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.