DHODH inhibition suppresses cutaneous squamous cell carcinoma growth by the induction of differentiation through perturbation of the cellular redox balance

Dihydroorotate dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis, has recently emerged as a therapeutic target in various cancers. We have previously identified a pivotal role of DHODH in the initiation of cutaneous squamous cell carcinoma (cSCC), the second most common type of non-melanoma skin cancer. We also showed that pharmacological inhibition of this enzyme suppresses ultraviolet (UV)-induced tumor formation. However, the key mechanisms driving the anticancer activity of DHODH inhibition remain unexplored in cSCC. We investigated the biological consequences of pharmacological and genetic DHODH inhibition in cSCC using xenograft models derived from two human cell lines, A431 and SCC13, implanted in immunodeficient NSG mice. DHODH activity was suppressed pharmacologically with leflunomide (LFN) and the potent DHODH inhibitor PTC299, or genetically via lentiviral shRNA-mediated DHODH silencing (shDHODH). Proteomic and metabolomic analyses were integrated with histopathological, immunohistochemical, and immunoblotting evaluations to delineate the downstream effects of DHODH blockade. Comprehensive proteomic and metabolomic profiling revealed that DHODH inhibition induces a coordinated adaptive program involving keratinization, differentiation, redox homeostasis, and metabolic stress responses. Histological and immunostaining analyses demonstrated marked reductions in Ki67-positive proliferating cells and a corresponding increase in pan-cytokeratin (PanCK) and keratin 10 (Krt10) expression, indicative of enhanced epithelial differentiation. These changes were most pronounced in PTC299-treated and shDHODH xenografts, whereas LFN displayed minimal or no efficacy in SCC13 tumors. DHODH inhibition drives tumor differentiation and suppresses proliferation in cSCC, highlighting metabolic dependency as a potential therapeutic vulnerability. PTC299 exhibited superior antitumor activity and differentiation-inducing capacity compared with LFN. These findings position DHODH as a promising target for bioenergetic vulnerability-based cancer therapy in advanced or treatment-resistant cSCC.