Structural Basis of DNA-Dependent Coactivator Recruitment by the Tuft Cell Master Regulator POU2F3 [DMS_Screen]

The transcription factor POU2F3 defines the identity of tuft cells and underlies a distinct molecular subtype of small cell lung cancer (SCLC). Although POU2F3 is considered undruggable, its activity critically depends on the coactivators OCA-T1 and OCA-T2. Here, we demonstrate that acute suppression of either POU2F3 or OCA-T1 induces regression of tuft cell-like SCLC xenografts in vivo. To explore the structural basis and druggability of this dependency, we solved crystal structures of POU2F3 bound to OCA-T1 or OCA-T2 in complex with DNA, revealing a tripartite, DNA-dependent interface. We further employed deep mutational scanning to systematically assess the functional impact of 4,218 missense variants in POU2F3 and OCA-T1, uncovering both mutation-sensitive hotspots and structurally constrained regions critical for tumor cell fitness. These findings define a unique transcriptional complex that integrates DNA recognition with coactivator recruitment and nominate POU2F3–OCA-T as a structurally tractable vulnerability in tuft cell-like carcinomas. Overall design: Deep Mutational Scanning (DMS) of CRISPR-resistant OCA-T1 and POU2F3 cDNA libraries co-expressing sgRNAs targeting the endogenous OCA-T1(sgOCA-T1) or POU2F3(sgPOU2F3) genes respectively. NCI-H211 Cas9+ cells were transduced with lentiviral DMS libraries at low MOI and cultured for 10 population doublings.