Milademetan is a highly potent MDM2 inhibitor in Merkel cell carcinoma

Abstract

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.

Keywords: Apoptosis; Oncology; Skin cancer; Therapeutics; p53.

Figure 1. MCCP cell lines with WT p53 are sensitive to milademetan treatment.

(A) MKL-1, WaGa, PeTa, and MS-1 cell lines and (B) MCC PDCLs MCC-301 and MCC 336 were treated with indicated doses of milademetan, and the Cell Titer Glo assay was performed to assess the effect on viability after 3 days of treatment. Each assay was performed in triplicate, and 3 biological replicates were performed. Data are shown as the mean ± SD. (C) MCC MKL-1, WaGa, and PeTa cell lines were treated with 100 nM milademetan, and p53 response was analyzed using WB analysis for the indicated proteins. TBP was used as a loading control. Representative WB of n = 2. PARP (cl.) indicates cleaved PARP.

Figure 2. MCC cell lines devoid of p53 or expressing dominant negative p53 are resistant to milademetan treatment.

(A) WB indicating levels of p53 in MKL-1 control or p53-KO cell lines. Vinculin was used as a loading control. (B) WB indicating levels of EGFP, WT p53, and dominant-negative p53 (p53 DD) with or without induction with doxycycline (dox). Vinculin was used as a loading control. (C and D) Cell lines were treated with milademetan followed by analysis of cell viability after 3 days of milademetan treatment. Each assay was performed in triplicate and 3 biological replicates were performed. Data are shown as the mean ± SD.

Figure 3. Antitumor activity of milademetan in an MKL-1 Xenograft tumor model.

(A) Schematic representation of the study carried out in the MCC MKL-1 xenograft model treated with either vehicle or 3 different doses of milademetan. (B) Effect of vehicle or indicated doses of milademetan treatment on individual tumor trajectories in the MCC MKL-1 xenograft study. Data were plotted until tumor volumes reached maximal permissible size or until study termination. (C) Mean xenograft tumor volumes of mice treated with vehicle or indicated doses of milademetan. Data were plotted until at least 8–9 mice per treatment group were alive. Data are shown as the mean ± SEM. (D) Kaplan-Meier survival curves of the mice in the vehicle- or milademetan-treated groups throughout the duration of the MCC MKL-1 xenograft study. (E) WB analysis of 16 different tumors obtained from 16 individual mice at the indicated time points after the last dose of vehicle or milademetan was administered showing levels of indicated proteins. PARP (cl.) indicates cleaved PARP.

Figure 4. Antitumor activity of milademetan in a WT p53 PDX model.

(A) MCC PDX 48396 individual tumor trajectories. Tumor volumes of mice treated with vehicle or indicated doses of milademetan during and after the course of treatment are shown. The shaded area indicates days when mice were treated with either vehicle or the indicated milademetan dose once a day. Data were plotted until study termination. (B) MCC PDX 48396 mean tumor volumes of mice treated with vehicle or indicated doses of milademetan during and after the course of treatment. Data were plotted for the period when all mice in every treatment group were alive. Data are shown as the mean ± SEM. (C) Kaplan-Meier survival curves of the mice in the vehicle- or milademetan-treated groups throughout the duration of the MCC PDX 48396 study.