PROJECT SUMMARY: It is now widely appreciated that lung adenocarcinoma (LUAD) is a collection of molecularly distinct diseases. Subsets are defined based on mutations in distinct oncogenes, which confer unique therapeutic vulnerabilities. Advanced LUAD patients who have a targetable oncogenic driver and receive appropriate genotype-directed therapy experience a median survival of 3.5 years, which compares favorably to the 2.5 year survival experienced by those patients who do not receive personalized medicine. More durable benefit of genotype- directed therapies is not achieved due to the inevitable development of acquired drug resistance. This highlights the need for agents that can be readily and safely implemented in combination with genotype- directed therapies to delay or prevent failure and disease progression. Based on our preliminary and published data, we hypothesize that vitamin D3 metabolites have genotype-dependent effects that can be exploited to prolong duration of benefit from standard of care therapies (SOCT). If proven, daily oral vitamin D3 supplementation could be implemented in well-defined subsets of aLUAD patients to simultaneously improve their nutritional status and safely prevent disease progression. To test our hypothesis, we focus on two distinct subsets of LUAD: those with EGFR mutations (develop commonly in never/light smokers and express relatively high levels of the vitamin D receptor (VDR)) and those with KRAS mutations (develop commonly in heavy smokers and express relatively low levels of VDR). In Aim 1, we establish the tumor intrinsic, genotype-dependent effects of vitamin D3 intake on SOCT. Contemporary, patient-derived organoids (PDOs) that represent KRAS or EGFR mutant LUADs are transplanted into vitamin D3-deficient mice, and the effect of subsequently modifying dietary vitamin D3 intake on genotype-directed SOCT is determined. In Aim 2, we use an existing set of patient biospecimens and data to determine the impact of 25D3 status on durability of SOCT in patients with advanced KRAS mutant LUAD. Results are compared with those recently obtained in patients diagnosed with EGFR mutant aLUAD: individuals who were classified as 25D3 sufficient (≥30 ng/mL) experienced a significantly longer benefit from SOCT (mean 506 days) than those who were 25D3 insufficient (<30 ng/mL; mean 292 days, p=0.026). Positive results will provide strong scientific rationale for the implementation of a genotype-directed clinical trial of vitamin D3 supplementation to prolong benefit of SOCT and prevent disease progression among individuals diagnosed with EGFR mutant LUAD. The potential of vitamin D3 metabolites in advanced LUAD patients has been assessed previously with negative results. However, past studies were conducted prior to widespread genomic testing and included few never-smokers (where EGFR mutations predominate). Consequently, vitamin D3 effects within the EGFR mutant subset of aLUADs (that we predict are most responsive...