Abstract 6011: Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo

Abstract Tumors carrying defects in DNA mismatch repair (MMR-d) display high tumor mutational burden (TMB) and increased tumor neoantigen levels. MMR-d leads to accumulation of single nucleotide variants (SNVs) and insertions or deletions (indels) amongst repetitive DNA sequences generating microsatellite instability (MSI). Cancers that harbor 40% microsatellite variations are described as MSI-high (MSI-H). These tumors display unique clinical features including better prognosis, increased immune infiltration and remarkable response to immune checkpoint blockade (ICB) therapy. Colorectal cancer (CRC) patients with MMR-d often respond to therapies based on immune checkpoint blockade, whilst those with mismatch repair proficient (MMR-p) tumors do not. Therefore, identifying strategies to enhance the efficacy of immunotherapeutic treatments and transform this prevalent subgroup of CRCs from immunologically cold to hot remains an urgent and an unmet clinical need. We discovered NP1867 a first-in-class, potent, selective, covalent small molecule inhibitor of the DNA Mismatch Repair protein PMS2. We show by biochemical, biophysical, and protein-ligand X-ray crystallographic methods that NP1867 binds irreversibly to the ATP binding site of the N-terminal ATPase domain of PMS2. NP1867 displays potent binding to PMS2 in a NanoBRET cellular target engagement assay and inhibits MMR activity in mechanistic and functional cell-based assays of MMR-dependent DNA repair. Continuous treatment of murine CRC cell line CT26 (MMR-p) with NP1867 increases TMB, specifically enriches gained mutations with typical MMR-d related COSMIC mutational signatures, and elicits MSI-H status in a time-dependent manner, demonstrating for the first time that inhibition of PMS2 with a small molecule phenocopies the inactivation of MMR observed in patients. Remarkably, inoculation of immune-competent mice with CT26 cells pre-treated with NP1867 elicits tumor regressions in anti-PD1-treated animals, recapitulating the unique clinical features of MMR-d/MSI-H tumors. Conversely, no response is observed in immune-competent animals inoculated with control-treated cells. The extent of in vivo response correlates with the duration of in vitro NP1867 treatment and with the MSI-H status of treated CT26 cells. In conclusion, small molecule NP1867 functionally inhibits DNA MMR by targeting PMS2, enriches gained mutations with MMR-d COSMIC signatures, elicits MSI-H status, and enhances immune surveillance in preclinical models. These findings pave the way for the development of orally bioavailable compounds suitable for long-term dosing in animal models. Citation Format: Eleonora Piumatti, Alexia Hervieu, Philippe Riou, Julian Blagg, Adam Peall, Sam Weeks, Maria T. Rodríguez-Plata, Giuseppe Rospo, Sasi Arunachalam, Bettina Meier, Paige Tongue, Tessa McLaren, Kalpesh Parmar, Pradip Patel, David Clark, Gareth Langley, Charles Nichols, Benoît Rousseau, Paul Winship, Matthew Baker, Martin Drysdale, Giovanni Germano, Alberto Bardelli. Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo 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 (6Suppl): Abstract nr 6011.