Project Grant F30CA310102

ROLE OF RIBOSOMAL DNA EPIGENETIC ABERRATIONS IN ACUTE MYELOID LEUKEMIA (AML) - PROJECT SUMMARY/ABSTRACT RIBOSOMAL DYSREGULATION IS A HALLMARK OF CANCER, DRIVEN BY INCREASED RIBOSOMAL OUTPUT NEEDED FOR RAPID CELL PROLIFERATION. RIBOSOME BIOGENESIS IS A COMPLEX PROCESS INITIATED IN THE NUCLEOLUS WITH TRANSCRIPTION OF RIBOSOMAL DNA (RDNA) INTO RRNA PRECURSORS THAT SUBSEQUENTLY MATURE INTO RRNA. AN ABNORMAL EPIGENETIC LANDSCAPE ALSO CHARACTERIZES CANCER, CONTRIBUTING TO GENOME INSTABILITY AND TRANSCRIPTIONAL DYSREGULATION. WHILE RECENT STUDIES HAVE HIGHLIGHTED TRANSCRIPTION FACTORS' ROLE IN SUPPORTING RDNA TRANSCRIPTION AND RRNA PRODUCTION, EPIGENETIC REGULATION OF RDNA REMAINS LARGELY UNCLEAR DUE TO ITS HIGHLY REPETITIVE NATURE AS TANDEM ARRAYS ACROSS THE GENOME, WHICH POSES SIGNIFICANT CHALLENGES FOR SHORT-READ SEQUENCING AND ALIGNMENT. TO ADDRESS THIS, A CUSTOMIZED GENOME-ASSEMBLY PIPELINE HAS BEEN DEVELOPED TO MAP HUMAN AND MOUSE RDNA USING HIGH-THROUGHPUT SEQUENCING DATA. USING THIS PIPELINE WITH VARIOUS DNA METHYLATION DATASETS, I OBSERVED A DISTINCT INCREASE IN DNA METHYLATION WITHIN RDNA REGIONS IN AML, WHICH MAY REFLECT DISEASE STATUS AND TREATMENT RESPONSE. I ALSO DETECTED A POSITIVE CORRELATION BETWEEN INCREASED DNA METHYLATION AND RNA POLYMERASE I (RNAPI) BINDING AT RDNA IN AML, SUGGESTING A POTENTIAL ROLE FOR METHYLATION IN REGULATING RRNA PRODUCTION. INTEGRATIVE ANALYSES USING MULTIPLE AML PATIENT DATASETS AND TF BINDING MOTIFS IDENTIFIED SEVERAL TF CANDIDATES WHOSE BINDING MAY BE INFLUENCED BY INCREASED RDNA METHYLATION, POTENTIALLY CONTRIBUTING TO LEUKEMIA CELL GROWTH. BASED ON THESE PRELIMINARY DATA, I HYPOTHESIZE THAT AML-SPECIFIC RDNA METHYLATION PATTERNS ASSOCIATE WITH DISEASE STATUS AND RESPONSE TO HYPOMETHYLATING AGENT (HMA) THERAPY, AND THAT ALTERED RDNA METHYLATION AFFECTS TF BINDING AND RRNA PRODUCTION, THEREBY PROMOTING LEUKEMOGENESIS. TO TEST THIS, I WILL ANALYZE PUBLICLY AVAILABLE AML DATASETS AND LONG-READ SEQUENCING DATA GENERATED IN THIS PROPOSAL TO CHARACTERIZE DYSREGULATED RDNA METHYLATION IN AML (AIM 1). THIS WILL ENABLE IDENTIFICATION OF AML-SPECIFIC METHYLATION SIGNATURES AND ASSESSMENT OF THEIR CORRELATION WITH TRANSCRIPTIONAL CHANGES, CLINICAL OUTCOMES, AND HMA RESPONSES, PROVIDING INSIGHTS INTO FUNCTIONAL CONSEQUENCES OF RDNA EPIGENETIC ALTERATIONS. IN PARALLEL, I WILL INVESTIGATE RDNA METHYLATION'S FUNCTIONAL ROLE IN REGULATING TF BINDING AND RRNA PRODUCTION (AIM 2). I HAVE IDENTIFIED KEY TFS WHOSE BINDING IS LIKELY AFFECTED BY ALTERED RDNA METHYLATION AND WILL EVALUATE THESE CANDIDATES ALONGSIDE RNAPI BINDING, RDNA METHYLATION LANDSCAPE, AND RDNA TRANSCRIPTIONAL ACTIVITY IN AML, CML, AND NORMAL HSPCS, WHICH EXHIBIT VARIOUS LEVELS OF RDNA METHYLATION, TO SUPPORT MECHANISTIC STUDIES. DCAS9-MEDIATED EPIGENOME EDITING WILL PROBE THE CAUSAL RELATIONSHIP BETWEEN RDNA METHYLATION CHANGES AND TF BINDING IN AML. SUCCESSFUL COMPLETION WILL PROVIDE A COMPREHENSIVE ANALYSIS OF PREVIOUSLY UNDER- RECOGNIZED EPIGENETIC MODIFICATIONS WITHIN RDNA IN REGULATING RRNA PRODUCTION AND MALIGNANT TRANSFORMATION, WITH POTENTIAL TO UNCOVER NEW EPIGENETIC BIOMARKERS FOR AML AND ESTABLISH A FRAMEWORK FOR STUDYING RDNA METHYLATION ACROSS HUMAN DISEASES.