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Aberrant DNA hypermethylation within promoter regions and subsequent gene silencing are near universal hallmarks of human cancer. Reversal of DNA methylation by a hypomethylating agent, such as decitabine (Dacogen) or azacytidine (Vidaza), has shown clinical benefit for the treatment of heme malignancies. However, these agents have several limitations that preclude their full potential from being realized such as the requirement for IV administration, poor PK properties and a mechanism that requires incorporation into replicating DNA. This indirect, irreversible inhibition of the DNA methyltransferase (DNMT) family (DNMT1, 3a and 3b) and subsequent DNA damage induces significant dose-limiting toxicity thus preventing sufficient target engagement required for maximal demethylation. A drug discovery effort focused on DNMT1, the key family member responsible for maintaining the DNA methylation pattern, was initiated based on the compelling nature of the target coupled with the need for improved agents. A high-throughput screen identified a single dicyanopyridine (DCP) series of reversible, non-DNA incorporating, highly selective inhibitors for DNMT1 over DNMT3a or DNMT3b. Ensuing structure-activity relationship (SAR) optimization of the series led to the discovery of potent tool compounds that in cancer cells induced robust decreases in global DNA methylation, transcriptional activation of many silenced genes, and inhibition of cancer cell growth. In contrast to decitabine where most cells showed a cytotoxic response, our DNMT1 tool inhibitors primarily elicited a cytostatic response. Furthermore, studies in a mouse tumor model revealed decreased DNA methylation and a dose-dependent (1-45 mg/kg, BID) decrease in tumor growth with regression at the highest doses. In summary, a series of potent, selective DNMT1 inhibitors were discovered and refined delivering tool compounds capable of eliciting changes in DNA methylation, transcriptional activation, and tumor regression at well-tolerated doses. Thus demonstrating that selective, non-covalent inhibitors of DNMT1 may provide benefit over traditional DNA incorporating hypomethylating agents. Citation Format: Melissa B. Pappalardi, Mark Cockerill, Jessica L. Handler, Alexandra Stowell, Kathryn Keenan, Christian S. Sherk, Elisabeth A. Minthorn, Charles F. McHugh, Charlotte Burt, Kristen Wong, David T. Fosbenner, Mehul Patel, Jacques Briand, Helai Mohammad, Lourdes Rueda, Andrew Benowitz, Rab Prinjha, Dirk Heerding, Ryan G. Kruger, Ali Raoof, Allan Jordan, Bryan W. King, Michael T. McCabe. Discovery of selective, noncovalent small molecule inhibitors of DNMT1 as an alternative to traditional DNA hypomethylating agents [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 2994.