Therapy Development for Genetic Disorders of the RAS/MAPK Pathway - ABSTRACT RASopathies, pleiomorphic genetic disorders altering RAS/mitogen-activated protein kinase (MAPK) signaling, are treated symptomatically. Recent data suggest that the MAPK pathway inhibitor trametinib can reverse severe heart and lymphatic involvement in some patients; not all patients respond to MEKis, and powerful inhibition of RAS/MAPK signaling has toxic side effects. Thus, development of alternative RASopathy drugs with favorable side effect profiles is an unmet medical need. To achieve that goal, we developed a RASopathy drug development platform using transgenic Drosophila, human induced pluripotent stem cell (iPSC), and mouse models. We identified the RAS binding domain (RBD) mimetic rigosertib as highly efficacious. From chemical screening, we identified a novel compound, M1, with modest activity and then improved its efficacy substantially. The two lead M1-logs are almost certainly not kinase inhibitors. The bona fide targets for rigosertib and the M1-logs in the RASopathies are unknown. Identifying their targets represents an exciting opportunity for advancing therapies for RASopathies and may have relevance for other disorders with altered RAS signaling. In Aim 1, we will take three complementary approaches to identify rigosertib's protein targets in RASopathies. Using Drosophila genetics, we will determine functional targets of rigosertib and assess if perturbation of microtubule biology contributes to rigosertib's RASopathy efficacy. Using targets identified that way, we will look for physical proximity of rigosertib with putative targets in iPSC-derived RASopathy cardiomyocytes (CMs) using bioluminescence resonance energy transfer with nanoluciferase (NanoBRET). Finally, we will determine the impact of knock-down of the best target genes on the CM phenotype using CRISPR interference. In Aim 2, we will assess existing rigosertib analogs (rigo-logs) against our RASopathy fly models. Analogs with improved efficacy will be tested for rescue of hypertrophy in RAF1-mutant CMs. The best rigo-log will be tested head-to-head with rigosertib and trametinib in the Raf1 mouse model, looking at hypertrophic cardiomyopathy regression. In Aim 3, we will take two complementary strategies to identify the targets of our lead M1-logs. First, we will use a photoaffinity strategy, derivatizing M1-logs with a minimalist tag and then using those to tag putative targets. Isolated proteins will be identified using mass spectrometry. Second, we will use un-derivatized M1-logs for a physicochemical approach to target identification called proteome integral solubility alteration (PISA). For both approaches, we will confirm target validity using Drosophila genetic approaches and NanoBRET. The proposed research will provide new insights into the therapeutic mechanisms of rigosertib and M1-logs for the RASopathies. This may include discovery of rigosertib analogs with efficacy exceeding rigosertib itself. As the M1-logs are novel and not kinase inhibitors, their targets are expected to provide new approaches for targeting RASopathies and other disorders with RAS- MAPK gain of function, including many cancers.
