Virus-positive Merkel Cell Carcinoma Research Articles

Durability of immune checkpoint inhibitor (ICI) response following treatment discontinuation in advanced merkel cell carcinoma (MCC).

9596 Background: Advanced MCC has a high response rate to ICI therapy, but the durability of responses once treatment is discontinued remains unclear. We therefore reviewed the long-term outcomes of advanced MCC patients at our institution who discontinued ICI treatment after achieving favorable initial response. Methods: In an IRB-approved single institution retrospective review of advanced MCC patients, we analyzed all patients who received at least one dose of anti-PD(L)1 monotherapy for unresectable or metastatic disease, achieved stable disease (SD) or better, and discontinued treatment for a reason other than disease progression. Duration of response and outcomes of subsequent therapy were assessed. Results: Of 195 advanced MCC patients treated with ICI, we identified 45 who met study criteria. Of these, 21 (47%) had a complete response (CR) to initial ICI treatment, 23 (51%) a partial response (PR) and 1 (2%) SD. Twenty-five (56%) patients discontinued ICI electively and 20 (44%) discontinued due to toxicity. In total, 21 of the 45 patients (47%) experienced disease progression at a median of 11.2 months (range 2.1-22.7 months) from ICI cessation. The median follow-up after ICI discontinuation for patients who have not experienced progression is 22.8 months (5.8-82.0 months). There was a lower rate of progression in patients who achieved CR vs. non-CR (24% vs 67%, p=0.006) and a trend towards a lower rate in those who discontinued electively vs. due to toxicity (36% vs 60%, p=0.14). For patients in whom the molecular subtype was known, there was a higher risk for progression in patients with viral MCC (12 of 16, 75%) compared to UV-associated MCC (4 of 13, 31%, p=0.02). Fifteen of the 21 patients (71%) who experienced progression were rechallenged with ICI therapy, including both single agent (12 patients, 57%) and combination (3, 14%) therapy. The remaining patients received locally-directed therapy only (3, 14%), chemotherapy (1, 5%), declined further treatment (1, 5%), or transferred care and were lost to follow up (1, 5%). Eleven of 15 ICI-retreated patients (73%) achieved an objective response to rechallenge, including all 3 patients who received combination therapy with ipilimumab+nivolumab. Twelve of the 45 patients have expired; 6 due to progressive MCC and remaining 6 known or suspected from unrelated causes. Conclusions: Patients with advanced MCC have a substantial risk of disease progression following treatment discontinuation despite initial favorable ICI response, 47% in our series with a median time to event of 11.2 months, particularly in those that achieve less than a complete response. In the setting of disease progression post-ICI discontinuation, most patients maintain sensitivity to retreatment with the same drug class. Virus-positive MCC may be a risk factor for post-discontinuation relapse, which should be validated in future studies.

The effect of selinexor on prostaglandin synthesis in virus-positive Merkel cell carcinoma cell lines.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with high rates of metastasis and mortality. In vitro studies suggest that selinexor (KPT-330), an inhibitor of exportin 1, may be a targeted therapeutic option for MCC. This selective inhibitor prevents the transport of oncogenic mRNA out of the nucleus. Of note, 80% of MCC tumors are integrated with Merkel cell polyomavirus (MCPyV), and virally encoded tumor-antigens, small T (sT) and large T (LT) mRNAs may require an exportin transporter to relocate to the cytoplasm and modulate host tumor-suppressing pathways. To explore selinexor as a targeted therapy for MCC, we examine its ability to inhibit LT and sT antigen expression in vitro and its impact on the prostaglandin synthesis pathway. Protein expression was determined through immunoblotting and quantified by densitometric analysis. Statistical significance was determined with t-test. Treatment of MCPyV-infected cell lines with selinexor resulted in a significant dose-dependent downregulation of key mediators of the prostaglandin synthesis pathway. Given the role of prostaglandin synthesis pathway in MCC, our findings suggest that selinexor, alone or in combination with immunotherapy, could be a promising treatment for MCPyV-infected MCC patients who are resistant to chemotherapy and immunotherapy.

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.

T antigen-specific CD8+ T cells associate with PD-1 blockade response in virus-positive Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

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.

An Investigation of Structure-Activity Relationships and Cell Death Mechanisms of the Marine Alkaloids Discorhabdins in Merkel Cell Carcinoma Cells.

A library of naturally occurring and semi-synthetic discorhabdins was assessed for their effects on Merkel cell carcinoma (MCC) cell viability. The set included five new natural products and semi-synthetic compounds whose structures were elucidated with NMR, HRMS, and ECD techniques. Several discorhabdins averaged sub-micromolar potency against the MCC cell lines tested and most of the active compounds showed selectivity towards virus-positive MCC cell lines. An investigation of structure-activity relationships resulted in an expanded understanding of the crucial structural features of the discorhabdin scaffold. Mechanistic cell death assays suggested that discorhabdins, unlike many other MCC-active small molecules, do not induce apoptosis, as shown by the lack of caspase activation, annexin V staining, and response to caspase inhibition. Similarly, discorhabdin treatment failed to increase MCC intracellular calcium and ROS levels. In contrast, the rapid loss of cellular reducing potential and mitochondrial membrane potential suggested that discorhabdins induce mitochondrial dysfunction leading to non-apoptotic cell death.

Insights into anti-tumor immunity via the polyomavirus shared across human Merkel cell carcinomas.

Understanding and augmenting cancer-specific immunity is impeded by the fact that most tumors are driven by patient-specific mutations that encode unique antigenic epitopes. The shared antigens in virus-driven tumors can help overcome this limitation. Merkel cell carcinoma (MCC) is a particularly interesting tumor immunity model because (1) 80% of cases are driven by Merkel cell polyomavirus (MCPyV) oncoproteins that must be continually expressed for tumor survival; (2) MCPyV oncoproteins are only ~400 amino acids in length and are essentially invariant between tumors; (3) MCPyV-specific T cell responses are robust and strongly linked to patient outcomes; (4) anti-MCPyV antibodies reliably increase with MCC recurrence, forming the basis of a standard clinical surveillance test; and (5) MCC has one of the highest response rates to PD-1 pathway blockade among all solid cancers. Leveraging these well-defined viral oncoproteins, a set of tools that includes over 20 peptide-MHC class I tetramers has been developed to facilitate the study of anti-tumor immunity across MCC patients. Additionally, the highly immunogenic nature of MCPyV oncoproteins forces MCC tumors to develop robust immune evasion mechanisms to survive. Indeed, several immune evasion mechanisms are active in MCC, including transcriptional downregulation of MHC expression by tumor cells and upregulation of inhibitory molecules including PD-L1 and immunosuppressive cytokines. About half of patients with advanced MCC do not persistently benefit from PD-1 pathway blockade. Herein, we (1) summarize the lessons learned from studying the anti-tumor T cell response to virus-positive MCC; (2) review immune evasion mechanisms in MCC; (3) review mechanisms of resistance to immune-based therapies in MCC and other cancers; and (4) discuss how recently developed tools can be used to address open questions in cancer immunotherapy. We believe detailed investigation of this model cancer will provide insight into tumor immunity that will likely also be applicable to more common cancers without shared tumor antigens.

Distinct Regulation of EZH2 and its Repressive H3K27me3 Mark in Polyomavirus-Positive and -Negative Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P= 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. Invitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.

Characterization of the Impact of Merkel Cell Polyomavirus-Induced Interferon Signaling on Viral Infection.

Merkel cell polyomavirus (MCPyV) has been associated with approximately 80% of Merkel cell carcinoma (MCC), an aggressive and increasingly incident skin cancer. The link between host innate immunity, viral load control, and carcinogenesis has been established but poorly characterized. We previously established the importance of the STING and NF-κB pathways in the host innate immune response to viral infection. In this study, we further discovered that MCPyV infection of human dermal fibroblasts (HDFs) induces the expression of type I and III interferons (IFNs), which in turn stimulate robust expression of IFN-stimulated genes (ISGs). Blocking type I IFN downstream signaling using an IFN-β antibody, JAK inhibitors, and CRISPR knockout of the receptor dramatically repressed MCPyV infection-induced ISG expression but did not significantly restore viral replication activities. These findings suggest that IFN-mediated induction of ISGs in response to MCPyV infection is not crucial to viral control. Instead, we found that type I IFN exerts a more direct effect on MCPyV infection postentry by repressing early viral transcription. We further demonstrated that growth factors normally upregulated in wounded or UV-irradiated human skin can significantly stimulate MCPyV gene expression and replication. Together, these data suggest that in healthy individuals, host antiviral responses, such as IFN production induced by viral activity, may restrict viral propagation to reduce MCPyV burden. Meanwhile, growth factors induced by skin abrasion or UV irradiation may stimulate infected dermal fibroblasts to promote MCPyV propagation. A delicate balance of these mutually antagonizing factors provides a mechanism to support persistent MCPyV infection. IMPORTANCE Merkel cell carcinoma is an aggressive skin cancer that is particularly lethal to immunocompromised individuals. Though rare, MCC incidence has increased significantly in recent years. There are no lasting and effective treatments for metastatic disease, highlighting the need for additional treatment and prevention strategies. By investigating how the host innate immune system interfaces with Merkel cell polyomavirus, the etiological agent of most of these cancers, our studies identified key factors necessary for viral control, as well as conditions that support viral propagation. These studies provide new insights for understanding how the virus balances the effects of the host immune defenses and of growth factor stimulation to achieve persistent infection. Since virus-positive MCC requires the expression of viral oncogenes to survive, our observation that type I IFN can repress viral oncogene transcription indicates that these cytokines could be explored as a viable therapeutic option for treating patients with virus-positive MCC.

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).

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.

ATTAC-MCC: Phase I/II study of autologous CD8+ and CD4+ transgenic T cells expressing a high affinity MCPyV-specific TCR combined with checkpoint inhibitors and class I MHC-upregulation in patients with metastatic MCC refractory to PD-1 axis blockade.

TPS9596 Background: Merkel cell carcinoma (MCC) is a highly aggressive skin cancer, with an incidence that has doubled in the last 20 years to approximately 3000 cases/year in the US. Over one-third of patients will develop widespread disease, and survival in these patients has been historically poor with a 5-year survival rate of < 10%. MCC is highly immune-sensitive due to the antigenicity of the cancer-causing Merkel cell polyomavirus (MCPyV) expressed in most MCC tumors. Although immune checkpoint inhibitors (ICIs) targeting the PD-(L)1 axis show promising efficacy, most MCC patients will eventually relapse. There is no standard of care for patients that become refractory to ICIs. We hypothesize that cellular immune therapies targeting MCPyV may provide additional clinical benefit to these patients. To test this, we have engineered high-affinity TCR T cells against MCPyV and initiated a clinical trial. Methods: NCT03747484 is an ongoing phase I/II, open label, investigator-initiated trial (IIT) of FH-MCVA2TCR in combination with an anti-PD-(L)1 checkpoint inhibitor and an agent to upregulate MHC-I expression on tumor cells. The trial is conducted in patients aged 18 years or older with metastatic or unresectable, histologically confirmed virus-positive MCC whose disease has progressed on or after treatment with a PD-(L)1 axis checkpoint inhibitor. Patients undergo leukapheresis to collect white blood cells (WBCs) for TCR T cell product manufacturing. The cell product is administered on day 0. Patients receive an agent to upregulate MHC-I on tumor cells and continue on an anti-PD-(L)1 checkpoint inhibitor for up to one year. In phase I, three patients receive up to two infusions of dose level 1 of the T cell product. The primary objectives of phase I are to determine safety and tolerability based on dose-limiting toxicities (DLT) during an observation period of 28 days after the first infusion. Phase II enrolls patients at dose level 2. The total sample size of phase II is 12 patients. The first three patients are enrolled in a staggered manner and observed for DLTs. If no DLTs occur, the remaining sample size is accrued in open enrollment. The main objectives of phase II are safety based on number of adverse events and preliminary efficacy based on tumor response according to RECISTv1.1 and iRECIST. Secondary/exploratory objectives include cellular kinetics of TCR T cells, T cell phenotype, tumor infiltration kinetics, and MHC-I expression dynamics over time. After the first 12 months, patients transition to a long-term follow-up (LTFU) study for up to 15 years as per FDA guidelines. Clinical trial information: NCT03747484.

Merkel cell polyomavirus large T antigen binding to pRb promotes skin hyperplasia and tumor development.

Clear evidence supports a causal link between Merkel cell polyomavirus (MCPyV) and the highly aggressive human skin cancer called Merkel cell carcinoma (MCC). Integration of viral DNA into the human genome facilitates continued expression of the MCPyV small tumor (ST) and large tumor (LT) antigens in virus-positive MCCs. In MCC tumors, MCPyV LT is truncated in a manner that renders the virus unable to replicate yet preserves the LXCXE motif that facilitates its binding to and inactivation of the retinoblastoma tumor suppressor protein (pRb). We previously developed a MCPyV transgenic mouse model in which MCC tumor-derived ST and truncated LT expression were targeted to the stratified epithelium of the skin, causing epithelial hyperplasia, increased proliferation, and spontaneous tumorigenesis. We sought to determine if any of these phenotypes required the association between the truncated MCPyV LT and pRb. Mice were generated in which K14-driven MCPyV ST/LT were expressed in the context of a homozygous RbΔLXCXE knock-in allele that attenuates LT-pRb interactions through LT’s LXCXE motif. We found that many of the phenotypes including tumorigenesis that develop in the K14-driven MCPyV transgenic mice were dependent upon LT’s LXCXE-dependent interaction with pRb. These findings highlight the importance of the MCPyV LT-pRb interaction in an in vivo model for MCPyV-induced tumorigenesis.

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.

Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus.

Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative cells. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and infected tissues and express proteins that have the potential to modulate the infectious and tumorigenic properties of these viruses.

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.

Virus-positive Merkel Cell Carcinoma Is an Independent Prognostic Group with Distinct Predictive Biomarkers.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma that can be divided into two classes: virus-positive (VP) MCC, associated with oncogenic Merkel cell polyomavirus (MCPyV); and virus-negative (VN) MCC, associated with photodamage. We classified 346 MCC tumors from 300 patients for MCPyV using a combination of IHC, ISH, and qPCR assays. In a subset of tumors, we profiled mutation status and expression of cancer-relevant genes. MCPyV and molecular profiling results were correlated with disease-specific outcomes. Potential prognostic biomarkers were further validated by IHC. A total of 177 tumors were classified as VP-MCC, 151 tumors were VN-MCC, and 17 tumors were indeterminate. MCPyV positivity in primary tumors was associated with longer disease-specific and recurrence-free survival in univariate analysis, and in multivariate analysis incorporating age, sex, immune status, and stage at presentation. Prioritized oncogene or tumor suppressor mutations were frequent in VN-MCC but rare in VP-MCC. TP53 mutation developed with recurrence in one VP-MCC case. Importantly, for the first time we find that VP-MCC and VN-MCC display distinct sets of prognostic molecular biomarkers. For VP-MCC, shorter survival was associated with decreased expression of immune markers including granzyme and IDO1. For VN-MCC, shorter survival correlated with high expression of several genes including UBE2C. MCPyV status is an independent prognostic factor for MCC. Features of the tumor genome, transcriptome, and microenvironment may modify prognosis in a manner specific to viral status. MCPyV status has clinicopathologic significance and allows for identification of additional prognostic subgroups.

Mutational Landscape of Virus- and UV-Associated Merkel Cell Carcinoma Cell Lines Is Comparable to Tumor Tissue.

Simple SummaryMerkel cell carcinoma (MCC) is an aggressive, rare skin cancer which is caused either by a virus or chronic UV exposure. For both forms, distinct genetic alterations have been described; however, these observations were mostly made in tumor tissue. Since cancer cell lines are frequently used as preclinical models to investigate biological function, we considered it necessary to establish the genomic landscape of MCC cell lines by whole-exome sequencing. We confirmed the presence of UV-induced DNA damage, a high number of mutations and several coding mutations in virus-negative cell lines which were absent in virus-positive cell lines; these, however, harbored characteristic copy number variations, suggesting some virally caused genetic instability. Knowing the genomic features of MCC cell lines validates previous, and facilitates upcoming, experimental studies to discover their biological and translational relevance.Merkel cell carcinoma (MCC) is a rare, highly aggressive cutaneous malignancy that is either associated with the integration of the Merkel cell polyomavirus or chronic UV exposure. These two types of carcinogenesis are reflected in characteristic mutational features present in MCC tumor lesions. However, the genomic characteristics of MCC cell lines used as preclinical models are not well established. Thus, we analyzed the exomes of three virus-negative and six virus-positive MCC cell lines, all showing a classical neuroendocrine growth pattern. Virus-negative cell lines are characterized by a high tumor mutational burden (TMB), UV-light-induced DNA damage, functionally relevant coding mutations, e.g., in RB1 and TP53, and large amounts of copy number variations (CNVs). In contrast, virus-positive cell lines have a low TMB with few coding mutations and lack prominent mutational signatures, but harbor characteristic CNVs. One of the virus-negative cell lines has a local MYC amplification associated with high MYC mRNA expression. In conclusion, virus-positive and -negative MCC cell lines with a neuroendocrine growth pattern resemble mutational features observed in MCC tissue samples, which strengthens their utility for functional studies.

Robust Production of Merkel Cell Polyomavirus Oncogene Specific T Cells From Healthy Donors for Adoptive Transfer.

Virus positive Merkel cell carcinoma (VP-MCC) is an aggressive but immunogenic skin malignancy driven by Merkel cell polyomavirus (MCPyV) T antigen (TAg). Since adoptive T cell transfer (ACT) can be effective against virus-driven malignancies, we set out to develop a methodology for generating MCPyV TAg specific T cells. MCPyV is a common, asymptomatic infection and virus-exposed healthy donors represent a potential source of MCPyV TAg specific T cells for ACT. Virus specific T cells were generated using monocyte-derived dendritic cells (moDCs) pulsed with MCPyV TAg peptide libraries and co-cultured with autologous T cells in supplemented with pro-inflammatory and homeostatic cytokines for 14 days. Specific reactivity was observed predominantly within the CD4+ T cell compartment in the cultures generated from 21/46 random healthy donors. Notably, responses were more often seen in donors aged 50 years and older. TAg specific CD4+ T cells specifically secreted Th1 cytokines and upregulated CD137 upon challenge with MCPyV TAg peptide libraries and autologous transduced antigen presenting cells. Expanded T cells from healthy donors recognized epitopes of both TAg splice variants found in VP-MCC tumors, and minimally expressed exhaustion markers. Our data show that MCPyV specific T cells can be expanded from healthy donors using methods appropriate for the manufacture of clinical grade ACT products.