Retinoic acid pathway regulates NECTIN4 expression and enhances NECTIN4-directed chimeric antigen receptor (CAR) T and antibody-drug conjugate therapies in bladder cancer

Urothelial carcinoma (UC) is the sixth most common malignancy in the United States, with 83,000 new cases and 17,000 deaths annually. Prognosis for metastatic UC remains poor, underscoring the need for new targeted therapies. NECTIN4, a cell adhesion molecule highly expressed in UC, is a target of an FDA-approved antibody-drug conjugate (ADC) enfortumab vedotin (EV) with 40% response rates. NECTIN4 genomic amplifications and expression levels correlate with response to NECTIN4-targeted therapies like EV, but regulators of NECTIN4 remain poorly defined. To identify regulators of NECTIN4 surface expression, we performed a CRISPR interference (CRISPRi) FACS-based screen in bladder cancer cell lines, followed by validation across multiple UC models and use of pharmacological modulators of genetic hits to assess effects on NECTIN4 expression and sensitivity to NECTIN4-directed ADCs and CAR T cells. Our screen revealed both positive and negative regulators of NECTIN4 expression, especially factors linked to luminal differentiation and epithelial-to-mesenchymal transition (EMT), which exert opposing effects. We found that activating retinoic acid (RA) signaling, alone or with PPARgamma modulation, transcriptionally induces NECTIN4 as part of the luminal program. Pharmacologic RA activation increased NECTIN4 surface levels and sensitized bladder cancer cells to both EV and NECTIN4-targeted CAR T cells. This RA-driven regulation reinforces luminal identity, acting alongside PPARgamma and counteracting EMT-driven therapeutic escape. Modulating RA signaling offers a promising strategy to overcome both, primary resistance due to low NECTIN4 levels and acquired resistance from NECTIN4 downregulation.