Project Grant R21AI186406

DISCRIMINATING PATHOGENIC FROM BENIGN ALLELES OF MYELODYSPLASTIC SYNDROME PREDISPOSITION GENES - PATIENT GENOME SEQUENCING HAS REVEALED GERMLINE GENETIC VARIATION IN DDX41 AS ONE OF THE MOST FREQUENT GENOMIC ALTERATIONS IMPLICATED IN CREATING A PREDISPOSITION TO MYELODYSPLASTIC SYNDROME (MDS) AND ACUTE MYELOID LEUKEMIA (AML). DDX41 ENCODES AN RNA HELICASE THAT REGULATES RNA SPLICING, SENSES DOUBLE-STRANDED DNA, OPERATES IN THE CGAS-STING PATHWAY AND PROMOTES INNATE IMMUNITY. MANY QUESTIONS REMAIN REGARDING DDX41 MECHANISMS AND HOW GENETIC VARIANTS IMPACT ITS FUNCTIONS. TO GAIN FUNDAMENTAL AND TRANSLATIONAL INSIGHTS, WE ENGINEERED THE GENOME OF HOXB8-IMMORTALIZED MURINE HEMATOPOIETIC PROGENITOR CELLS, WHICH RECAPITULATE THE PHENOTYPE OF PRIMARY PROGENITORS, TO YIELD DDX41+/- CELLS. WE INNOVATED A RESCUE SYSTEM TO COMPARE HUMAN DDX41 ACTIVITY WITH THAT OF CLINICAL VARIANTS. USING AN UNBIASED GENOMIC STRATEGY, WE IDENTIFIED DDX41-REGULATED MRNAS AND MEMBRANE PROTEINS AS ACTIVITY METRICS. WE WILL USE OUR FOUNDATION AND MACHINE LEARNING TO CONSTRUCT A MATRIX THAT INFORMS THE RELATIONSHIP BETWEEN DDX41 AND CLINICAL VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) OR THOSE DEEMED PATHOGENIC. ALTHOUGH DDX41 REPRESENTS ONE OF >60 DEAD BOX DOMAIN (DDX) PROTEINS, UNIFYING PRINCIPLES ARE NOT ESTABLISHED. AIM 1 WILL INNOVATE A SYSTEM TO DISCRIMINATE PATHOGENIC FROM BENIGN HUMAN DDX41 CLINICAL GENETIC VARIANTS. USING A PRIORITIZATION STRATEGY INVOLVING GENETIC VARIATION ATTRIBUTES, AN ENSEMBLE OF VARIANTS WAS ASSEMBLED FOR ANALYSIS. WE WILL USE OUR DDX41 ACTIVITY METRICS TO CREATE A MATRIX THAT ESTABLISHES THE FUNCTIONAL SIGNATURE OF ANY VARIANT. THIS WILL ENABLE A CLASSIFICATION STRATEGY TO PREDICT WHETHER A VARIANT RESEMBLES DDX41 ("DDX41-LIKE") OR PATHOGENIC VARIANTS ("PATH-DDX41"). ACTIVITY METRICS WILL BE EXTENDED BY QUANTITATIVE PROTEOMICS TO IDENTIFY ADDITIONAL DDX41-REGULATED PROTEINS AND ADVANCED RNA-SEQ ANALYSES TO IDENTIFY TRANSCRIPT ISOFORMS. LOSS-OF-FUNCTION AND RESCUE STUDIES WILL DETERMINE IF ACTIVITY METRICS CAN BE EXTRAPOLATED TO PRIMARY HEMATOPOIETIC STEM/PROGENITOR CELLS. AIM 2 WILL CONDUCT PILOT/EXPLORATORY STUDIES ON A MECHANISM INVOLVING DDX41-DEPENDENT ALTERNATIVE SPLICING AT A LOCUS ENCODING AN RNA SPLICING FACTOR-REGULATORY KINASE. OUR RESULTS REVEALED THAT DDX41, BUT NOT A PATHOGENIC VARIANT, PROMOTES INTRON RETENTION IN CLK3 RNA, AND DDX41 ELEVATES THE CDC-LIKE KINASE-3 (CLK3) PROTEIN LEVEL IN MYELOID CELLS. THE CLK3 KINASE PHOSPHORYLATES SPLICING FACTOR (SR) PROTEINS SRSF1-12, SOME OF WHICH ARE IMPLICATED IN MDS AND AML. WE HYPOTHESIZE THAT DDX41-INDUCED INTRON RETENTION AND ELEVATED CLK3 PROTEIN HAVE IMPORTANT FUNCTIONAL CONSEQUENCES. WE WILL TEST MODELS TO EXPLAIN THE CONSEQUENCES OF CLK3 INTRON RETENTION AND CLK3 PROTEIN ELEVATION. THE STUDIES WILL ESTABLISH A FOUNDATION TO UNDERSTAND THE DDX41-CLK3 MECHANISM, WHICH MAY HAVE CONSIDERABLE PHYSIOLOGICAL AND PATHOLOGICAL IMPACT. THE RULES GOVERNING DDX41 FUNCTION AND DYSFUNCTION THAT EMERGE WILL ADVANCE PATIENT GENETIC CURATION AND MECHANISTIC LOGIC TO INFORM FUTURE LINES OF BIOLOGICAL (E.G., HEMATOLOGY- AND IMMUNOLOGY-FOCUSED) AND PATHOLOGICAL (E.G., BONE MARROW FAILURE/MDS) INVESTIGATIONS IN THE CONTEXTS OF ERYTHROID AND MYELOID BIOLOGY AND MORE BROADLY.