contains approved drugs with gene level information. Level 2 contains drugs currently in oncology clinical trials. Level 3 provides pharmacogenetic associations between approved drugs and genes. CONCLUSION: IMPACT Web Portal allows for sequencing data to be linked to actionable therapeutics for translational and drug repurposing research. The IMPACT Web Portal online resource allows users to query genes and variants to approved and investigational drugs. We envision that this resource will be a valuable database for personalized medicine and drug repurposing. IMPACT Web Portal is freely available for non-commercial use at http://tanlab.ucdenver.edu/IMPACT .

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma.

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Combining a GSI and BCL-2 inhibitor to overcome melanoma's resistance to current treatments.

Major limitations of current melanoma treatments are for instances of relapse and the lack of therapeutic options for BRAF wild-type patients who do not respond to immunotherapy. Many studies therefore focus on killing resistant subpopulations, such as Melanoma Initiating Cells (MICs) to prevent relapse. Here we examined whether combining a GSI (γ-Secretase Inhibitor) with ABT-737 (a small molecule BCL-2/BCL-XL/BCL-W inhibitor) can kill both the non-MICs (bulk of melanoma) and MICs. To address the limitations of melanoma therapies, we included multiple tumor samples of patients relapsed from current treatments, with a diverse genetic background (with or without the common BRAF, NRAS or NF1 mutations) in these studies. Excitingly, the combination treatment reduced cell viability and induced apoptosis of the non-MICs; disrupted primary spheres, decreased the ALDH+ cells, and inhibited the self-renewability of the MICs in multiple melanoma cell lines and relapsed patient samples. Using a low-cell-number mouse xenograft model, we demonstrated that the combination significantly reduced the tumor initiating ability of MIC-enriched cultures from relapsed patient samples. Mechanistic studies also indicate that cell death is NOXA-dependent. In summary, this combination may be a promising strategy to address treatment relapse and for triple wild-type patients who do not respond to immunotherapy.

Long term storage of dry versus frozen RNA for next generation molecular studies.

The standard method for the storage and preservation of RNA has been at ultra-low temperatures. However, reliance on liquid nitrogen and freezers for storage of RNA has multiple downsides. Recently new techniques have been developed for storing RNA at room temperature utilizing desiccation and are reported to be an effective alternative for preserving RNA integrity. In this study we compared frozen RNA samples stored for up to one year to those which had been desiccated using RNAstable (Biomatrica, Inc., San Diego, CA) and stored at room temperature. RNA samples were placed in aliquots and stored after desiccation or frozen (at -80°C), and were analyzed for RNA Integrity Number (RIN), and by qPCR, and RNA sequencing. Our study shows that RNAstable is able to preserve desiccated RNA samples at room temperature for up to one year, and that RNA preserved by desiccation is comparable to cryopreserved RNA for downstream analyses including real-time-PCR and RNA sequencing.

The RET G691S polymorphism is a germline variant in desmoplastic malignant melanoma.

The RET protooncogene was originally identified in 1985. It encodes for a receptor tyrosine kinase. The RET receptor is activated by its ligand glial cell-derived neurotrophic factor. A polymorphism, RETp (G691S), in the intracellular juxtamembrane domain of RET, which enhances signaling by glial cell-derived neurotrophic factor has been described and studied previously in pancreatic cancer, medullary thyroid cancer, the multiple endocrine neoplasia 2 syndromes, and recently in cutaneous malignant melanoma. In particular, it has been shown that desmoplastic melanomas, which have neurotrophic features, have a high frequency of this polymorphism. In previous studies, however, it was not clear whether this was a germline or somatic change. Previous studies on pancreatic cancer indicated that both mechanisms may occur. To clarify this further we examined peripheral blood cell DNA from 30 patients with desmoplastic melanomas and 30 patients with nondesmoplastic melanoma for the RETp. In this study, a germline polymorphism was found in 30% of the patients with desmoplastic melanomas and 21% of the patients with nondesmoplastic melanoma. These findings indicate that the RETp may be a genetic risk factor for the development of desmoplastic melanoma.