A CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that can be targeted for therapeutic intervention in AML

DNA methylation is an epigenetic mechanism that regulates gene expression during many stages of development, including genomic imprinting, stem cell regulation, and X-chromosome inactivation. Moreover, aberrant DNA methylation patterns, characterized by genome-wide hypomethylation and promoter-specific hypermethylation, are a prominent feature of cancer. Methylation of DNA at the 5-position of cytosines is mediated by the DNA methyltransferase (DNMT) protein family, which regulates both maintenance methylation (DNMT1) and de novo methylation (DNMT3A and DNMT3B). Loss-of-function mutations of DNMT3A have been identified in hematological malignancies including acute myeloid leukemia (AML), where DNMT3A is mutated in approximately 25% of known cases. Published reports suggest the existence of a synthetic lethal interaction between DNMT3A and DNMT1/3B. To further study this potential genetic interaction, we are performing a CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that are synthetic lethal with DNMT3A. We generated a lentiviral library containing 777 and 421 single guide RNAs (sgRNAs) that tile the coding region of DNMT1 and DNMT3B, respectively and performed viability screens in AML cell lines that are either wild-type or mutant for DNMT3A. This screen was designed to identify in-frame alterations within functional domains that lead to effects on cell viability. Next generation sequencing of sgRNAs identified three functional domains of DNMT1 which, when mutated, leads to decreases in cell viability. Current efforts are focused on verifying the essentiality of these functional domains using CRISPR-Cas9-based approaches as well as mutagenesis by integrated tiles (MITE)-seq analyses. Citation Format: Balpreet Bhogal, Barbara Weir, Ramona Crescenzo, Min Chul Kwon, Ulrike Philippar, Ricardo Attar, Glenn Cowley, David Pocalyko. A CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that can be targeted for therapeutic intervention in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1383.