Chromosomal rearrangements of the proto-oncogene ROS1 produce constitutively active ROS1 kinase-fusion
proteins that are established as druggable pathogenic drivers in human cancer. Currently, this is the only
validated mechanism of aberrant ROS1 activation in oncogenesis. ROS1 is targetable with multiple tyrosine
kinase inhibitors (TKI), and significant tumor regression is observed in ROS1-fusion positive patients who are
treated with targeted TKI. Cancer genome sequencing studies reveal numerous ROS1 somatic mutations but
their impact on catalytic function has not been tested, prompting new questions about ROS1-driven cancer
pathogenesis. Further, the mechanistic role of ROS1 carboxy terminus in governing intra- or
intermolecular regulation is unknown. Broadly, these gaps in knowledge, regarding regulation of ROS1
and its structure-function relationships, impede meaningful utilization of accumulating cancer genome data.
Our overall hypothesis is that characterization of ROS1 tyrosine kinase and carboxy-terminal domains through
the lens of somatic mutations will unveil biological underpinnings of receptor regulation, and contribute to
translation of cancer genomic data. We will experimentally address this hypothesis by answering the following
(1) Do cancer-associated ROS1 tyrosine kinase domain (TKD) mutations activate catalytic function, and
is this sufficient for neoplastic transformation or metastatic dissemination, either alone or in cooperation
with hotspot oncoproteins? Preliminary data offer proof of concept that an engineered activating ROS1 TKD
mutation transforms cells, and is targetable with ROS1-TKI. We will test if twenty prioritized somatic ROS1 TKD
mutations enhance catalytic function, induce cellular transformation and tumor formation, and assess their drug
(2) How does the carboxy-terminal domain (CTD) of ROS1 regulate protein stability and TKI-induced
protein downregulation? Our pilot data show that (a) ROS1 is ubiquitinated, (b) catalytically inactive ROS1
undergoes proteasome-assisted degradation, and (c) regulatory motifs within ROS1 CTD are likely involved.
Engineered ROS1 CTD truncations retain catalytic activity but exhibit longer protein half-life under steady state
and TKI-treated conditions. In Aim 2, we will map ubiquitination sites, track the cellular fate of ubiquitinated
ROS1, and assess impact of somatic CTD mutations on ROS1 function.
Cumulatively, I am confident that these studies will provide novel mechanistic insight into ROS1, and potentially
facilitate clinical translation of genomic sequencing data for expanded impact of ROS1-TKI.