Virus-negative Merkel Cell Carcinoma Research Articles

Abstract 3042: Unraveling the mechanism of YAP toxicity in neuroendocrine solid tumors: Insights from Merkel cell carcinoma

Abstract The Hippo pathway, a pivotal regulator orchestrating organ size and tissue growth, has emerged as a central player in cancer biology. The downstream transcription factor YAP within this pathway has been implicated in diverse oncogenic processes. YAP activation contributes to drug resistance, metastasis, and poor clinical outcomes in many solid tumors. Conversely, YAP exhibits growth-suppressive effects in neuroendocrine (NE) solid cancers, a paradox that remains poorly understood. Analysis of RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) revealed a distinct classification of tumors into YAP1-expressing (YAPon) and non-expressing (YAPoff) subgroups, with hematopoietic malignancies and NE solid tumors featured prominently in the latter. Merkel cell carcinoma (MCC) is a rare, high-grade, NE carcinoma of the skin with two distinct etiologies. Virus-positive MCC contains integrated Merkel cell polyomavirus that expresses the viral T antigens and virus-negative MCC caused by sunlight exposure and extensive UV damage. Bulk RNAseq performed with more than 60 MCC tumors revealed that YAP expression displayed an inverse correlation with an NE gene expression signature. To gain insight into why YAP expression is so low in MCC, we generated MCC cell lines with inducible expression of YAP. Expression of YAP led to growth arrest and cell death. We performed a genome-wide CRISPR-Cas9 screen to identify genes that when lost enable MCC cells to tolerate YAP expression. Functional enrichment analysis of the positively selected genes uncovered a variety of signaling pathways. Intriguingly, the CRISPR screen revealed a positive enrichment of non-canonical BAF complex members, including BRD9 and SMARCD1. To validate this finding, we treated MKL-1 cells with a BRD9 inhibitor, observing a substantial reduction in YAP toxicity. These findings open avenues for the development of targeted therapeutic strategies for high grade NE cancers, addressing a critical gap in our understanding of YAP biology in the context of MCC and beyond. Citation Format: Furkan Bahar, Thomas C. Frost, Varsha Ananthapadmanabhan, Julia L. Schnabel, Manisha Thakuria, James A. DeCaprio. Unraveling the mechanism of YAP toxicity in neuroendocrine solid tumors: Insights from Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3042.

Abstract B046: Exploring the potential of commensal polyomavirus as a promising approach for breast cancer treatment

Abstract Cancer is a leading cause of mortality worldwide, accounting for nearly one in six deaths. Our understanding of the immune system role in cancer control has increased dramatically in recent decades, facilitating the recent development of effective cancer immunotherapies, which have significantly improved the survival of cancer patients. However, current immunotherapies can induce overactive immune responses with severe even fatal outcomes. Moreover, immunotherapy may not work in every scenario, especially in ‘cold’ tumors that lack immune cell infiltration. Thus, more effective immunotherapies with fewer side effects are under active investigation. Viruses are one of the most abundant biological entities in the world; about 12% to 20% of human cancers are associated with viral infections. Viral antigens can increase tumor immunogenicity, triggering T cell immunity against tumor cells. Polyomaviruses are small, nonenveloped double-stranded DNA viruses that are widespread. In immunocompetent hosts, polyomaviruses remain at a low persistent level in the host after the primary infection, which appears to cause little or no symptoms. Previous studies have shown high polyomavirus seroprevalence and the frequent presence of polyomavirus-specific T cells in healthy adult blood donors. In the elderly population, Merkel cell polyomavirus (MCPyV) infection is linked to Merkel cell carcinoma (MCC), a rare, aggressive skin cancer associated with immunosuppression. MCPyV integration into the genome is the primary oncogenic event in ~80% of MCC cases, and the remaining virus-negative MCC cases are associated with ultraviolet (UV) radiation. MCPyV-induced MCCs are associated with more intratumor immune cells and better responses to cancer therapy. Moreover, BK and JC polyomavirus positive lymphoma patients showed a trend toward significantly better overall survival. These clinical data suggest that polyomavirus in tumors may increase tumor immunogenicity. Thus, polyomavirus presents an intriguing opportunity to develop a novel strategy to enhance cancer immunogenicity. We utilized mouse polyomavirus (MPyV) to colonize typically ‘cold’ breast tumor cell line E0771 breast cancer cells in mice. MPyV infection blocked the growth of tumors in immunocompetent mice and promoted immune cell infiltration when intratumorally delivered to the tumor. The tumor rejection activity of polyomavirus is depend on CD4 and CD8 T cells. Unlike conventional oncolytic viruses which directly lyse the infected cells and trigger a nonspecific inflammatory response, polyomavirus does not lyse the colonized cells, and it induces a relatively low level of inflammation. Instead, polyomavirus stimulates viral antigen expression in tumors that boosts T cell response leading to epitope spreading to tumor-associated antigens. The outcomes of this research will establish the utility of commensal polyomavirus as a novel agent for cancer immunotherapy. Citation Format: YUN XIA. Exploring the potential of commensal polyomavirus as a promising approach for breast cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B046.

Abstract C134: The MDM2 degrader KTX-049 is highly potent in TP53 wild-type (p53 WT) Merkel cell carcinoma (MCC)

Abstract Background and aims: MCC is a highly aggressive neuroendocrine carcinoma of the skin with a high fatality rate. Clonal integration of Merkel cell polyomavirus (MCPyV) occurs in about 80% of all MCCs. These virus positive MCC (MCCP) tumors have a low tumor mutational burden and usually express wild type (WT) p53 (TP53). In contrast, virus negative MCC (MCCN) tumors present a predominantly UV mutational signature, a high mutational burden and often have alterations in p53. MCCP tumors express MCyV small T antigen (ST) and a truncated form of large T antigen (LT). The MCyV ST in MCCP tumors, recruits MYCL and EP400 to form the SLaP complex that specifically trans-activates several genes. A direct target of the SLaP complex is MDM2, an E3 ubiquitin ligase of p53 that inhibits p53-mediated tumor suppression. The use of MDM2 inhibitors can stabilize p53 in p53 WT tumors. However, currently, no MDM2 inhibitors are approved for treatment of MCC. Furthermore, MDM2 degraders have not been studied in the context of MCC. Here, we aim to analyze the efficacy of KTX-049, a potent MDM2 degrader, in MCC and compare its efficacy to the previously reported and highly potent MDM2 inhibitor, DS-3032. Experimental procedures: Established MCCP cell lines with functional p53, non-functional p53 and two p53 WT MCCP patient derived cell lines (PDCLs) were treated with KTX-049 or DS-3032 and the effect on cell viability was analyzed using a luminescence-based assay. Apoptosis induction was studied using the caspase 3-7 Glo assay. The p53 response in MCC cells post KTX-049 or DS-3032 treatment was assessed by western blot (WB) analysis of p53 and its downstream effectors. Results: KTX-049 and DS-3032 reduced cell viability of p53 WT MCCP cell lines but not cell lines with non-functional p53. Notably, all sensitive cell lines had ≥100-fold lower absolute IC50 values for KTX-049 as compared to DS-3032. Additionally, KTX-049 effectively reduced cell viability of the sensitive cell lines even after brief exposures. KTX-049 also triggered a rapid and sustained p53 response and apoptosis induction in p53 WT MCC cells. Conclusions: KTX-049 is a promising MDM2 degrader effective against p53 WT MCC cell lines. These results provide evidence for in vivo exploration of KTX-049 in models of MCC. Citation Format: Varsha Ananthapadmanabhan, Yogesh Chutake, Jessica Filiatrault, Stefanie Schalm, Alice McDonald, James A DeCaprio. The MDM2 degrader KTX-049 is highly potent in TP53 wild-type (p53 WT) Merkel cell carcinoma (MCC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C134.

The effect of GP-2250 on cultured virus-negative Merkel cell carcinoma cells: preliminary results.

Even in the novel immunotherapy era, Merkel cell carcinoma (MCC) remains challenging in its treatment. Apart from Merkel cell polyomavirus (MCPyV) associated MCC, this cancer is linked in about 20% of cases to ultraviolet-induced mutational burden frequently causing aberrations in Notch and PI3K/AKT/mTOR signalling pathways. The recently developed agent GP-2250 is capable to inhibit growth of cells of different cancers, including pancreatic neuroendocrine tumors. The objective of the present study was to investigate the effects of GP-2250 on MCPyV-negative MCC cells. MethodsWe employed three cell lines (MCC13, MCC14.2, MCC26) which were exposed to different GP-2250doses. GP-2250's effects on cell viability, proliferation, and migration were evaluated by means of MTT, BrdU, and scratch assays, respectively. Flow cytometry was performed for the evaluation of apoptosis and necrosis. Western blotting was implemented for the determination of AKT, mTOR, STAT3, and Notch1 protein expression. Cell viability, proliferation, and migration decreased with increasing GP-2250 doses. Flow cytometry revealed a dose response to GP-2250 in all three MCC cell lines. While the viable fraction decreased, the share of necrotic and in a smaller amount the apoptotic cells increased. Regarding Notch1, AKT, mTOR, and STAT3 expression a comparatively time- and dose-dependent decrease of protein expression in the MCC13 and MCC26 cell lines was observed. By contrast, Notch1, AKT, mTOR, and STAT3 expression in MCC14.2 was scarcely altered or even increased by the three dosages of GP-2250 applied. The present study indicates GP-2250 having anti-neoplastic effects in MCPyV-negative tumor cells in regard to viability, proliferation, and migration. Moreover, the substance is capable of downregulating protein expression of aberrant tumorigenic pathways in MCPyV-negative MCC cells.

Chromosomal Aberrations Accumulate during Metastasis of Virus-Negative Merkel Cell Carcinoma

Merkel cell carcinoma is a rare, aggressive skin tumor initiated by polyomavirus integration or UV light DNA damage. In New Zealand, there is a propensity toward the UV-driven form (31 of 107, 29% virus positive). Using archival formalin-fixed, paraffin-embedded tissues, we report targeted DNA sequencing covering 246 cancer genes on 71 tumor tissues and 38 nonmalignant tissues from 37 individuals, with 33 of 37 being negative for the virus. Somatic variants of New Zealand virus-negative Merkel cell carcinomas partially overlapped with those reported overseas, including TP53 variants in all tumors and RB1, LRP1B, NOTCH1, and EPHA3/7 variants each found in over half of the cohort. Variants in genes not analyzed or reported in previous studies were also found. Cataloging variants in TP53 and RB1 from published datasets revealed a broad distribution across these genes. Chr 1p gain and Chr 3p loss were identified in around 50% of New Zealand virus-negative Merkel cell carcinomas, and RB1 loss of heterozygosity was found in 90% of cases. Copy number variants accumulate in most metastases. Virus-negative Merkel cell carcinomas have complex combinations of somatic DNA-sequence variants and copy number variants. They likely carry the small genomic changes permissive for metastasis from early tumor development; however, chromosomal alterations may contribute to driving metastatic progression.

The Antineoplastic Effect of Dimethyl Fumarate on Virus-Negative Merkel Cell Carcinoma Cell Lines: Preliminary Results.

Merkel cell carcinoma (MCC) is a rare, difficult-to-treat skin cancer once immunotherapy has failed. MCC is associated either with the clonal integration of the Merkel cell polyomavirus (MCPyV) or mutagenic UV-radiation. Fumaric acid esters, including dimethyl fumarate (DMF), have been shown to inhibit cell growth in cutaneous melanoma and lymphoma. We aimed to explore the effects of DMF on MCPyV-negative MCC cell lines. Three MCC cell lines (MCC13, MCC14.2, and MCC26) were treated with different doses of DMF. The cytotoxic effects and cell proliferation were assessed by the MTT cytotoxicity assay and BrdU proliferation assay at different time points. A significant reduction in cell viability and proliferation were demonstrated for all the cell lines used, with DMF proving to be effective.

458 Tertiary lymphoid structures improve survival in virus-negative Merkel cell carcinoma

Tertiary lymphoid structures (TLSs) are useful biomarkers for predicting the prognosis and assessing the response to immunotherapy in many cancers. In Merkel cell carcinoma (MCC), however, TLSs have only been examined in MCPyV-positive cases and were reported not to correlate with the prognosis. Here, we examined the prognostic value of TLSs in 61 patients with MCC, including both MCPyV-positive and MCPyV-negative cases. TLS-positive samples had a significantly better prognosis than TLS-negative samples.

Defining myeloid cells in the tumor microenvironment of Merkel cell carcinoma and their role in resistance to immunotherapy

Background and aims: Immunotherapy has changed the landscape for advanced Merkel cell carcinoma (MCC) with over 50% of patients initially responding to therapy. However, for patients who do not respond (or later recur), there is a need for new therapies. MCC is highly immunogenic cancer, as it contains numerous potent non-self-viral antigens (in tumors driven by Merkel cell polyomavirus) or UV-induced neoantigens (in virus negative MCC). Historically, studies have been focused on adaptive immunity, and little is known about innate immunity in MCC which may play an important role in immune evasion.

Milademetan is a highly potent MDM2 inhibitor in TP53 wild-type (p53 WT) models of Merkel cell carcinoma (MCC)

Background and aims: MCC is a highly aggressive neuroendocrine carcinoma of the skin with a high fatality rate. Merkel cell polyomavirus (MCPyV) integrates in about 80% of all MCCs. These virus positive MCC (MCCP) tumors have few somatic mutations and usually express wild type (WT) p53 (TP53). In contrast, virus negative MCC (MCCN) tumors present a high mutational burden and a predominantly UV mutational signature. MCCP tumors express MCyV small T antigen (ST) and a truncated form of large T antigen (LT).

Distinct Radiation Responses in Virus-Positive and Virus-Negative Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin neuroendocrine cancer (Ahmed et al., 2021). Nonviral MCC (MCCN) is caused by excessive exposure to UVR, whereas polyomavirus-associated MCC (MCCP) is caused by clonal integration of Merkel cell polyomavirus (Feng et al., 2008). MCCP tumors constitutively express the viral large T and small T antigens (Ahmed et al., 2021). TP53 is frequently wild type (WT) in MCCP and often altered in MCCN (Knepper et al., 2019).

Milademetan is a highly potent MDM2 inhibitor in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin with 2 etiologies. Merkel cell polyomavirus (MCPyV) integration is present in about 80% of all MCC. Virus-positive MCC (MCCP) tumors have few somatic mutations and usually express WT p53 (TP53). By contrast, virus-negative MCC (MCCN) tumors present with a high tumor mutational burden and predominantly UV mutational signature. MCCN tumors typically contain mutated TP53. MCCP tumors express 2 viral proteins: MCPyV small T antigen and a truncated form of large T antigen. MCPyV ST specifically activates expression of MDM2, an E3 ubiquitin ligase of p53, to inhibit p53-mediated tumor suppression. In this study, we assessed the efficacy of milademetan, a potent, selective, and orally available MDM2 inhibitor in several MCC models. Milademetan reduced cell viability of WT p53 MCC cell lines and triggered a rapid and sustained p53 response. Milademetan showed a dose-dependent inhibition of tumor growth in MKL-1 xenograft and patient-derived xenograft models. Here, along with preclinical data for the efficacy of milademetan in WT p53 MCC tumors, we report several in vitro and in vivo models useful for future MCC studies.

Tertiary Lymphoid Structures and Chemokine Landscape in Virus-Positive and Virus-Negative Merkel Cell Carcinoma.

Tertiary lymphoid structures (TLSs) are used as biomarkers in many cancers for predicting the prognosis and assessing the response to immunotherapy. In Merkel cell carcinoma (MCC), TLSs have only been examined in MCPyV-positive cases. Here, we examined the prognostic value of the presence or absence of TLSs in 61 patients with MCC, including MCPyV-positive and MCPyV-negative cases. TLS-positive samples had a significantly better prognosis than TLS-negative samples. MCPyV-positive samples had a good prognosis with or without TLSs, and MCPyV-negative/TLS-positive samples had a similarly good prognosis as MCPyV-positive samples. Only MCPyV-negative/TLS-negative samples had a significantly poor prognosis. All cases with spontaneous regression were MCPyV-positive/TLS-positive. We also performed a comprehensive analysis of the chemokines associated with TLS formation using next-generation sequencing (NGS). The RNA sequencing results revealed 5 chemokine genes, CCL5, CCR2, CCR7, CXCL9, and CXCL13, with significantly high expression in TLS-positive samples compared with TLS-negative samples in both MCPyV-positive and MCPyV-negative samples. Only 2 chemokine genes, CXCL10 and CX3CR1, had significantly different expression levels in the presence or absence of MCPyV infection in TLS-negative samples. Patients with high CXCL13 or CCL5 expression have a significantly better prognosis than those with low expression. In conclusion, the presence of TLSs can be a potential prognostic marker even in cohorts that include MCPyV-negative cases. Chemokine profiles may help us understand the tumor microenvironment in patients with MCPyV-positive or MCPyV-negative MCC and may be a useful prognostic marker in their own right.

Abstract P203: Milademetan is a potent, murine double minute 2 (MDM2) inhibitor, highly active in TP53 wild-type (p53WT) Merkel cell carcinoma (MCC) cell lines

Abstract Background: MCC is a highly aggressive neuroendocrine carcinoma of the skin with a poor overall prognosis. Current treatment options include surgery and radiation therapy for local MCC tumor and checkpoint blockade therapy for advanced disease. However, primary and acquired resistance can reduce response to therapy. Around 80% of all MCCs have integrated copies of Merkel cell polyomavirus (MCV). Virus-Positive MCC (MCCP) tumors typically contain few somatic mutations and express wild type (WT) p53 (TP53). In contrast, Virus-Negative MCC (MCCN) tumors have a high mutational burden, with a predominantly UV mutational signature. MCCP express two viral proteins: MCV small T antigen (ST) and a truncated form of large T antigen (LT). The MCV ST recruits MYCL and EP400 to form the SLaP complex that specifically activates several genes contributing to oncogenesis. A direct target of the SLaP complex is MDM2, an E3 ligase for p53. In p53WT MCCP, SLaP-dependent activation of MDM2 inhibits the tumor suppressive functions of p53. Here, we analyzed the efficacy of milademetan (RAIN-32), a potent, selective, and orally available MDM2 inhibitor, in MCC. Experimental procedures: Established MCCP cell lines with WT (MKL-1, WaGa, PeTa) or mutant p53 (MS-1), were treated with vehicle or several concentrations of milademetan or another MDM2 inhibitor AMG232 and cell viability was analyzed. Similar viability assays were also performed using MKL-1 p53 knockout (KO) cells and two newly established p53WT primary MCC cell lines. The p53 response in MCC cells treated with vehicle, milademetan or AMG232 was assessed by western blot (WB) analysis of p53 and its downstream effectors p21, PUMA and PARP-1. For in vivo testing, an initial tolerability study was conducted with once daily administration of milademetan by oral gavage in NSG mice. Milademetan activity was evaluated in MKL-1 xenograft and patient-derived xenograft (PDX) models in NSG mice. Pharmacodynamic markers of response in tumor samples from mice treated with vehicle or milademetan is analyzed by q-PCR and WB analysis. Summary of Results: Nanomolar concentrations of milademetan reduced cell viability of p53WT but not p53mutantMCCP MCC cell lines. Milademetan treatment increased levels of p21, PUMA and cleaved PARP-1 in MCCP cell lines MKL-1 and WaGa. Using p53 KO MKL-1 cells, we show that the effect of milademetan on MCC cell viability is p53 dependent. In vitro data show that milademetan is more potent than AMG232 in the context of MCC. Tolerability studies show that mice safely tolerate 100 mg/kg of milademetan. Milademetan treatment in the MKL-1 xenograft tumor model shows a dose-dependent response in tumor growth inhibition. In the DFMC-33043 PDX model, milademetan significantly inhibited tumor growth. Conclusion: Milademetan is a promising drug effective against p53WT MCC cell lines, xenograft, and PDX models. Ongoing in vivo testing of the anti-cancer cell activity of milademetan will provide evidence for clinical exploration of milademetan in MCC refractory to current therapies. Citation Format: Varsha Ananthapadmanabhan, Aine Knott, Kara M. Soroko, Prafulla C. Gokhale, Vijaya Tirunagaru, Robert Doebele, James A. DeCaprio. Milademetan is a potent, murine double minute 2 (MDM2) inhibitor, highly active in TP53 wild-type (p53WT) Merkel cell carcinoma (MCC) cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P203.

Merkel Cell Polyomavirus‒Negative Merkel Cell Carcinoma Originating from In Situ Squamous Cell Carcinoma: A Keratinocytic Tumor with Neuroendocrine Differentiation

Although virus-negative Merkel cell carcinoma (MCC) is characterized by a high frequency of UV-induced mutations, the expression of two viral oncoproteins is regarded as a key mechanism driving Merkel cell polyomavirus‒positive MCC. The cells in which these molecular events initiate MCC oncogenesis have yet not been identified for both MCC subsets. A considerable proportion of virus-negative MCC is found in association with squamous cell carcinoma (SCC), suggesting (i) coincidental collision, (ii) one providing a niche for the other, or (iii) one evolving from the other. Whole-exome sequencing of four combined tumors consisting of SCC in situ and Merkel cell polyomavirus‒negative MCC showed many mutations shared between SCC and MCC in all cases, indicating a common ancestry and thereby a keratinocytic origin of these MCCs. Moreover, analyses of the combined cases as well as of pure SCC and MCC suggest that RB1 inactivation in SCC facilitates MCC development and that epigenetic changes may contribute to the SCC/MCC transition.

Merkel Cell Carcinoma of the Head and Neck: Epidemiology, Pathogenesis, Current State of Treatment and Future Directions.

Simple SummaryMerkel cell carcinoma of the head and neck is a relatively uncommon cutaneous malignancy with distinct treatment management from Merkel cell carcinoma occurring in other anatomic locations. The efficacy of immunotherapy has markedly changed prognosis for patients with locally advanced or metastatic Merkel cell carcinoma of the head and neck. However, patients with primary or acquired resistance to immunotherapy remain therapeutically challenging, with novel treatment being vital to improving outcomes. Given the novel therapeutic options available for these patients, as well as increasing characterization regarding the differing oncogenesis of Merkel cell polyoma virus-positive vs. -negative tumors, up to date information regarding epidemiology; oncogenesis; current standards of treatment; and future therapeutic avenues are important to improving outcomes for patients with Merkel cell carcinoma of the head and neck.Merkel cell carcinoma (MCC) is a rare, cutaneous neuroendocrine malignancy with increasing incidence. The skin of the head and neck is a common subsite for MCC with distinctions in management from other anatomic areas. Given the rapid pace of developments regarding MCC pathogenesis (Merkel cell polyoma virus (MCPyV)-positive or virus-negative, cell of origin), diagnosis, staging and treatment, and up to date recommendations are critical for optimizing outcomes. This review aims to summarize currently available literature for MCC of the head and neck. The authors reviewed current literature, including international guidelines regarding MCC pathogenesis, epidemiology, diagnosis, staging, and treatment. Subsequently recommendations were derived including the importance of baseline imaging, MCPyV serology testing, primary site surgery, nodal evaluation, radiotherapy, and the increasing role of immune modulating agents in MCC. MCPyV serology testing is increasingly important with potential distinctions in treatment response and surveillance between virus-positive and virus-negative MCC. Surgical management continues to balance optimizing local control with minimal morbidity. Similarly, radiotherapy continues to have importance in the adjuvant, definitive, and palliative setting for MCC of the head and neck. Immunotherapy has changed the paradigm for advanced MCC, with increasing work focusing on optimizing outcomes for non-responders and high-risk patients, including those with immunosuppression.

491 Functional genomics screen identifies alternative targetable pathways for Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer. Most MCC tumors have Merkel cell polyomavirus integrated into the host genome (virus-positive MCC; VP-MCC), whereas virus-negative MCC (VN-MCC) tumors bear UV-induced mutations. Immune checkpoint inhibitors (ICI) are the first-line treatment for metastatic MCC, but the majority of patients are either not candidates for ICI or have disease progression while on treatment. In order to identify alternative therapeutic targets for these patients, we performed a druggable genome RNAi screen in MKL-2 (VP-MCC) and MCC26 (VN-MCC) cell lines. We screened >25,000 siRNA for their ability to reduce MCC viability at 72 hours. The main gene functions identified as essential to MCC viability included cell proliferation (VP- and VN-MCC) and apoptosis (VP-MCC). These results were corroborated by a follow-up RNAi screen including the two original cell lines, WaGa (VP-MCC), MCC13 (VN-MCC), and UISO (VN-MCC). Interestingly, many essential gene products identified in our screen were targets of compounds that selectively reduced MCC viability in an independent high-throughput MCC viability screen of ∼4000 small molecules. High-priority lead compounds supported by both screens showed efficacy in preclinical xenograft mouse models of MCC. Overall, our discovery and validation of novel targetable pathways have identified alternative treatment approaches for metastatic MCC.

Distinct Signatures of Genomic Copy Number Variants Define Subgroups of Merkel Cell Carcinoma Tumors.

Simple SummaryCancer results from genetic changes in cells. These changes are often mutations that alter the DNA sequence of critical genes. However, duplications and deletions in cancer-related genes can also contribute to malignant transformation. In this study we use Nanostring technology to assess DNA copy number changes in samples of Merkel cell carcinoma (MCC), a rare and aggressive neuroendocrine skin tumor. We were able to identify recurrent amplifications and deletions in cancer-related genes. We also found that MCC tumors grouped into three distinct copy number variant profiles. The first group consisted of tumors with multiple deletions. The second group contained tumors with low levels of genomic structural alterations. The last group comprised tumors containing multiple amplifications. Our study suggests that most MCC tumors are associated with deletions in cancer-related genes or are lacking in copy number changes, whereas a small percentage of tumors are associated with genomic amplifications.Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated Merkel cell polyomavirus DNA (virus-positive MCC, VP-MCC) and carry a low somatic mutation burden whereas virus-negative MCC (VN-MCC) possess numerous ultraviolet-signature mutations. In contrast to viral oncogenes and sequence mutations, little is known about genomic structural variants in MCC. To identify copy number variants in commonly altered genes, we analyzed genomic DNA from 31 tumor samples using the Nanostring nCounter copy number cancer panel. Unsupervised clustering revealed three tumor groups with distinct genomic structural variant signatures. The first cluster was characterized by multiple recurrent deletions in genes such as RB1 and WT1. The second cluster contained eight VP-MCC and displayed very few structural variations. The final cluster contained one VP-MCC and four VN-MCC with predominantly genomic amplifications in genes like MDM4, SKP2, and KIT and deletions in TP53. Overall, VN-MCC contained more structure variation than VP-MCC but did not cluster separately from VP-MCC. The observation that most MCC tumors demonstrate a deletion-dominated structural group signature, independent of virus status, suggests a shared pathophysiology among most VP-MCC and VN-MCC tumors.

Sex Differences in Overall Survival and the Effect of Radiotherapy in Merkel Cell Carcinoma-A Retrospective Analysis of a Swedish Cohort.

Simple SummaryMerkel cell carcinoma (MCC) is a rare and aggressive skin cancer which is believed to be partially caused by a virus or ultraviolet exposure. Most previous studies have shown that MCC is more common in men compared to women, virus associated MCC has a better prognosis and surgery followed by radiotherapy gives a better outcome. In this article, we explore these traits in a Swedish cohort of 113 patients and find that MCC is more common in women and female patients have a longer survival compared to male patients. In addition, we found that virus negative MCC has a worse outcome in male patients and radiotherapy after surgery gives a better outcome for patients who are treated with a curative dosage, irrespective of sex.Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer where Merkel cell Polyomavirus (MCPyV) contributes to the pathogenesis. In an adjuvant setting, radiotherapy (RT) is believed to give a survival benefit. The prognostic impact of sex related to MCPyV-status and adjuvant RT were analyzed in patients referred to Karolinska University Hospital. Data were collected from 113 patients’ hospital records and MCPyV analyses were made in 54 patients (48%). We found a significantly better overall survival (OS) for women compared to men and a significant difference in OS in patients receiving adjuvant RT. Furthermore, we found that men with virus negative MCC have an increased risk for earlier death (HR 3.6). This indicates that MCPyV positive and negative MCC act as two different diseases, and it might be due to different mechanism in the immune response between male and female patients. This could have significance in tailoring treatment and follow-up in MCC patients in the future.

Immunologic Characteristics of Nonmelanoma Skin Cancers: Implications for Immunotherapy.

In this review, we summarize the immunology of nonmelanoma skin cancers (NMSCs) and the clinical data with immunotherapy in this heterogeneous group of cancers that include basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (CSCC), and Merkel cell carcinoma (MCC). NMSCs are exceedingly common, and their treatment consumes substantial health care resources. Annual global mortality from NMSCs is comparable to that from malignant melanoma. Although the majority of NMSCs are localized at diagnosis and are treated effectively with surgery, metastases (nodal and distant) can sometimes arise and require systemic therapy. Given the success of immunotherapy in treating cutaneous melanoma, there has been an increasing interest in studying the immunology of NMSCs. Immunocompromised patients have a substantially higher risk of developing NMSCs (particularly CSCC and MCC), suggesting a role of the immune system in the pathogenesis of these cancers. Similar to cutaneous melanoma, the pathogenesis of BCC, CSCC, and virus-negative MCC is related to DNA damage from ultraviolet radiation exposure, and these cancers have a very high tumor mutational burden, which likely results in higher levels of tumor neoantigens that may be targets for the immune system. Viral antigens in virus-positive MCC are also strongly immunogenic. Emerging data from clinical trials of immune checkpoint inhibitors in NMSCs look very promising and are rapidly changing the treatment landscape of these cancers. Specifically, pembrolizumab and avelumab are U.S. Food and Drug Administration-approved for treatment of metastatic MCC and cemiplimab for metastatic CSCC. Several ongoing trials are investigating novel immunotherapies (monotherapies as well as combination) for treatment of NMSCs.

Unique Role of Histone Methyltransferase PRDM8 in the Tumorigenesis of Virus-Negative Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a deadly skin cancer, and about 80% of its cases have been shown to harbor integrated Merkel polyomavirus in the tumor cell genome. Viral oncoproteins expressed in the tumor cells are considered as the oncogenic factors of these virus-positive Merkel cell carcinoma (VP-MCC). In contrast, the molecular pathogenesis of virus-negative MCC (VN-MCC) is less well understood. Using gene expression analysis of MCC cell lines, we found histone methyltransferase PRDM8 to be elevated in VN-MCC. This finding was confirmed by immunohistochemical analysis of MCC tumors, revealing that increased PRDM8 expression in VN-MCC is also associated with increased H3K9 methylation. CRISPR-mediated silencing of PRDM8 in MCC cells further supported the histone methylating role of this protein in VN-MCC. We also identified miR-20a-5p as a negative regulator of PRDM8. Taken together, our findings provide insights into the role of PRDM8 as a histone methyltransferase in VN-MCC tumorigenesis.