Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer 1 . Elevated levels of the metabolites, 2-hydroxyglutarate (2HG), succinate, and fumarate, occur in human malignancies due to somatic mutations in the isocitrate dehydrogenase-1/2 (IDH1/2) genes or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase (SDH) genes, respectively 2 – 4 . Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR) 5 , 6 and confer an exquisite sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors that is being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we elucidate the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of Tip60 and ATM, two key proximal HDR factors, is significantly impaired at DNA breaks, with reduced end-resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Retinal dopamine is a critical modulator of high acuity, light-adapted vision and photoreceptor coupling in the retina. Dopaminergic amacrine cells (DACs) serve as the sole source of retinal dopamine, and dopamine release in the retina follows a circadian rhythm and is modulated by light exposure. However, the retinal circuits through which light influences the development and function of DACs are still unknown. Intrinsically photosensitive retinal ganglion cells (ipRGCs) have emerged as a prime target for influencing retinal dopamine levels because they costratify with DACs in the inner plexiform layer and signal to them in a retrograde manner. Surprisingly, using genetic mouse models lacking specific phototransduction pathways, we find that while light influences the total number of DACs and retinal dopamine levels, this effect does not require ipRGCs. Instead, we find that the rod pathway is a critical modulator of both DAC number and retinal dopamine levels.

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Cancer associated mutations in citric acid cycle enzymes cause overproduction of 2-hydroxyglutarate (2HG), fumarate, or succinate, commonly referred to as oncometabolites. These oncometabolite producing mutations are prevalent in 70% of gliomas, >76% of hereditary leiomyomatosis and renal cell carcinoma, and >25% of paraganglioma and pheochromocytomas, respectively. These oncometabolite producing cancers harbor vulnerabilities in multiple cellular pathways because oncometabolites competitively inhibit alpha ketoglutarate dependent dioxygenases. This group of enzymes includes the ten eleven translocation (TET) family of enzymes, which catalyze locus specific DNA demethylation. Thus, oncometabolite producing cancers have been shown to exhibit global DNA hypermethylation, which drastically alters gene expression in the cell and is thought to occur via TET inhibition. Cells with isocitrate dehydrogenase 1 (IDH1) mutations, which produce 2HG, have loss of expression of a critical NAD biosynthesis enzyme, nicotinate phosphoribosyltransferase (NAPRT). We show that in addition to IDH1 mutant cells, fumarate hydratase (FH)-deficient isogenic cell lines, which overproduce fumarate, show loss of NAPRT expression, which can be targeted by nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. The goal of our work is to determine if this NAPRT silencing is present in many oncometabolite producing cancers, elucidate the mechanism of silencing, and test therapies to target this vulnerability in NAD biosynthesis. FH-deficient cancer cell lines show reduction of NAPRT mRNA levels by qRT-PCR and absence of protein by western blot, suggesting a reduction in NAPRT transcription. In addition, FH-deficient cell lines exhibit synthetic lethality in short term cell viability assays when treated with NAMPT inhibitor, FK866, which inhibits a parallel NAD biosynthesis pathway. Cell viability is not rescued by nicotinic acid supplementation in FH-deficient lines while parental lines are rescuable, validating the loss of functional NAPRT in only FH-deficient lines. Furthermore, patient samples of FH-deficient renal cell carcinomas show higher NAPRT promoter methylation compared to healthy tissue via methylation array. Ongoing studies are investigating the role of TET1/2/3 enzymes in NAPRT promoter methylation. Together, these results suggest that hypermethylation of the NAPRT promoter is associated with silencing of NAPRT expression in FH-deficient cancers, which can be therapeutically targeted through NAMPT inhibition. Citation Format: Katelyn Noronha, Jiayu Liang, Ranjit Bindra. Oncometabolites confer vulnerabilities in NAD biosynthesis via NAPRT gene silencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2970.