Project Summary The American Cancer Society estimates that 238,000 Americans will be diagnosed with lung cancer in 2023 and over 127,000 Americans will die of lung cancer complications. Non-small cell lung cancer (NSCLC) accounts for the vast majority (~82%) of all lung cancer diagnoses and can be caused by a number of distinct mechanisms. One such mechanism is an inversion of part of Chromosome 2p that leads to the fusion of the tyrosine kinase domain of the Anaplastic Lymphoma Kinase (ALK) gene with coiled-coil domain of echinoderm microtubule-associated protein-like 4 gene (EML4), leading to a soluble, dimerized, constitutively active tyrosine kinase. While a series of competitive ALK inhibitors have been approved by the FDA in the last decade, spontaneous suppressor mutations in the ALK active site limit their efficacy, with median duration of response of 11 months. Rather than continue to develop ALK inhibitors, we have developed a series of proteolysis-targeting chimeras (PROTACs) to induce the degradation of the EML4-ALK protein. These heterobifunctional PROTACs are small molecules that contain an EML4-ALK targeting motif, a flexible linker, and a ligand that recruits an E3 ubiquitin ligase. This complex induces the ubiquitination and subsequent proteasome-mediated degradation of the target protein. In cellular assays, we see that our early stage PROTACs induce degradation of transformed, NSCLC-causing EML4-ALK fusions, as well as EML4-ALK with suppressor mutations that block the efficacy of currently approved therapeutics. Importantly, we observe EML4-ALK degradation for concentrations at or lower than those used for current inhibitors. In the initial stages of this proposal, we will perform iterative synthesis and testing of additional PROTAC molecules to increase both their selectivity and effectiveness in in vitro and cellular studies. Once we have obtained the most promising candidates for further development, we will test them for a comprehensive set of pharmaceutical criteria including desirable ADME and safety pharmacology profile, pharmacodynamics, pharmacokinetics, in vivo efficacy, and oral bioavailability. PROTACs that meet such criteria will then be tested in clinically relevant xenograft ALK-positive NSCLC tumor models to evaluate in vivo efficacy and pharmacodynamics, as well as additional ADME and PK studies. This work will develop a first-in class, orally active ALK degrader that can be used for patients that have become resistant to first-line inhibitor treatment. In addition, ALK degraders have the potential to be used as a first-line treatment as well, since they have already shown efficacy in degrading non-resistant forms of EML4-ALK fusions.