Selpercatinib (LOXO-292) is a highly active selective RET inhibitor explored on an ongoing registrational program (LIBRETTO-001 phase 1/2 trial) in RET-dependent cancers (US FDA approval in 2020 for RET fusion-positive lung/thyroid cancer and RET-mutant thyroid cancer). Unfortunately, resistance is uncharacterized and remains a liability. This proposal anticipates and addresses this unmet need by identifying and functionally characterizing mechanisms of intrinsic and acquired genomic and non-genomic resistance to selective RET inhibition in RET-dependent cancers. To accomplish this, we will leverage unique clinical, computational, and translational resources at our disposal. Aim 1 will identify determinants of intrinsic resistance to RET inhibition in RET-dependent cancers. Pre-treatment biopsies of selpercatinib responders and non-responders will undergo targeted/whole exome sequencing. Computational analysis will explore the role of clonality, allelic imbalance, and co-mutational signatures relative to selpercatinib response and progression-free survival. Aim 2 will establish the mechanisms of acquired resistance to selective RET inhibition. Paired pre-treatment and post-progression tumor biopsies and longitudinal cell-free (cf)DNA (baseline, on-treatment, at/post- progression) from LIBRETTO-001 patients will be profiled. Utilizing paired samples will allow for the identification of emergent genomic and non-genomic (including histologic/EMT transformation) resistance mechanisms. In addition, plasma profiling will allow for a dynamic assessment of selpercatinib resistance that captures the consequences of serial genomic evolution. Aim 3 will functionally characterize resistance to selective RET inhibition. A unique and rich library of patient-derived models of treatment-naïve and RET inhibitor resistant RET-dependent cancers will be augmented by ongoing prospective collection and model development from LIBRETTO-001 and commercial use. In these models, on-target (secondary RET mutations) and off-target (MET/PI3K/KRAS/MDM2 activation) resistance mechanisms will be functionally characterized in terms of cell/tumor viability, receptor tyrosine kinase activation, and downstream signaling dependencies. Novel therapeutic strategies, specifically RET tyrosine kinase inhibitor type switching (on-target resistance) and combinatorial therapies (off-target resistance), will be explored in vitro and in vivo. Optimal combination therapies will then be employed in compassionate use programs to confirm their effectiveness and provide tailored, life-saving treatment to patients. In addition, patients with on-target resistance will be treated on-protocol (Drilon PI) with the next-generation RET inhibitor, TPX-0046. This proposal has both immediate and long-term relevance considering that about 400 patients have been treated with selpercatinib around the world on trial and the potential approval and rapid adoption of this drug by multiple regulatory agencies. Thes...