DNA methylation is a key epigenetic signature playing mediating role in gene expression. Numerous studies have established a link between aberrant genomic methylation and cancers. In Myelodysplastic Syndromes (MDS), a heterogeneous group of malignant hematopoietic disorders, DNA methylation abnormalities play a pivotal role during the progression to Acute Myeloid Leukemia (AML) occurring in 30 percent of the cases. To date the cause of a deleterious genomic methylation remains elusive. Recently we identified a novel class of RNAs able to interact with DNA methyltransferase 1 (DNMT1), DNMT1-interacting RNAs (DiRs) and inhibit DNMT1 enzymatic activity thus regulating genomic methylation patterns and expression of the corresponding genes. We have shown that DiR originating within the locus of the methylation sensitive gene CEBPA - the extra-coding CEBPA (ecCEBPA) - regulates CEBPA expression by preventing CEBPA gene locus methylation through its interaction with DNMT1. Furthermore, by deep sequencing of the transcripts associated with DNMT1, combined with genome-scale methylation and expression profiling we have extended the generality of this finding to numerous gene loci. Here, we hypothesize that dysregulation of transcriptional profile triggers DNA methylation changes promoting secondary leukemogenesis. The two aims of this project are: 1) to identify DiRs involved in the establishment of aberrant DNA methylation patterns in MDS evolution to AML: and 2) to validate their potential as a tool to reset aberrant genomic methylation. Aim 1 will be accomplished during the K99 mentored phase, under the guidance of Prof. Daniel G Tenen (Harvard Medical School) and Prof. John L. Rinn (Harvard University), world-leading experts in the field of leukemogenesis and RNA biology, respectively. By correlating DiRs and DiRs-regulated genes' transcription profiles of primary paired MDS (diagnosis)/AML (progression) bone marrow mononuclear cells with DNA methylation status of the correspondent genomic regions, DNA methylation and gene expression profiles will be linked to the presence or absence of RNA-DNMT association. This will lead to the identification of DiRs associated with aberrant DNA methylation patterns in MDS-AML transformation (Aim 1). Chosen candidates will be functionally validated during the R00 phase, in vitro and in vivo systems (Aim 2). This approach will allow correcting gene-specific aberrant DNA methylation using RNA molecules. Currently, the only demethylating agents approved for therapeutic applications are Azacitidine and Decitabine. However major downsides for using these drugs include cytotoxic effects and global non-specific demethylation. This study holds promise for a genuine, rather than nominal, targeted therapy. While in the short terms this research will lead to the identification of novel ky regulators contributing to leukemogenesis; in the long terms it will pave the way for the development of a long-awaited gene-specific demethylating tool for the treatment of cancer and other diseases triggered by DNA methylation abnormalities.