Abstract 1360: Targeting the ubiquitin reading function of DNMT1 as a next-generation approach for epigenetic cancer therapy.

Abstract DNA methyltransferase 1 (DNMT1) is the key enzyme supporting the epigenetic inheritance of DNA methylation patterns during DNA replication, a process frequently dysregulated across nearly all human cancers. Abnormal DNA methylation patterning is an appreciated mechanism of tumor suppressor gene (TSGs) silencing and immune evasion, motivating the clinical development of DNA methyltransferase inhibitors (DNMTi’s). While nucleoside analog DNMTi’s are the most clinically advanced epigenetic cancer therapies, their utility has been limited due to pharmacokinetic barriers and adaptive resistance mechanisms. New approaches to inhibit abnormal DNA methylation patterning are therefore needed. Here, we define the DNMT1 ubiquitin interacting motifs (UIMs), which recognize UHRF1-dependent histone H3 mono-ubiquitination, as targetable domains to allosterically inhibit DNMT1 catalytic activity, and we identify a pharmacophore that can be used as a starting point for UIM inhibitor development. To characterize the functional significance of the DNMT1 UIMs, we developed a genetic complementation system in two human colon cancer cell lines to cover the loss of endogenous DNMT1 with wild-type or loss-of-function mutants with disrupted catalytic or UIM activities. Disruption of DNMT1 UIM function mirrored DNMT1 knockdown, catalytic-dead mutant cover, and exposure to nucleoside- and non-nucleoside catalytic DNMT inhibitors. These effects included widespread DNA hypomethylation that correlated with the re-expression of epigenetically silenced TSGs and transposable elements, reduced colony formation, impaired cell proliferation, and impaired growth of xenografted tumors. A pilot screen of a chemical library revealed four compounds with a shared pharmacophore able to disrupt the interaction of DNMT1 with ubiquitinated nucleosomes. Collectively, these studies demonstrate that the oncogenic properties of DNMT1 are supported by its ubiquitin reading function and provide a roadmap for developing next-generation DNMT1 inhibitors targeting this allosteric site. Citation Format: Joshua A. Kuleape, Yanqing Liu, Joel A. Hrit, Rochelle Tiedemann, Scott B. Rothbart, . Targeting the ubiquitin reading function of DNMT1 as a next-generation approach for epigenetic cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1360.