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
Authors: Ananthapadmanabhan Varsha , Knott Aine , Soroko Kara M. , Gokhale Prafulla C. , Tirunagaru Vijaya , Doebele Robert , DeCaprio James A. 
Published: December 2021

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.