Abstract Combined MEK and CDK4/6 inhibition (MEKi + CDK4i) has shown promising clinical outcomes in patients with NRAS-mutant melanoma. Here, we interrogated longitudinal biopsies from a patient who initially responded to MEKi + CDK4i therapy but subsequently developed resistance. Whole-exome sequencing and functional validation identified an acquired PIK3CAE545K mutation as conferring drug resistance. We demonstrate that PIK3CAE545K preexisted in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multiregion sampling due to PIK3CAE545K being nonuniformly distributed. This resistant population rapidly expanded after the initiation of MEKi + CDK4i therapy and persisted in all successive samples even after immune checkpoint therapy and distant metastasis. Functional studies identified activated S6K1 as both a key marker and specific therapeutic vulnerability downstream of PIK3CAE545K-induced resistance. These results demonstrate that difficult-to-detect preexisting resistance mutations may exist more often than previously appreciated and also posit S6K1 as a common downstream therapeutic nexus for the MAPK, CDK4/6, and PI3K pathways. Significance: We report the first characterization of clinical acquired resistance to MEKi + CDK4i, identifying a rare preexisting PIK3CAE545K subpopulation that expands upon therapy and exhibits drug resistance. We suggest that single-region pretreatment biopsy is insufficient to detect rare, spatially segregated drug-resistant subclones. Inhibition of S6K1 is able to resensitize PIK3CAE545K-expressing NRAS-mutant melanoma cells to MEKi + CDK4i. Cancer Discov; 8(5); 556–67. ©2018 AACR. See related commentary by Sullivan, p. 532. See related article by Teh et al., p. 568. This article is highlighted in the In This Issue feature, p. 517