Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

We studied lysosomal Ca²⁺ in inflammasome. Lipopolysaccharide (LPS) + palmitic acid (PA) decreased lysosomal Ca²⁺ ([Ca²⁺]_Lys) and increased [Ca²⁺]_i through mitochondrial ROS, which was suppressed in Trpm2-KO macrophages. Inflammasome activation and metabolic inflammation in adipose tissue of high-fat diet (HFD)-fed mice were ameliorated by Trpm2 KO. ER→lysosome Ca²⁺ refilling occurred after lysosomal Ca²⁺ release whose blockade attenuated LPS + PA-induced inflammasome. Subsequently, store-operated Ca²⁺entry (SOCE) was activated whose inhibition suppressed inflammasome. SOCE was coupled with K⁺ efflux whose inhibition reduced ER Ca²⁺ content ([Ca²⁺]_ER) and impaired [Ca²⁺]_Lys recovery. LPS + PA activated KCa3.1 channel, a Ca²⁺-activated K⁺ channel. Inhibitors of KCa3.1 channel or Kcnn4 KO reduced [Ca²⁺]_ER, attenuated increase of [Ca²⁺]_i or inflammasome activation by LPS + PA, and ameliorated HFD-induced inflammasome or metabolic inflammation. Lysosomal Ca²⁺ release induced delayed JNK and ASC phosphorylation through CAMKII-ASK1. These results suggest a novel role of lysosomal Ca²⁺ release sustained by ER→lysosome Ca²⁺ refilling and K⁺ efflux through KCa3.1 channel in inflammasome activation and metabolic inflammation.