Project Grant R21ES035196

NOVEL ROLE OF MELANIN-CARBONYLS IN PROGRESSION OF NRAS MUTANT MELANOMA - WE DISCOVERED MELANIN-CHEMIEXCITATION (MECH) WHERE NITRIC OXIDE SYNTHASE (NOS) OXIDIZES MELANIN INTO MELANIN-CARBONYLS (MCS) THAT HAVE TRIPLET ENERGY EQUIVALENT TO A UV PHOTON. THE MCS GENERATE ~50% OF THE TOTAL CYCLOBUTANE PYRIMIDINE DIMERS (CPDS), UV SPECIFIC DNA ADDUCTS, IN COMPLETE ABSENCE OF UV. THE CHEMICAL EVENTS THAT CONSPIRE DURING AND AFTER MECH, AND SPECIFICALLY THE ROLE OF MCS REMAIN UNKNOWN IN MELANOMA. WE PROPOSE TO IDENTIFY AND CHARACTERIZE THE ROLE OF MELANIN-CARBONYL-DNA (MCD) AND MELANIN- CARBONYL-PROTEIN (MCP) ADDUCTS IN MELANOMA PROGRESSION AND RESISTANCE AGAINST CURRENT THERAPEUTIC APPROACHES. THE RATIONALE IS THAT NOS IS MOSTLY HYPERACTIVE, AND ASSOCIATED WITH POOR PROGNOSIS OF MELANOMA PATIENTS, AND THE ROLE OF PIGMENTATION IS STILL CONTROVERSIAL IN MELANOMA. WE HYPOTHESIZE THAT HIGH NOS INDUCES MECH, LEADING TO CHRONIC MC PRODUCTION WHICH DYSREGULATES MELANOCYTIC PHYSIOLOGY, AND PROMOTES MELANOMA PROGRESSION AND THERAPY RESISTANCE THROUGH "MCD AND MCP ADDUCTS" OR OTHER UNKNOWN MECHANISMS. THE HYPOTHESIS RELATES TO THREE UNIQUE ARMS IN MELANOMA. FIRST IS PIGMENTATION. MELANIN PROMOTES MELANOMA BY ACTIVATING HIF-1A. CONTRARILY, MELANIN'S PHYSICAL EXISTENCE INHIBITS METASTASIS THROUGH ELASTICITY MODULATIONS, BUT REMAINS TO BE TESTED STRINGENTLY. SECOND IS MECH WHICH IS PROPOSED TO BE A CENTRAL AXIS IN MELANOMA BIOLOGY THOUGH NEVER TESTED EXPERIMENTALLY. THIRD IS THE NOS ENZYME, HYPERACTIVE IN MELANOMA, CORRELATES STRONGLY WITH POOR PROGNOSIS, AND EXCLUSIVELY ASSOCIATED WITH MELANIN. INTEGRATING THESE THREE ARMS WITH THE MECHANISMS OPERATED BY MCD AND MCP ADDUCTS, WE WILL IDENTIFY A POSITIVE LOOP IN MELANOMA THAT IS STEERED BY NOS, MECH, AND MCS IN THE FOLLOWING TWO AIMS: IN AIM 1, WE HYPOTHESIZE THAT OWING TO HYPERACTIVE NOS, THE CHRONIC MC PRODUCTION LEADS MCD AND MCP ADDUCTS. LABELING THE CELLULAR MELANIN USING CLICK-CHEMISTRY- ANALOGS (CCA) OF MELANIN-MONOMERS (PROTOCOLS ESTABLISHED), WE WILL INDUCE MECH CHEMICALLY OR WITH UV AND PURIFY AND CHARACTERIZE CCA LABELED GENOMIC AND PROTEOMIC FRACTION USING HPLC-MS/MS. FOCUS WILL BE ON CPD HOTSPOTS IN THE GENOME AND LYSINE AND CYSTEINE RICH PROTEINS FROM PROLIFERATIVE MELANOMA SIGNALING SINCE -NH2 AND -SH GROUPS ARE PRIME TARGETS FOR CARBONYL ADDUCTION IN AIM 2, WE HYPOTHESIZE THAT MCD AND MCP ADDUCTS PROMOTE MELANOMA PROGRESSION AND THERAPY RESISTANCE. WE SENSITIZED PATIENT DERIVED MELANOMA CELLS TO TARGETED THERAPIES BY SCAVENGING CARBONYLS AND INHIBITING NOS. ANALYSES ACROSS MELANOCYTES, DYSPLASTIC NEVI, AND MELANOMA, AND THE SELF-ESTABLISHED PIGMENTED AND NON-PIGMENTED, PATIENT DERIVED MELANOMA CELLS WILL IDENTIFY PROBABLE MECHANISMS BEHIND THIS SENSITIZATION WHICH IN OUR PRELIMINARY OBSERVATIONS, WAS INDEPENDENT OF THE DRIVER MUTATIONS LIKE BRAF OR NRAS. AN INTEGRATED ANALYSIS OF AIM 1 AND 2 WILL IDENTIFY MC-MEDIATED, NON-CLASSICAL REGULATION OF MELANOMA PROGRESSION AND THERAPY RESISTANCE THAT COULD BE USED AS A NOVEL VULNERABILITY AGAINST MELANOMA.