Gene-specific demethylation and re-activation of tumor suppressors in acute myeloid leukemia via a novel RNA-mediated platform
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Abstract
Aberrant DNA methylation is considered to be a key epigenetic feature of cancer. More specifically, abnormal DNA methylation of the promoter regions of tumor suppressor genes commonly leads to gene silencing and the loss of cell cycle checkpoints, paving the way for uncontrolled proliferation of cancerous cells. Acute myeloid leukemia is one such cancer that exhibits this epigenetic mechanism of transcriptional inhibition, notably at the CDK inhibitor and tumor suppressor gene CDKN2B. Currently available demethylating agents, namely 5-azacytidine and 5-aza-2’-deoxycytidine, are used to treat acute myeloid leukemia and inhibit DNA methyltransferases either by direct binding or by incorporation into DNA as nucleoside analogs. While effective, they are significantly limited by their non-specific impacts across the genome and their potential for DNA damage and cytotoxicity. These unintended side effects consequently create an urgent need for novel DNMT-inhibiting mechanisms with improved accuracy.
In this thesis, we therefore designed and developed CRISPR-DiR (DNMT1-interacting RNA), an RNA-based system to localize DNMT1-binding RNA structures to specific gene loci. We established a previously discovered long non-coding RNA from the CEBPα gene as the foundation of CRISPR-DiR; studies have identified critical secondary structures from this long non-coding RNA that inhibit DNA methyltransferase 1. We then hybridized these secondary structures to the CRISPR guide RNA system. CRISPR-DiR thus has the ability to localize DNMT1-inhibition to specifically designated gene targets. This thesis details a step-by-step strategy for application of the novel technology as a therapeutic against acute myeloid leukemia. We validated CRISPR-DiR’s ability to demethylate known hypermethylated targets, elucidated the methylation profile of the CDKN2B gene, and subsequently generated viable CRISPR guide RNA targets of interest. Finally, we designed a method for the construction of sgDiR-dCas9 cell lines and relevant assays to assess CRISPR-DiR efficacy as both a therapeutic and a tool to examine novel mechanisms of epigenetic regulation.