A genome-wide CRISPR-Cas9 knockout screen reveals FSP1 as warfarin-resistant vitamin K reductase

Abstract Vitamin K is a vital micronutrient implicated in a variety of human diseases1. Warfarin, a vitamin K antagonist, is the most commonly prescribed oral anticoagulant2. Warfarin exerts its effect by inhibiting vitamin K epoxide reductase (VKOR) leading to the depletion of reduced vitamin K, a cofactor of gamma-glutamyl carboxylase (GGCX) required for the post-translational modification of proteins with diverse functions. Patients overdosed on warfarin can be rescued by administering high doses of vitamin K because of the existence of a warfarin-resistant vitamin K reductase (VKR)3,4. Despite the functional discovery of VKR over eight decades ago, the identity of VKR remained elusive. Here, we report the identification of warfarin-resistant VKR using a genome-wide CRISPR-Cas9 knockout screening with a novel vitamin K-dependent (VKD) apoptotic reporter cell line. We found that ferroptosis suppressor protein 1 (FSP1) is responsible for vitamin K reduction in a warfarin-resistant manner. Knocking out FSP1 in HEK293 cells dramatically decreased VKR activity, while HEK293 cells overexpressing FSP1 exhibited robust VKR activity that is resistant to warfarin inhibition. Inhibitor of FSP1 that inhibited ubiquinone reduction and thus triggering cancer cell ferroptosis, displayed strong inhibition of VKD carboxylation. Intriguingly, dihydroorotate dehydrogenase (DHODH), another ubiquinone-associated ferroptosis suppressor protein parallel to the function of FSP15, does not support VKD carboxylation and its inhibitors have no effect on carboxylation. These findings provide new insights into selectively controlling the physiological and pathological processes involving electron transfers mediated by vitamin K and ubiquinone.