Project Grant R44CA268146

SIMULTANEOUS PHARMACOLOGICAL PROFILING OF ONCOGENIC GENE FUSION PROTEINS IN CANCER - RESEARCH SUMMARY THE HUMAN GENOME ENCODES MORE THAN 1,600 TRANSCRIPTION FACTORS (TFS), ALONG WITH ADDITIONAL COFACTORS, CHROMATIN REGULATORS, AND STRUCTURAL PROTEINS THAT COLLECTIVELY EXECUTE THE REGULATORY INSTRUCTIONS ENCODED WITHIN THE NUCLEAR DNA. DYSFUNCTIONS OF THESE PROTEINS, COLLECTIVELY KNOWN AS GENE REGULATORY PROTEINS (GRPS), ARE KNOWN TO DRIVE MULTIPLE DISEASES SUCH AS CANCER, INFLAMMATION-RELATED, AND NEUROLOGICAL CONDITIONS. IN CANCER, THESE PROTEINS ARE FREQUENTLY REARRANGED AND FUSED TO CREATE NEW PROTEINS WHICH CAUSE THE INITIATION AND PROGRESSION OF VARIOUS TYPES OF LEUKEMIA, SARCOMA AND OTHER TUMORS. DESPITE THE IMPORTANCE OF THESE PROTEINS, GRPS HAVE BEEN CONSIDERED UNDRUGGABLE DUE TO CHALLENGES IN MODELING THEIR ACTIVITY IN VITRO. WE HAVE SOLVED THESE SHORTCOMINGS BY IMPLEMENTING AN IN-CELL FUNCTIONAL PROTEOMICS DRUG DISCOVERY PLATFORM THAT QUANTIFIES THE EFFECTS OF SMALL-MOLECULES ON THE ABUNDANCE OF GRPS BOUND TO THE GENOME IN A DIVERSITY OF CELL AND TISSUE TYPES. THE PLATFORM IS BASED ON CHROMATIN EXTRACTION BY SALT SEPARATION, COUPLED TO DATA INDEPENDENT ANALYSIS MASS SPECTROMETRY (CHESS-DIA), WHICH WAS RECENTLY REPORTED. IN THIS PROPOSAL, WE WILL APPLY THIS TECHNOLOGY FOR DRUG DEVELOPMENT OF ONCOGENIC FUSION PROTEINS. FIRST, WE WILL USE MIXED LINEAGE LEUKEMIA (MLL) REARRANGED LEUKEMIA TO PERFORM TECHNOLOGY DEVELOPMENT OF THE ONCOGENIC FUSION PROTEIN PROTEOMICS STRATEGY. MLL REARRANGEMENTS ARE FOUND IN A SUBSET OF AML AND ALL PATIENTS, COMMONLY IN CHILDREN, AND REMAIN CHALLENGING TO TREAT WITH EXISTING THERAPEUTIC OPTIONS. SEVERAL DRUG CANDIDATES FOR MLL-REARRANGED LEUKEMIA ARE CURRENTLY IN CLINICAL TRIALS, AND THESE WILL BE USED TO VALIDATE THE ACCURACY OF THE CHESS-DIA ASSAY FOR REPORTING THE ABILITY OF MLL-TARGETING COMPOUNDS TO DISRUPT THE MLL COMPLEX IN LIVE CELLS. WITH A VALIDATED MLL CHESS-DIA ASSAY, WE WILL THEN CONDUCT A PILOT SCREEN TO PROVE THE ASSAY'S UTILITY IN A SCREENING SETTING, USING THE NATIONAL CANCER INSTITUTE'S MECHANISTIC DIVERSITY COMPOUND SET SUPPLEMENTED WITH KNOWN INHIBITORS OF THE MLL COMPLEX. THESE COMPOUNDS CONTAIN A DIVERSE ARRAY OF BIOACTIVITIES, MANY OF WHICH ACT THROUGH UNKNOWN MECHANISMS. THIS PROVIDES AN OPPORTUNITY TO FIND NEW COMPOUNDS CAPABLE OF DISRUPTING THE MLL COMPLEX. LASTLY, WE WILL USE THE ROADMAP DEVELOPED FOR MLL TO DEVELOP CHESS-DIA ASSAYS FOR MANY OF THE COMMON ONCOGENIC FUSIONS, THEN DEVELOP A METHOD TO UNIFY THESE ASSAYS TOGETHER IN A SINGLE, UNIFIED, IN-CELL ASSAY FOR ONCOGENIC FUSION PROTEINS.