NAPRT-mediated deamidated NAD biosynthesis enhances colon tissue resiliency and suppresses tumorigenesis

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: major_revision

Abstract
Nicotinamide adenine dinucleotide (NAD) is synthesized through both amidated salvage and deamidated pathways. Although NAD-producing enzymes are often overexpressed in cancer cells to meet the high metabolic demands of rapid proliferation and are considered oncogenic, we report that physiological levels of nicotinic acid phosphoribosyl transferase (NAPRT), the first enzyme in the Preiss-Handler arm of the deamidated pathways, suppress tumorigenesis. We show that NAPRT is enriched in gut epithelial cells, where it sustains the NAD pool for an efficient response to stress-induced acute NAD depletion. Consequently, NAPRT deficiency impairs the activity of poly-(ADP-ribose) polymerases and DNA repair, sensitizes mice to chemical-induced colitis and tumorigenesis as well as to age-associate spontaneous tumor development. Moreover, low NAPRT expression correlates with poor prognosis in several human cancer types. Thus, homeostatic levels of deamidated NAD biosynthesis contributes to tumor suppression, and boosting this pathway may offer a strategy for cancer prevention.

Introduction
Nicotinamide adenine dinucleotide (NAD), a cofactor for a variety of electron-exchange-dependent biochemical redox reactions, is central in energy metabolism and a molecule essential for life 1. It also serves as an indispensable co-substrate for the sirtuin protein deacylases, the mono and poly-(ADP-ribose) polymerases (PARPs) involved in DNA repair, and cellular surface cyclic ADP ribose hydrolase CD38, and thereby NAD is important in regulation of genome stability, immune responses, circadian rhythms, and aging 2. Mammalian cells have multiple synthetic pathways for NAD, including the de novo pathway from tryptophan, the Preiss-Handler pathway (i.e., deamidated salvage pathway) from nicotinic acid (NA), and the amidated salvage pathway from nicotinamide (NAM), as well as additional pathways using precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) 3, 4 (Supplementary Fig. 1a). Among these pathways, the amidated salvage whereby NAM is converted to NAD by nicotinamide phosphoribosyl transferase (NAMPT), is considered the primary source of NAD in most mammalian cells and tissues 5, 6.

Cellular NAD homeostasis is tightly maintained by its consumption and biosynthesis. Disruption of this balance is associated with various pathological conditions. For instance, reduced cellular NAD levels are observed in various diseases and aging 7. Consequently, different therapeutic strategies aimed at boosting NAD levels are being actively investigated, including supplementation with NAD precursors such as NAM, NR and NMN, activation of NAMPT, or by inhibition of CD38 8. On the other hand, actively proliferating cancer cells and active immune cells require high NAD flux, and NAMPT has been shown to be upregulated in many human cancers as well as various inflammatory conditions, such as rheumatoid arthritis and inflammatory bowel disease (IBD) 9-14. Nicotinic acid phosphoribosyl transferase (NAPRT), the first enzyme in the deamidated salvage pathway, is also reported to have a high frequency of amplification in tumors derived from tissues that normally have high expression of NAPRT 9. Therefore, inhibitors of the amidated or the deamidated NAD biosynthesis pathways are under development for potential treatment of cancer 9, 10, 15-18 and inflammation 12, 14.

Interestingly, although the amidated NAD salvage pathway is considered the main pathway of NAD production in mammals 19, recent studies uncovered an important role of the deamidated NAD biosynthesis pathway in mediating the host-microbe NAD metabolic interaction 20. Particularly, following oral administration of amidated precursors such as NAM or NR, gut microbiota can convert these dietary amidated NAD precursors to NA and enable the deamidated NAD biosynthesis 21. In several organs, including the liver, colon, and kidney, this microbiota-dependent deamidated biosynthesis accounts for the majority of newly synthesized NAD 21. Moreover, in the absence of dietary consumption of NAD precursors, gut microbiome can convert host-derived NAM in the gut lumen into NA, maintaining host NAD biosynthesis through the deamidated pathway 22. However, in contrast to the well-studied NAMPT-mediated amidated NAD salvage pathway, the (patho)physiological importance of this microbiota-enabled highly efficient NAD biosynthesis pathway is largely understudied, and the functional difference between these two NAD biosynthesis pathways remains unclear.

In the present study, we investigate the functional impacts of the deamidated NAD biosynthesis pathway on animal (patho)physiology and stress responses and identify an unexpected suppressive role of this pathway in both cancer and inflammation.

Results
NAPRT is enriched and functionally important in gut epithelial cells
To explore the potential function of deamidated NAD biosynthesis, we decided to focus on the gut, the direct interface for host-microbe metabolic interaction. Single cell RNA-seq (scRNA-seq) analysis of genes encoding key enzymes of the three major NAD biosynthesis pathways, including Nampt, Naprt, and quinolinate phosphoribosyl transferase (Qprt), the rate-limiting enzyme in the de novo synthesis pathway, in normal mouse gut revealed that both small intestine and colon have very low expression of Qprt (Fig. 1a). As a comparison, all three enzymes were highly expressed in the liver, particularly in hepatocytes (Supplementary Fig. 1b, using a publicly available mouse hepatic scRNA-seq dataset 23). Among two highly expressed NAD biosynthesis enzymes in the gut, Nampt was widely expressed in most cells (including immune cells), whereas Naprt was highly expressed in gut epithelial cells, particularly in enterocytes in the small intestine and colonocytes in the colon (Fig. 1a). The enrichment of NAPRT in the mouse colonocytes was confirmed by immunofluorescence staining using two independent anti-NAPRT antibodies (Fig. 1b). Similar expression patterns of these enzymes were also observed in human colon based on the scRNA-seq datasets from The Human Protein Atlas (www.proteinatlas.org) (Supplementary Fig. 1c). Enterocytes and colonocytes are the most abundant intestinal epithelial cells responsible for nutrient absorption and transport 24. They are also in direct contact with gut microbiota in the gut lumen and immune cells inside the gut lamina propria. The enrichment of NAPRT in these gut epithelial cells strongly suggests that NAPRT-mediated deamidated NAD biosynthesis is critical in mediating diet-microbe-host interaction that influences gut physiology and pathology.

To test the direct functional impacts of NAPRT and deamidated NAD biosynthesis in gut epithelial cells, we knocked out NAPRT in a human colon cancer cell line CRC119 25 using CRISPR/Cas9-mediated gene editing (Supplementary Fig. 1d). NAPRT KO CRC119 cells displayed normal morphology and proliferation rates when cultured in a regular growth medium containing 8 M NAM but no NA (Supplementary Fig. 1e and 1f). Supplementation of 100 M NA, a concentration comparable to the serum NA range after oral supplementation with NAD boosting precursors 22, markedly induced deamidated NAD intermediates NAMN and NAAD and significantly increased total NAD abundance in WT but not in NAPRT KO CRC119 cells (Fig. 1c and 1d). Further analysis of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) revealed that the defective deamidated NAD biosynthesis observed in NAPRT KO CRC119 cells cultured in NA-containing medium impaired their glycolysis but not oxidative phosphorylation (OXPHS) (Fig. 1e), suggesting that NAPRT-mediated deamidated NAD biosynthesis contributes to cytosolic NAD pool. However, this reduced glycolysis in NAPRT KO CRC119 cells cultured in the NA-containing medium was not associated with any reduction in ATP production or increase in cell death unless NAMPT was inhibited by a specific inhibitor STF-118804 (Fig. 1f and 1g, STF+NA). Therefore, NAPRT deficiency impairs cellular metabolism and increases the sensitivity to NAMPT inhibition-induced NAD depletion and subsequent cell death.

NAPRT KO cells accumulate DNA damage
DNA damage is a common environmental stress that induces NAD consumption. Upon the induction of DNA damage, the PARP DNA repair enzymes consume large amounts of NAD as a substrate to synthesize poly-(ADP–ribose) (PAR) and mono-ADP-ribose (MAR) at or near DNA damage lesions to initiate DNA repair 26. DNA damaging reagents, therefore, are reported to induce nuclear and cellular NAD depletion 27-31. Indeed, treatment with methyl methanesulfonate (MMS), a DNA alkylating agent that adds methyl groups to DNA preferentially at the N7 position of guanine 32, induced a quick reduction of total cellular NAD in WT CRC119 cells cultured in medium supplemented with NA (Fig. 2a, WT, NAD). This acute reduction of NAD was coupled with a transient increase followed by reduction of intracellular NAMN but not NMN (Fig. 2a, WT), suggesting that deamidated NAD biosynthesis, particularly NAPRT, was activated in response to MMS treatment. The level of NAAD followed the same trend as NAD (Fig. 2a, WT). Consistent with the observation in Fig. 1c and 1d, NAPRT KO CRC119 cells cultured in the same NA-containing medium had undetectable levels of the deamidated NAD precursors NAMN and NAAD, and a significantly reduced NAD content compared to WT cells before treatment (Fig. 2a, 0 minute). MMS treatment in these cells also induced an acute reduction of NAD. However, the reduction was slower than that in WT cells (Fig. 2a, KO, NAD). Moreover, WT cells cultured in medium without NA supplementation, thereby possessing only amidated NAD synthesis, had a slow NAD depletion rate comparable to that in NAPRT KO cells (Supplementary Fig. 2a, bottom).

In agreement with their reduced NAD consumption upon DNA damage in NA containing medium (Fig. 2a and Supplementary Fig. 2a), NAPRT KO cells had reduced induction of protein PARylation, including histone PARylation, after MMS treatment and recovery (Supplementary Fig. 2b and 2c). Moreover, when cultured in a medium containing 20 M NA, a dose resulting in the maximal increase of NAD in CRC119 cells (Supplementary Fig. 2d), MMS-induced auto- Mono-ADP-ribosylation (MARylation) of PARP1 (upper band) and MARylation of histones (lower band) were reduced in NAPRT KO cells (Fig. 2b). Histone PARylation and MARylation are important for efficient DNA repair 33, 34. Consistently, the reduction of protein PARylation and MARylation in NAPRT KO cells after MMS treatment was associated with increased DNA damage as assessed by H2AX immunoblotting (Fig. 2b, H2AX) and immunofluorescence staining (Fig. 2c and Supplementary Fig. 2e). Importantly, transient re-expression of NAPRT in NAPRT KO cells was able to significantly suppress H 2AX levels upon MMS treatment (Fig. 2d and Supplementary Fig. 2f). Furthermore, when cultured in a medium containing 1 M NA, which is equivalent to the plasma concentration of NA in mice on a regular chow diet 22, NAPRT KO CRC119 cells had increased H2AX levels and decreased histone MARylation following MMS treatment compared to WT cells (Fig. 2e, KO+MMS vs WT+MMS). Stably adding-back HA-tagged mouse NAPRT (HA-mNAPRT) restored MARylation and reduced H2AX levels (Fig. 2e). Together, our observations indicate that NAPRT deficiency in cells reduces the activation of PARP upon DNA damage, impairing DNA repair and increasing the sensitivity to DNA damage.

As both deamidated and amidated NAD biosynthesis were active in CRC119 cells (Fig. 1), we sought to test the relative importance of these two pathways for cell viability following DNA damage using two different experimental strategies. In the first strategy, we used STF treatment (100 nM STF for 24 hours) in WT cells to inhibit the amidated pathway (WT STF), and NAPRT KO to inactivate the deamidated pathway, and STF treatment in NAPRT KO cells (KO STF) to inactivate both amidated and deamidated pathways. We then challenged these cells with MMS for 1 hour to induce transient DNA damage. Cells were subsequently washed, and long-term cell viability was assessed using colony formation assay. Inhibition of amidated NAD biosynthesis sensitized WT cells to MMS-induced cell death (Fig. 2f, WT STF+MMS vs WT MMS), whereas inactivation of the deamidated synthesis failed to do so (Fig. 2f, KO MMS vs WT MMS). Inactivation of both pathways completely suppressed cell survival (Fig. 2f, KO STF+MMS vs WT MMS). In the second strategy, we generate NAMPT KO CRC119 cells (Supplementary Fig. 2g), and cultured WT, NAPRT KO, and NAMPT KO cells in a medium containing 20 M NA and 8 M NAM. We then treated them with or without MMS for 4 hours or 72 hours (Fig. 2g). Despite a significant NAD reduction (Fig. 2g, NA; NAD (4 hours)), either the amidated or the deamidated pathways were sufficient to maintain cellular ATP levels in untreated cells cultured in this full medium (NA; ATP (4 hours)). Treatment with MMS for 4 hours depleted cellular NAD levels by 90% in all cells and markedly reduced cellular ATP levels. Yet compared to WT cells, NAPRT KO cells displayed comparable cellular ATP levels whereas NAMPT KO cells had significantly reduced cellular ATP (NA+MMS; ATP (4 hours)). Similar results were observed after cells were treated with MMS for 72 hours (NA+MMS; ATP (72 hours)). These observations are consistent with our colony formation assay in Fig. 2f, suggesting that NAPRT KO CRC119 cells have a comparable survival ability as the WT cells in response to the genotoxic stress. Therefore, in contrast to NAMPT-mediated amidated NAD salvage, the deamidated pathway is not essential for cell survival in response to genotoxic stress. Taken together, our results indicate that NAPRT-mediated deamidated NAD biosynthesis significantly contributes to DNA repair upon DNA damage. However, despite the accumulation of more severe DNA damage compared to WT cells, a comparable fraction of NAPRT-deficient cells survives the stress.

NAPRT is important for NAD biosynthesis and stress response in mice
To further investigate the functional importance of deamidated NAD biosynthesis in vivo, particularly in the gut, we generated a mutant mouse model in which a 21-bp fragment encoding 7 amino acids in the catalytic domain of NAPRT was deleted in all cells using the CRISPR/Cas9-mediated gene editing technology (Supplementary Fig. 3a). This deletion did not affect total Naprt mRNA levels in multiple tested tissues (Supplementary Fig. 3b) but led to slightly increased electrophoretic mobility and reduced intensity of NAPRT protein bands, particularly in the liver (Supplementary Fig. 3c). These observations suggest that the 7-amino acid deletion in this mutant line destabilizes the NAPRT protein. In mouse embryonic fibroblasts (MEFs) and primary macrophages differentiated from bone marrow of this mouse line, cellular NAD biosynthesis stimulated by NA in the presence of NAMPT inhibitor STF-118804 was completely blocked (Supplementary Fig. 3d), indicating that this mutant line is a true NAPRT null (KO) allele.

NAPRT KO mice were born normally and did not display clear phenotypes in unstressed conditions at a young age, which contrasts with the embryonic lethality of mice lacking NAMPT 35, indicating that deamidated NAD biosynthesis is not essential for development or normal functions of the experimental mice. The steady-state tissue NAD levels in several tested organs of NAPRT KO mice were normal compared to WT control mice (Supplementary Fig. 4a). However, stable isotopic tracing experiments following oral gavage of deuterium-labeled NAM (D4-NAM) (Supplementary Fig. 4b) demonstrated that deletion of NAPRT leads to a dramatic depletion of the deamidated intermediates NAMN and NAAD, along with a decrease in the levels of newly synthesized NAD and nicotinamide adenine dinucleotide phosphate (NADP) in the colon and liver 3 hours after dosing (Fig. 3a and Supplementary Fig. 4c, M+4/M+3/M+2; the M+3 labeling is the result of rapid turnover of the redox-active hydrogen; the M+2 labeling is the result of spontaneous hydrogen-deuterium exchange combined with redox turnover 36). As we and others previously elucidated, the induction of the deamidated NAD precursors NAMN and NAAD in WT mice following supplementation with NAM is a result of gut microbiota-mediated deamidation of NAM to NA 21, 22. Systematic investigation of additional tissues revealed that at the 3-hour time point, NAPRT-mediated deamidated NAD biosynthesis occurs in almost all tested tissues (Fig. 3b, as indicated by production of NAMN and NAAD) and significantly contributes to the levels of newly synthesized NAD in the small intestine and pancreas in addition to the colon and liver (Fig. 3b and Supplementary Fig. 4d, NAD).

Further kinetic analysis of tissue concentrations of NAD metabolites following oral gavage of D4-NAM confirmed that NAPRT-mediated deamidated biosynthesis is a major route of NAD biosynthesis in the liver, colon, kidney and pancreas and significantly contributes to NAD biosynthesis in the small intestine, heart and lung (Fig. 3c). The massive accumulation of NA in all tested NAPRT KO mouse tissues further indicates that NAD synthesis using NA as a substrate is active in all these tissues in WT mice. Moreover, in WT mice, robust NAAD and NAD biosynthesis occurred without significant accumulation of NA, even at the earliest time point examined (except for the colon) (Fig. 3c, NA), indicating that NAPRT-mediated NA assimilation is swift and highly efficient. Colon in WT mice had an extremely high level of NA due to its direct contact with gut microbiota, many of which bear a nicotinamidase (PncA) activity that converts NAM to NA as we previously reported 21. Interestingly, in NAPRT KO mice, the accumulation of NA in all tested tissues was transient and peaked at or prior to 3 hours (Fig. 3c, NA), possibly due to the catabolism of NA into nicotinuric acid (NUA) in the kidney of these mice (Supplementary Fig. 4e, KO). Of note, NUA was undetectable in WT mice (Supplementary Fig. 4e, WT), suggesting that this catabolite may serve as a potential urinary biomarker for defective deamidated NAD biosynthesis. Recent studies have shown that NA can also be converted to NAR in an NAPRT-dependent manner 37. NAR then acts as the primary circulating deamidated NAD precursor for certain tissues, such as the kidney 37, 38. Further isotope tracing experiments using D4-labeled NA revealed that in WT mice, oral gavage of D4-NA at 80 mg/Kg elevated plasma NA levels from below the detection limit of our platform (1-2 M) to ~300 M, along with a significant increase in plasma NAR levels (from 0.028 M to 0.202 M) (Supplementary Fig. 4f, WT). In NAPRT KO mice, the rise of NAR was almost completely abolished despite a higher increase of circulating NA (Supplementary Fig. 4f, KO), confirming the NAPRT-dependent conversion of NA to NAR. Additionally, D4-NA gavage elevated plasma levels of both basal (M+0) and labeled (M+3) NAM, peaking between 1-3 hours in WT but not in KO mice (Supplementary Fig. 4f, NAM), suggesting increased NAD consumption following NA-induced NAD boosting in WT mice. Similar results were observed with a higher dose of D4-NA (400 mg/Kg) (Supplementary Fig. 4f, right panels). All together, these results from NAPRT KO mice indicate the key role of the microbiota- and NAPRT-dependent deamidated pathway in mediating the NAD-boosting efficacy of various NAD precursors.

To confirm our observations in CRC119 cells that NAPRT deficiency increases the sensitivity of cells to NAD-depleting stresses, we cultured colonic and small intestinal organoids from WT and NAPRT KO mice then treated them with an NAMPT inhibitor STF-118804 and/or DNA damaging reagent MMS. Consistent with our findings in CRC119 cell line, primary NAPRT KO colonic and small intestinal organoids were more sensitive to STF-induced ATP depletion and death in the presence of NA compared to WT organoids (Fig. 3d and 3e, Supplementary Fig. 4g and 4h). Moreover, we observed that although NAPRT KO colonic organoids cultured in NA-containing medium were sensitive to STF treatment (Fig. 3e, KO SITF vs WT STF), they were not more sensitive to MMS induced death than WT colonic organoids (Fig. 3f, KO MMS vs WT MMS). Instead, STF-treated WT colon organoids had reduced survival compared to WT colonic organoids after MMS treatment (Fig. 3f, WT MMS+STF vs WT MMS). These observations support our conclusion from the CRC119 cells that deamidated pathway, although important for tissue NAD biosynthesis, is not essential for gut epithelial cell survival after genotoxic stress.

NAPRT deficiency sensitizes mice to intestinal DNA damage and inflammation
We next directly assessed the importance of NAPRT-mediated deamidated NAD biosynthesis in DNA damage response of gut epithelium in vivo using WT and NAPRT KO mice. In unstressed conditions, NAPRT KO mice had normal gut morphology and normal expression of intestinal cell marker genes compared to WT mice (Supplementary Fig. 5a and 5b). We treated WT and NAPRT KO mice with azoxymethane (AOM), a chemical procarcinogen commonly used in experimental models of colon carcinogenesis. When AOM is intraperitoneally injected into rodents, DNA alkylation induced by its metabolites in the colon peaks at 6-12 hours 39. We found that in the colon of WT mice supplemented with NA in their drinking water, this peak of DNA damage was coupled with an approximately 50% reduction in NAD which partially recovered at 48 hours (Fig. 4a, NAD, WT). These changes of NAD were associated with a massive depletion followed by a partial restoration of deamidated NAD metabolites NA and NAAD (Fig. 4a, WT). NAPRT KO mice, on the other hand, displayed reduced basal NAD levels and experienced minimal alterations in NAD metabolism after AOM injection within the observed timeframe (Fig. 4a, KO). Additional metabolomic analysis confirmed that NAPRT KO mice had altered metabolism, particularly defective NAD metabolism, in the colon both before and 48 hours after the AOM injection compared to WT mice (Fig. 4b, Supplementary Fig. 5c, and Supplementary Data 1), and NAPRT KO mice had minimal NAD metabolic changes in response to the AOM injection (Supplementary Fig. 5c, red box).

The metabolomic analysis further revealed that AOM treatment significantly induced three deoxyribonucleosides, including 2’-deoxycytidine, 2’-deoxyuridine, and thymidine, in both WT and NAPRT KO colons (Fig. 4c and Supplementary Fig. 5d). Moreover, their induction peaked at 12 hours post-injection when DNA damage was at its highest (Fig. 4c), suggesting that DNA damaged-induced DNA turnover and repair may be the sources of these nucleosides 40. Notably, 2’-deoxyuridine levels were significantly higher in NAPRT KO colons than in WT at all time points and did not show substantial recovery at 48 hours (Fig. 4c, 2’-deoxyuridine), suggesting increased and sustained DNA damage in the absence of NAPRT. Consistently, NAPRT KO colon epithelium had a significantly elevated level of H2AX compared to those in WT mice both before and after AOM injection (Fig. 4d). Altogether, these results suggest that deamidated NAD biosynthesis is important for efficient DNA repair in the gut.

To better understand the diminished NAD metabolic response of NAPRT KO mice to AOM-induced DNA damage at the molecular level, we carried out genome-wide transcriptomic analysis of the whole colon tissue from WT and NAPRT KO mice at 0, 12, and 48 hours after AOM injection. Despite their normal morphology, colons from NAPRT KO mice had significantly different expression of 2,502 genes compared to those from WT mice before AOM injection (0 h), among which genes mediating immune response were increased while genes involved in type I interferon-mediated signaling pathway were decreased (Supplementary Fig. 6a and Supplementary Data 2). In WT colons, AOM injection suppressed the expression of genes associated with nutrient metabolism and membrane trafficking (Supplementary Fig. 6b and 6c, clusters 1 and 2) while inducing those involved in immune response, ROS production, cell migration, DNA and RNA catabolism, and stress response (Supplementary Fig. 6b and 6c, clusters 3-6). In NAPRT KO colons, these transcriptomic changes were more pronounced compared to WT (Supplementary Fig. 6b and 6c). Comparisons between NAPRT KO vs WT colons at different timepoints after AOM injection further revealed that during the 48-hour timeframe after AOM injection, a cluster of 382 genes enriched in immune response pathways, including numerous immunoglobulin genes, several macrophage markers, and inflammatory chemokines/cytokines, was significantly more induced in the colon of KO mice than that in WT mice at the same time point (Cluster 1 in Fig. 4e, 4f, and Supplementary Data 3). This result suggests that NAPRT deficiency enhances the recruitment of various immune cells into the colon in response to AOM treatment. Further qPCR analysis showed that many of these inflammatory genes were induced at 12 hours and reduced at 48 hours after the AOM injection in the colon of WT mice, whereas their levels were elevated in the colon of KO mice before the AOM injection and remained high 48 hours after the injection (Supplementary Fig. 6d). Moreover, two clusters of genes involved in metabolic functions and interferon signaling, respectively, were significantly reduced in the colon of KO mice before and after the AOM treatment (Fig. 4e and 4f, and Supplementary Fig. 6d, Cluster 2 and Cluster 3). Another cluster of genes related to maintenance of cellular structure, protein quality control, and metabolism were induced in both WT and KO at 12 hours and remained elevated in KO but not in WT mice at 48 hours (Fig. 4e and 4f, Cluster 4). Collectively, the hypersensitivity to AOM-induced DNA damage is associated with an elevated inflammatory response yet reduced metabolic function and interferon signaling in the colon of NAPRT KO mice.

NAPRT deficiency increases sensitivity to DSS-induced colitis in female mice
To directly test whether NAPRT deficiency may indeed enhance chemically induced inflammation in the gut, we challenged WT and NAPRT KO female mice with dextran sulfate sodium (DSS)-induced colitis, a well-established experimental model of IBD characterized by compromised mucosal barrier function, infiltration of inflammatory immune cells into the lamina propria, epithelial ulceration, and focal crypt damage 41. In a DSS-induced colitis protocol where mice were first challenged with 2.5% DSS in their drinking water for 7 days and then allowed to recover with regular drinking water for 2 days (Supplementary Fig. 7a, Protocol 1), NAPRT KO mice experienced earlier and more severe rectal bleeding during the 7-day DSS treatment period and reduced recovery during the 2-day regular water recovery period (Fig. 5a). Accordingly, at the end of the procedure, NAPRT KO mice had aggravated colonic shortening (Fig. 5b) and more severe colonic epithelial tissue damage (Fig. 5c and 5d), including loss of mucin-positive goblet cells (Fig. 5e and 5f). These elevated measures of the morphological and pathological damage in the colon of NAPRT KO mice were coupled with elevated levels of IL6 cytokine in the plasma and increased induction of proinflammatory cytokine/chemokine genes in the colon tissues (Fig. 5g and 5h). In another DSS-colitis protocol in which mice were treated with 2.5% DSS together with 3 g/L NAM in drinking water for 7 days without recovery (Supplementary Fig. 7a, Protocol 2), we also observed an increased sensitivity of NAPRT KO mice to DSS-induced rectal bleeding, colonic shortening, and gut epithelial damage (Supplementary Fig. 7b-7e). Furthermore, when analyzed at the transcriptomic level, DSS treatment significantly repressed the expression of a cluster of genes involved in epithelial transporter activities in the colon of WT mice and such repression was significantly stronger in the colon of NAPRT KO mice (Supplementary Fig. 7f and 7g, Cluster 1), suggesting more severe damage of colon epithelial function in NAPRT KO mice. On the other hand, DSS-induced expression of proinflammatory genes was markedly higher in the colon of NAPRT KO mice than in WT mice (Supplementary Fig. 7f and 7g, Cluster 2). The increased induction of several proinflammatory cytokines and chemokines was further confirmed by qPCR (Supplementary Fig. 7h). Therefore, NAPRT deficiency sensitizes mice to chemically induced tissue damage and inflammation in the gut.

To better understand the potential mechanisms through which NAD depletion leads to increased inflammation in the DSS colitis in NAPRT KO mice, we analyzed the populations of immune cells in the lamina propria of the colon and blood before and after five-day treatment of DSS by FACS analysis. Consistent with the literature reports 41, the major immune cells responding to DSS-induced colitis were innate immune cells (Fig. 5i and Supplementary Fig. 7i). In WT mice, five-day treatment with DSS increased the abundance of blood myeloid cells, primarily neutrophils (Supplementary Fig. 7i, WT). This increase was coupled with an elevated infiltration of neutrophils into the colonic lamina propria (from 0.07% to 0.86%) (Fig. 5i, WT). Deletion of NAPRT did not significantly affect immune cell abundance in either blood or colonic lamina propria under the basal condition (Fig. 5i and Supplementary Fig. 7i, D0). However, DSS-induced infiltration of neutrophils into the colonic lamina propria was significantly enhanced (from 0.06% to 2.22%) in these mice (Fig. 5i, KO), whereas their blood neutrophil abundance was slightly lower than that of WT mice after DSS treatment (Supplementary Fig. 7i, D5). DSS-treatment also significantly increased infiltrated Ly6G negative myeloid cells (Fig. 5i, Ly6G-CD11b+), which include macrophages, monocytes, and dendritic cells. Together with our observations in Fig. 5a-5h, these findings suggest that NAPRT deficiency increases the recruitment of myeloid cells, including neutrophils, into colonic lamina propria in response to DSS-induced epithelial damage.

To further understand how deletion of NAPRT increases the recruitment of myeloid cells upon DSS-induced colitis at the molecular level, we analyzed transcriptomics of all colonic cell types before and after five-day treatment of DSS at the single cell level by scRNA-seq. Based on the individual cell transcriptome and the expression patterns of known marker genes (Supplementary Fig. 8a), the colonic cells could be categorized into 8 functional groups, including 6 epithelial cell groups and 2 immune cell groups (Fig. 6a and Supplementary Data 4). The two immune cell groups could be further subclustered into 9 clusters, including 7 immune cell types and two contaminating non-immune cell types (IECs and fibroblasts) (Fig. 6b, Supplementary Fig. 8b, and Supplementary Data 4). Consistent with the FACS results in Fig. 5i, DSS-treated KO mice had increased abundance of neutrophils in the colon compared to DSS-treated WT mice (Supplementary Data 4, Neutrophils). In contrast, the abundance of colonic T cells, particularly a subcluster of T cells expressing some exhaustion markers (Supplementary Fig. 8b, T2), was markedly reduced in KO mice (Supplementary Data 4, T and T2 cells). Interestingly, in the majority of cell types, the expression of Nampt was significantly induced by DSS treatment, whereas the expression of Naprt was not significantly affected (Fig. 6c and Supplementary Fig. 8c, WT D5 vs WT D0), suggesting the differential functions of these two NAD biosynthesis pathways in response to DSS-induced colitis. Transcriptomic analysis of colonocytes, the cell type with the highest expression level of Naprt (Fig. 6c, CC), showed that DSS treatment induced the expression of genes associated with membrane trafficking, G-protein coupled cell signaling, and adherens junctions, but reduced the expression of genes involved in lipid transport, digestive system, and lipid metabolism (Supplementary Fig. 8d). Venn-diagram analysis further revealed that among the genes induced by DSS in colonocytes of WT mice, deletion of NAPRT further enhanced the expression of genes involved in the defense response, particularly those related to antigen processing and presentation, but suppressed the induction of genes responsible for intracellular structure maintenance and protein quality control (Fig. 6d and 6e). These key transcriptomic changes were consistently observed across all annotated cell types, including both gut epithelial cells and immune cells (Fig. 6f and Supplementary Fig. 8e), suggesting that the enhanced intestinal inflammation observed in DSS-treated KO mice is associated with increased anti-microbial defense response and reduced maintenance of tissue integrity. Taken together, in agreement with our pathological, biochemical, and qPCR evaluations in Fig. 5a-5h, our FACS and scRNA-seq analyses indicate that inactivation of the deamidated NAD biosynthesis in NAPRT-deficient colon leads to reduced maintenance of tissue integrity and elevated anti-microbial response upon DSS challenge, which enhance recruitment of myeloid cells (particularly neutrophils) and induce intestinal inflammation.

To further explore the clinical significance of deamidated NAD biosynthesis in regulation of gut inflammation and tissue integrity, we analyzed the expression of two key deamidated NAD metabolic enzymes, NAPRT and NADSYN1, in a publicly available RNA-seq dataset from the intestine of human IBD patients and non-IBD controls 42. Consistent with previous reports 11, 43, the mRNA level of NAMPT was significantly elevated in the intestine, including the colon, of both ulcerative colitis (UC) and Crohn’s disease (CD) patients (Fig. 6g and Supplementary Fig. 8f, NAMPT), and NAMPT expression was strongly correlated with that of genes involved in response to extracellular stimuli, particularly many inflammatory and immune cell genes (SOCS3, CD274, LILRA5, and S100A9) (Fig. 6h, NAMPT, Supplementary Fig. 8g, and Supplementary Data 5). In contrast, neither NAPRT nor NADSYN1 were significantly altered in the intestine of IBD patients in this study (Fig. 6h and Supplementary Fig. 8f). Moreover, the mRNA level of NAPRT was positively correlated with that of genes involved in metabolism, mitochondrial function, and nutrient transport regardless of the patient IBD status (Fig. 6h, NAPRT, Supplementary Fig. 8h, and Supplementary Data 5). Collectively, our findings indicate that NAPRT is important for metabolism and overall functions of gut epithelial cells in both mice and humans.

NAPRT deficiency promotes tumorigenesis
The increased sensitivity of NAPRT KO mice to chemically induced DNA damage and inflammation in the colon raises the possibility that they may have enhanced propensity to tumorigenesis in this tissue. To test this possibility, we challenged WT and NAPRT KO female mice with an AOM/DSS-induced inflammation-associated colorectal cancer (CRC) model, in which mice were first injected with 10 mg/Kg AOM to induce DNA damage and then treated with 2 % DSS in drinking water for 7-8 days to induce inflammation and tissue damage (Supplementary Fig. 9a). In support of our hypothesis, when analyzed at the end of this procedure (D90 after AOM injection and around 11 weeks after DSS treatment), NAPRT KO mice developed more and bigger colorectal tumors, thereby significantly increasing tumor burden in the colon (Fig. 7a). Further pathological evaluation indicated that colorectal tumors developed in NAPRT KO mice were at the more advanced adenoma stage compared with those in WT mice (Fig. 7b and Supplementary Fig. 9b). More specifically, 80% NAPRT KO mice had colon tumors at the adenoma stage whereas WT mice had none (Fig. 7c, left). Moreover, while all tumors developed in WT mice were at a less advanced atypical hyperplasia stage, half of colorectal tumors in NAPRT KO mice were adenomas (Fig. 7c, right). Furthermore, the colon tissue from NAPRT KO mice displayed increased expression of multiple proinflammatory genes and immune cell markers at the end of the AOM/DSS CRC procedure (Fig. 7d and Supplementary Fig. 9c).

To confirm the increased susceptibility of NAPRT KO mice to intestinal tumorigenesis in an additional model, we bred WT and NAPRT KO mice into the Apcmin+/- background, a genetic intestinal tumor model that develops spontaneous intestinal adenomas driven by dysregulated WNT/-catenin pathway 44, 45. When analyzed at the age of 16 weeks, NAPRT KO male but not female mice had increased colon tumor burden on this Apcmin+/- background (Fig. 7e, 7f, and Supplementary Fig. 9d and 9e). Both male and female mice displayed expected defects in deamidated NAD biosynthesis (Supplementary Fig. 9f). Finally, monitoring the mortality of WT and NAPRT KO male and female mice during the first 24 months of age showed that female but not male NAPRT KO mice had significantly reduced symptom-free survival compared with age-matched WT females (Fig. 7g). Among the 16 KO females removed/lost from the aging cohort, five were removed due to visible large abdominal mass or rectal prolapse, whereas none of the six removed/lost WT females displayed these tumor-related symptoms, suggesting that NAPRT KO mice may have increased risk of developing spontaneous tumors. Indeed, detailed analysis of two independent cohorts of 22 to 24-month-old male and female mice revealed that 10 out of 19 male and 2 out of 5 female aged NAPRT KO mice developed spontaneous tumors in various organs, including gut, gut-associated tissues, liver, and lung (Fig. 7h, 7i, and Supplementary Fig. 9g). In contrast, none of the 12 male and only one out of 11 female aged WT mice carried a tumor (Fig. 7h). Further histopathological evaluation by a board-certified pathologist indicated that the majority of tumors developed in aged NAPRT KO mice were lymphomas and sarcomas (Supplementary Fig. 9g), suggesting that the tumor suppressor function of NAPRT is not limited to epithelial cancers.

NAPRT is found to be frequently amplified and/or overexpressed in various human cancer types and its knockdown slowed growth of xenograft tumors 9, 17, suggesting tumor-promoting role of this gene. However, our observations in NAPRT KO mice suggest that although high levels of NAD may promote tumor progression, maintaining an endogenous level of the deamidated NAD biosynthesis is critical for efficient DNA repair and subsequent suppression of tumorigenesis. In support of this hypothesis, AOM/DSS-induced tumorigenesis in WT mice was coupled with reduced expression of key deamidated NAD biosynthesis genes (Fig. 8a). Compared to normal colon tissues from age- and gender-matched untreated mice, tumor adjacent colon tissues from AOM/DSS treated WT mice had significantly reduced expression of both Naprt and Nadsyn but not Nampt (Fig. 8a, Adjacent vs Normal). The mRNA levels of these two key deamidated NAD biosynthesis enzymes were further reduced in AOM/DSS-induced tumors (Fig. 8a, Tumor vs Adjacent). Moreover, analysis of TCGA datasets showed that the mRNA levels of NAPRT are reduced in a significant proportion of tumors from seven analyzed human cancer types, including colon adenocarcinoma (COAD), all three types of kidney cancers, kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), as well as two types of lung cancers, lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (Fig. 8b). Similar results were observed when all primary tumor samples with expression values were analyzed (Supplementary Data 6).

To gain deeper insight into the role of NAMPT and NAPRT in colonic tumorigenesis, we stratified the colorectal cancer (CRC) data in Fig. 8b based on CMS subtypes 46. As shown in Fig. 8c, all four CRC subtypes had more tumors with an elevated expression of NAMPT than those with low expression of this gene. Notably, CMS1, the subtype characterized by increased immune infiltration and strong activation of immune evasion pathways 46, had the highest proportion of NAMPT-high tumors. On the other hand, CMS4, the subtype that shows upregulation of epithelial mesenchymal transition (EMT) and activation of transforming growth factor β (TGF β) 46, had the highest proportion of NAPRT-low tumors (Fig. 8c, NAPRT). These findings were consistent with our observations that NAMPT was enriched in immune cells whereas NAPRT was predominantly expressed in epithelial cells (Fig. 1a, 1b, 6c, and Supplementary Fig. 2b, 2c, 8c). Intriguingly, the expression levels of NAMPT and NAPRT were negatively correlated in CRC, especially in the CMS4 subtype that displays worse overall and relapse-free survival compared to other subtypes 46 (Fig. 8d), highlighting the subtype-specific roles of these two NAD biosynthesis pathways in intestinal tumorigenesis. In colorectal polyps, alterations in the expression of NAMPT and NAPRT also showed distinct patterns. Notably, the mRNA levels of NAMPT were significantly reduced in colorectal polyps compared to normal colonic tissues, whereas the mRNA levels of NAPRT remained largely unchanged (Supplementary Fig. 9h).

Further analysis of TCGA PanCancer Altas revealed that low expression of NAPRT in liver and kidney cancers, particularly in KICH, is significantly associated with reduced patient survival (Fig. 8e and 8f). Interestingly, the impact of NAPRT expression on the prognosis of CRC was stage dependent. Stage 1, but not stage 3 and 4, CRC patients with low NAPRT levels displayed worse prognosis compared to stage-matched patients with high NAPRT expression (Fig. 8g). Collectively, our findings indicate that NAPRT-mediated deamidated NAD biosynthesis is important for normal tissue integrity and tumor suppression in response to environmental stress.

Methods
Ethics statement
All animal experiments in this study were approved by the NIEHS/NIH Animal Care and Use Committee under the Animal Study Proposal # 07-03 and 2014-0016, MCBL.
For DSS-colitis and AOM/DSS CRC model, any mouse with three of the clinical signs in Category A or any one clinical sign in category B was euthanized: Category A: ruffled hair coat, hunched posture, lethargy, weight loss of 20%, rectal prolapse greater than 2 mm. Category B: weight loss of greater than or equal to 25% that does not improve or stabilize in 24h of close observation, inability to move about the cage, inability to right itself, labored breathing, pale extremities, bloating, rectal prolapse greater than 5mm, rectal bleeding score of 3.
For Apcmin+/- intestinal tumor model, mice displaying clinical signs of 10% loss of body weight compared to non Apcmin+/- littermates, hunched posture, rough coat, slower activity compared to cage mates, pinched face, rectal protrusion, rectal bleeding were evaluated by the veterinary staff for euthanasia.
For mice in aging study, any mouse that displays 20% weight loss with hunched posture and rough coat, or any of the following: labored breathing, incoordination, seizures, lethargy, or becoming prostrate, was euthanized.

Cells
The CRC119 human colon cancer cell line derived from a metastatic female CRC patient was acquired as a generous contribution from Dr. David Hsu at Duke University25. These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin and were incubated at 37°C and 5% CO2.
MEFs were isolated from E13.5 embryos and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin and streptomycin.
Bone marrow-derived macrophages were isolated from femurs of 8-week-old female mice and grown in DMEM supplemented with 10% fetal calf serum, 20% L929 conditioned medium and 1% penicillin and streptomycin.

NAPRT deletion and ectopic expression, and NAMPT deletion in CRC119 cells
To generate NAPRT or NAMPT-deficient CRC119 cells using CRISPR/Cas9-mediated gene editing, specific guide RNAs (gRNAs) targeting NAPRT or NAMPT were designed.
For NAPRT deletion, the forward and reverse sequences of the gRNA were 5′-CACCGACAAAGGCTGCCCGCTCGCC-3′ and 5′-AAACGGCGAGCGGGCAGCCTTTGTC-3′, respectively. For NAMPT deletion, the forward and reverse sequences of the gRNA were 5′-CACCGGGCCACTGTGATTGGATACC-3′ and 5′-AAACGGTATCCAATCACAGTGGCCC-3′, respectively. The gRNAs were cloned into a lentiCRISPRv2 vector via BsmBI restriction enzyme sites and subsequently transfected into CRC119 cells. After two days selection with 2 g/ml puromycin, single colonies were isolated. To identify NAPRT KO clones, each colony was split in half: one half was expanded, while the other was seeded in a 96-well plate and cultured for 2 days in a medium supplemented with 100 nM STF-118804 and 100 M NA. Clones failed to survive in this selection medium were selected for further validation of NAPRT inactivation. Similar strategy was used to identify NAMPT KO clones, but using regular growth medium for selection and NA-containing medium for maintenance. Cellular viability was determined by CellTiter-Glo® kit (Promega). The efficiency of NAPRT or NAMPT depletion was validated by immunoblotting analysis.
For transient NAPRT add-back in NAPRT-deficient CRC119 cells, cells were transfected with pcDNA3-HA-hNAPRT.
For stable NAPRT add-back, pLV-mCherry:T2A:Puro-EFHA-HA-mNAPRT was custom built by Vector Builder. NAPRT-expressing and control lentiviruses were packaged in 293T cells and used to infect CRC119 NAPRT-deficient cells followed by puromycin selection.

Experimental animals
All mice at the NIEHS facility were housed in individualized ventilated cages (Techniplast, Exton, PA) with a combination of autoclaved nesting material (Nestlet, Ancare Corp., Bellmore, NY and Crink-l’Nest, The Andersons, Inc., Maumee, OH) and housed on hardwood bedding (Sani-chips, PJ Murphy, Montville, NJ). Mice were maintained on a 12:12-h light:dark cycle at 220.5 °C and relative humidity of 40% to 60%. Mice were provided ad libitum autoclaved plant-based chow diet supplemented with 83 mg/Kg of NA (NIH-31, cat# 7017, Harlan Laboratories, Madison, WI) and deionized water treated by reverse osmosis. Mice were negative for mouse hepatitis virus, Sendai virus, pneumonia virus of mice, mouse parvovirus 1 and 2, epizootic diarrhea of infant mice, mouse norovirus, Mycoplasma pulmonis, Helicobacter spp., and endo- and ectoparasites upon receipt and no pathogens were detected in sentinel mice during this study.
The experimental mice were acclimatized for a week before being randomly assigned to experimental groups. Clinical health assessments were performed twice weekly during the experiments.

Generation of whole-body NAPRT KO (Naprt-/-), Naprt+/+Apcmin/+ and Naprt-/-Apcmin/+ mice
All mice utilized in the study were on the C57BL/6J background. The global NAPRT knockout (KO) mice (Naprt-/-) were generated by CRISPSR-cas9 technology by the Gene Editing and Mouse Model Core Facility at National Institute of Environmental Health Sciences (NIEHS). To generate the NAPRT mutant line, single cell C57BL/6J embryos were microinjected with 100 ng/l Cas9 protein (New England BioLabs EnGen Spy Cas9 NLS - M0646M) and 125 ng/l sgRNA targeting exon 9 ORF at GGCAGTGAGGTGAATGTCATNGG[PAM] (synthesized by IDTDNA). Injected single cell embryos were surgically transferred to SWISS pseudo-pregnant mice. F0 founders were screened at weaning using primers that amplify the entire target region (Fwd: GACCATGGACCTATTTCAGGGG; Rev: AGGTCCAGTAGCAAAGACCCTAT). Several independent founders with a NHEJ alleles of interest were crossed to C57BL/6J wild-type mice, and their F1 offspring were screened by genomic DNA sequencing to confirm transmission of the mutant NAPRT alleles. Among several different alleles generated, a catalytic inactive allele containing a 21 bp NHEJ deletion, which resulted in the in-frame loss of coding sequence of 376-IGIGTSV-382 disrupting the catalytic site in exon 9 (Supplementary Fig. 3a, d(CAT or d(21)), was selected for future studies. The d(21) mouse colony was subsequently genotyped by Transnetyx (Cordova, TN) using primer probe assays specific for the wild-type allele and d(21) catalytically-inactive allele. All experimental mice from the d(21) allele were further backcrossed into the C57BL/6J background for at least 4 generations.
Naprt+/+Apcmin/+ and Naprt-/-Apcmin/+ mice were generated by crossing wild-type (WT) and NAPRT KO mice with Apcmin/+ mice (C57BL/6J-ApcMin/J, Jax: 002020, both sexes) respectively. Age- and gender-matched mice were used for all experiments. Both males and females were analyzed in all experiments, and sex-specific findings were observed and reported.

Isotopic tracing
For D4-NAM isotopic tracing experiment, male WT and NAPRT KO mice, aged between eight to thirteen weeks, were orally administered either 80 mg/kg of nicotinamide labeled with four deuterium atoms on the pyridine ring (D4-NAM) or received a PBS gavage in the control group. To prevent any bias, gavages were staggered to keep the time between gavage and sacrifice constant and were administered alternately between the WT and NAPRT KO mice. The mice were euthanized at 0, 3, 6, or 24 hours after gavage administration, and small intestines and colons were flushed and flash-frozen in liquid nitrogen along with blood, liver, pancreas, kidneys, spleen, heart, lungs, quadriceps muscles, brain, white and brown adipose tissues. These samples were then preserved at -80 degrees until further processing. Each group receiving a specific treatment consisted of 4-5 mice.
For D4-NA isotopic tracing experiment, male WT and NAPRT KO mice, aged between eight to thirteen weeks, were orally administered with either 80 or 400 mg/kg of nicotinic acid labeled with four deuterium atoms on the pyridine ring (D4-NA) or received a PBS gavage in the control group. To prevent any bias, gavages were staggered to keep the time between gavage and sacrifice constant and were administered alternately between the WT and NAPRT KO mice. The mice were euthanized at 1, 3, 6 hours after gavage of 80 mg/kg D4-NA or 3 hr after 400 mg/kg D4-NA administration, and plasma was isolated by centrifugation of systemic blood at 2000g for 10 minutes and kept at -80 degrees until further processing.

AOM-induced DNA damage and DSS-induced colitis
For the AOM-induced DNA damage model, 9 to 16-week-old female WT and NAPRT KO mice were supplemented with 3 g/L NA in the drinking water. Twenty-four hours later, they were intraperitoneally (i.p.) injected with one dose of 10 mg/Kg AOM with a continuous supply of the NA water for the rest of the experiment. Colon tissues were collected either 12 hours or 48 hours post-injection.
For the DSS-induced colitis model, 8 to 12-week-old female WT and NAPRT KO mice were treated with 2.5% DSS in drinking water for 7 days, then recovered with regular drinking water for 2 days (protocol 1). Alternatively, mice were treated with 2.5% DSS in drinking water containing 3 g/L NAM for 7 days without the recovery period (protocol 2) Throughout these experiments, the body weight, rectal bleeding, and diarrhea of experimental mice were monitored daily, and the severity of rectal bleeding was scored based on a score system as previously described 41.

AOM/DSS, Apcmin/+, and spontaneous aging tumorigenesis models
For the chronic inflammation-related colon cancer model (AOM/DSS-colon cancer model), 9 to 16-week-old WT and NAPRT KO female mice were initially i.p. injected with 10 mg/kg AOM. One week later, they were treated with 2% DSS in drinking water for 8 days. Tumors were analyzed 13 weeks after the AOM injection. Colonic tumor burden was quantified by determining the ratio between the area occupied by tumors and the total area of the colon using ImageJ 2.14.0 (Fiji) software.
For a genetic intestinal tumor model, Naprt+/+Apcmin/+ and Naprt-/-Apcmin/+ mice were utilized. Small intestines and colons were collected when the mice were 16 weeks old. Tumors were analyzed and tumor burden was quantified by ImageJ 2.14.0 (Fiji) software. The different tumor burdens between Naprt+/+Apcmin/+ and Naprt-/-Apcmin/+ were only observed in males.
For the spontaneous aging model, male and female WT and NAPRT KO mice were allowed to age to 24 months, dissected and examined for tumors in different organs. All tumors were collected, fixed in formalin and paraffin embedded, and hematoxylin and eosin sections were examined.

Immunofluorescence staining
Colonic tissues from WT and NAPRT KO mice treated with AOM were formalin fixed, embedded in paraffin, and sectioned into slides. The slides were then subjected to a series of steps: deparaffinization in xylene and rehydration through graded ethanol. Antigen retrieval was performed using citrate-based unmasking solution in a microwave for 20 min at 10% power. The Squenza Slide Rack and Coverplate System was used for staining the slides with a mouse anti-rabbit phospho-histone H2AX (H2AX, Cell Signaling Technology, 9718) antibody or anti-NAPRT antibodies (NAPRT #1, Proteintech, 13549-1-AP), NAPRT #2 (Abcam, ab211529) overnight at 4 C followed by goat anti-rabbit or anti-mouse IgG H&L (Alexa Fluor® 594) for one hour at room temperature. Nuclei were stained with 5 g/ml DAPI for 5 minutes, followed by mounting with Prolong Diamond Antifade. Images from five different fields of view per condition were acquired with the confocal Zeiss LSM 880 or 980 microscope. Total nuclear fluorescent intensity from the H2AX channel from each cell was quantified using a custom ImageJ 2.14.0 (Fiji) macro.
CRC119 cells seeded on coverslips in the 12-well plate were fixed with cold methanol for 10 minutes and permeabilized with 0.5% Triton X-100 in PBS at room temperature for 10 minutes. Coverslips were incubated with anti-H2AX (1:200) for 1 hour at room temperature. After washing with PBS for 4-5 times, goat anti-rabbit IgG H&L (Alexa Fluor® 594) was applied for 1 hour at room temperature. Nuclei were stained with 5 g/ml DAPI for 5 minutes, followed by mounting the samples using Prolong Diamond Antifade. Imaging and analysis were performed as described for colonic tissues.
For co-staining of HA-NAPRT and H2AX, cells were grown in 8-chamber slides (Thermo Fisher), fixed with 4% formaldehyde for 10 minutes at room temperature, permeabilized with 0.5% Triton X-100 in PBS for 10 minutes, and stained with a mixture of anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-H2AX antibodies, followed by goat anti-mouse IgG H&L (Alexa Fluor® 594) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) antibodies, respectively. Nuclei were stained with 5 ug/ml DAPI. Images from five different fields of view per condition were acquired with the confocal Zeiss LSM 880 microscope. Total nuclear fluorescent intensity from both channels in each cell was quantified using a custom ImageJ 2.14.0 (Fiji) macro.

Histopathological evaluation
For the colon tissues from mice treated with DSS, 0.5 cm distal colon was collected and fixed in formalin. After paraffin embedding, sectioned slides were stained with hematoxylin and eosin (H&E), and evaluated by a certified pathologist. The evaluation included identification and quantification of inflammation, ulceration, edema, goblet cell loss, and granulation tissue/fibrosis.
For the colon tissues from mice treated with AOM/DSS, Naprt+/+Apcmin/+ and Naprt-/-Apcmin/+ mice, Swiss roll sections of the colon were prepared, stained with H&E, and assessed by a board-certified pathologist. The evaluation comprised identifying tumors, assessing atypical hyperplasia and adenomas. Detailed descriptions of lesions and their morphological diagnoses were provided. Atypical hyperplasia exhibited distinctive features including crowded epithelial cells lining crypts with retained polarity and some dysplasia. Adenomas, on the other hand, displayed proliferations of branching tubules or finger-like projections in the lamina propria, often presenting various degrees of dysplasia characterized by disrupted cell layering and irregular nuclear features. Carcinoma was identified as an invasive proliferation of epithelial cells invading the underlying lamina propria and submucosa.
For spontaneous tumors in aged mice, H&E-stained tumor slides were evaluated by a board-certified pathologist.

RNA isolation, RT-PCR and RNA-seq
Total RNA was isolated using TRIzol™ Plus RNA Purification Kit (Thermo Fisher Scientific) from frozen colon tissue after pulverization in liquid nitrogen with mortar and pestle. Total RNA from cultured cells was extracted using the RNeasy kit (Qiagen). For RT-PCR analysis, RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Real-time PCR assays were conducted on a CFX96 or CFX Opus 384 real-time PCR instruments (Bio-Rad) utilizing iQ SYBR Green SuperMix (Bio-Rad). Primer sequences can be found in Supplementary Data 7.
To conduct RNA-seq analysis, libraries were prepared using the TruSeq Stranded Total RNA kit (Illumina, San Diego, CA) with Ribo-Zero technology. The indexed samples were sequenced on the Nova-seq 6000 (Illumina) employing a 75-bp single-end protocol as per the manufacturer's guidelines. Subsequently, reads (ranging from 40 to 80 million reads per sample) were aligned to mm10 reference genome separately using the STAR aligner (version 2.6)56. Gene quantification based on GENCODE annotation release (GRCh37, p13) was conducted utilizing Subread featureCounts (version 1.4.6)57. Principal Component Analysis (PCA) aided in detecting any potential sample outliers. Comparisons between the vehicle and treatment samples were conducted to detect differentially expressed genes. To adjust the false discovery rate (FDR), the Benjamini and Hochberg method was employed. A gene was deemed significantly differentially expressed if the FDR-adjusted p-value for differential expression was below 0.05. The heatmaps were generated utilizing Partek Genomic Suite (Version 6.6, Partek, St. Louis, Missouri).
The RNA-seq data are available in the Gene Expression Omnibus repository at the National Center for Biotechnology Information (RNA-seq dataset of the AOM experiment: GSE271834; RNA-seq dataset of the DSS-colitis experiment: GSE271250).

Pathway Enrichment Analysis
All pathway enrichment analysis were preformed using generated differential gene lists in https://biit.cs.ut.ee/gprofiler/gost.

Western blotting
Cellular pellets were lysed in RIPA buffer supplemented with Complete Mini protease inhibitor cocktail (Roche Diagnostics). Subsequently, the proteins were separated on 4–20% gradient SDS-PAGE gel (BioRad), followed by their transfer onto PVDF membranes. Primary antibodies targeting specific proteins-NAPRT #1 (Proteintech, 13549-1-AP), NAPRT #2 (Abcam, ab211529), PAR (Sigma, MABE1016; Sigma, AM80), PARP (Cell Signaling Technology, 9542), MAR (Bio-Rad, HCA355), H2AX (Cell Signaling Technology, 9718), HA (Santa Cruz Biotechnology, sc-7392) and Actin (Sigma, MAB1501) were used followed by incubation with fluorescently labeled secondary antibodies and detection on the Odyssey (LI-COR) or ChemiDoc (Bio-Rad) imaging systems.

Mucin high-iron diamine and Alcian blue staining
Thin sections of formalin-fixed paraffin-embedded tissue (8 μm) were deparaffinized in xylene for five minutes and then sequentially passed through various alcohol concentrations (100, 90, 75, 50, and 0 percent) for five minutes each. These sections were subjected to treatment with a high-iron-diamine (HID) solution for 16 hours at Room Temperature. The HID solution composition comprised 120 mg of NN-dimethyl-meta-phenylenediamine-dihydrochloride and 20 mg of NN-dimethyl-paraphenylenediamine-dihydrochloride dissolved in distilled water up to 50 ml. To this solution, 1.4 ml of freshly prepared 60% (w/v) ferric chloride solution was added (all from Sigma). Following treatment, sections were rinsed multiple times in tap water and counterstained using 1% Alcian blue in 3% acetic acid (pH 2.5) for 20 minutes. After rinsing with water, the sections were mounted. Sulfated mucins were distinguished by displaying a brown/black color, while sialomucins exhibited a blue coloration. Mucin positive area in the colon were quantified by Fiji.

IL-6 ELISA
Measurement of IL-6 protein levels in plasma of DSS-treated mice was performed using IL-6 Mouse ELISA Kit (Thermo Fisher Scientific, KMC0061).

Colony formation assay
Pools of six single cell clones of WT or NAPRT KO CRC119 cells were seeded at 500 cells/well in 6-well plates in triplicates. 24 hours later, medium was replaced to fresh growth medium with 100 M NA and/or 100 nM STF-118804 for 24 hours. Then, medium was replaced to fresh growth medium with corresponding NA/STF with or without 1 mM MMS for 1 hour. Subsequently, cells were washed twice with PBS and medium was replaced to fresh growth medium with 100 M NA.
Colonies were stained with Crystal Violet 7 days later and visible colonies were counted.

Primary mouse organoids
Small intestinal and colon organoids were isolated from 6-8 weeks-old WT or NAPRT KO female mice according to IntestiCult™ protocol (StemCell). Organoids were grown in 3D in 25 l Matrigel (Sigma) domes per well of 48-well plates for maintenance and in 6 l Matrigel per well of 96-well plates for assays. Small intestinal organoids were grown in IntestiCult™ Organoid Growth Medium (Mouse) (StemCell). Colon organoids were grown in DMEM/F12 (ThermoFisher Scientific) supplemented with 20% R-Spondin conditioned medium, 10% Noggin-conditioned medium, 50 ng/ml human EGF (R&D Systems), 0.15 nM Wnt3a-Surrogate (ThermoFisher Scientific), 1:500 B27-Vit A (ThermoFisher Scientific), 1 mM N-acetylcysteine (Sigma), and 100 ug/ml Primocin (ThermoFisher Scientific). R-Spondin and Noggin-expressing cells for production of the conditioned media were obtained from Johns Hopkins University.

Real time metabolic (Seahorse) analysis
Cells were seeded at 6 × 104 cells per well (n = 4-5) on XF24 cell plates with 100 M NA in 250 μL RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin. 48 hours later, for glycolysis stress test, medium in each well was replaced with 500 μL XF RPMI assay medium (pH 7.4) supplemented with 2 mM glutamine, 100 M NA and cells were incubated for two hours at 37 °C without CO2 before running the assay. The Seahorse measurements on Seahorse XFe24 Analyzer (Seahorse Biosciences) included sequential injections of glucose (10 mM), oligomycin (1 μM), and 2-deoxy glucose (2-DG, 50 mM).
For mitochondrial stress test, medium in each well was replaced with 500 μL XF RPMI assay medium (pH 7.4) supplemented with 100 M NA, 2 mM glutamine, 10 mM glucose, 1 mM pyruvate and cells were incubated for two hours at 37 °C without CO2 before running the assay. The Seahorse measurements on Seahorse XFe24 Analyzer (Seahorse Biosciences) included sequential injections of oligomycin (1.5 μM), FCCP (1 μM), and rotenone (0.5 μM). Following the Seahorse assay, assay medium was carefully removed without disturbing the cells, and protein concentration was determined using BCA assay (ThermoFisher Scientific). The raw ECAR and OCR measurements were normalized to the total protein content.

scRNA-seq analysis of colonic epithelial cells
Isolation of colonic epithelial cells
Mouse colons were freshly dissected. Following the removal of the mesentery/fat and connective tissue, colons from three mice per group were combined, flushed with ice-cold CMF-PBS, longitudinally opened, and rinsed with ice-cold CMF-PBS. The colons were then cut into 0.5 to 1 centimeter pieces and washed once in rinse buffer containing 5.6 mM Na2HPO4 x2H2O, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM D-sorbitol. Then, the colon pieces were resuspended in isolation buffer (2mM EDTA and 0.5mM DTT in rinse buffer) and incubated on a rotation platform for 20 min at room temperature. Subsequently, supernatants were removed, and the remaining tissue was resuspended in 10 ml ice-cold rinse buffer and pipetted 25 times up and down with a 25 ml pipette. Following the settling of the large tissue pieces, the supernatants containing crypts were transferred to a new tube. After additional pipetting 25 times up and down with a 25 ml pipette and settling of the tissue pieces, the supernatants were centrifuged at 300 g, 4°C for 3 min, and the pellets were resuspended in 10 ml room-temperature single-cell-isolation-buffer (1 mg/ml DNase I, 5 mM MgCl2, 20 μM Y27 in TrypLE Express) and incubated on 100 RPM at 37°C for 20 min. Then, the cell solution was passed through the 40 m cell strainers. Digestion was stopped by the addition of 35 ml ice-cold CMF-PBS, and centrifugation at 500 g, 4°C for 4 min. The cell pellets were resuspended in ice-cold PBS with 2% FBS.
scRNA-seq library preparation and sequencing
Cell counts and viability assessments were performed using fluorescent AO/PI (Logos Biosystems, F23001) staining via a Luna-FX7 cell counter (Logos Biosystems, L70001). Subsequently, approximately 10,000 viable cells exhibiting over 50% viability were utilized and loaded into the Single Cell Chip for the creation of single-cell emulsion in the Chromium Controller by 10x Genomics (120263) employing the Chromium Single Cell 3’ Library & Gel Bead Kit v3.1 (10x Genomics, 1000268). Following the manufacturer's guidelines (10x Genomics, 1000268), the process encompassed reverse transcription of mRNA and subsequent cDNA amplification. The amplified cDNA underwent fragmentation to generate NGS libraries, which were sequenced by the NIEHS Epigenomics and DNA Sequencing Core Laboratory.
scRNA-seq data processing
Initial processing of raw reads involved the utilization of the Cell Ranger Single-Cell Software Suite (version 6.0.1, 10x Genomics Inc., CA). The demultiplexed FASTQ files, composed of paired-end reads (Read 1: 30bp, Read 2: 100bp), were generated using the CellRanger mkfastq command. Primary data analyses, including alignment, filtering, barcode counting, and quantification of unique molecular identifiers (UMIs) to determine gene transcript counts per cell, were conducted using CellRanger count command referencing the genome “refdata-gex-mm10-2020-A”. This process resulted in the creation of a gene-barcode matrix.
The raw gene expression matrices produced per sample by CellRanger were imported into R (version 4.3.1) and transformed into a Seurat object using the Seurat R package (version 4.3.2)58. The function decontXR from the R package "celda" was employed to eliminate RNA contamination59. Removal of deceased cells, doublets, and low-quality cells was performed systematically. This involved evaluating the total UMIs, total genes, and the percentage of UMIs originating from the mitochondrial genome for each cell. Cells with over 10% UMIs derived from the mitochondrial genome were excluded. Additionally, cells falling outside the upper and lower bounds—calculated as the mean plus or minus two standard deviations (SD) for both total UMIs and genes—were further eliminated based on this criterion.
Single-cell gene expression quantification and major cell type classification
The cleaned cells underwent normalization of gene expression, identification of highly variable genes (HVGs), and scaling using the Seurat SCTransform function. Normalization involved adjusting gene expression data to a standardized total cellular read count (set at 10,000 per cell), while the top 2000 highly variable genes were selected as features for subsequent dimensionality reduction and clustering. Additionally, Cell-Cycle scores were computed using the Seurat CellCycleScoring function, utilizing specific cell-cycle-related gene pairs to classify cells into G1, S, and G2/M phases. Any cell cycle-related effects were addressed through regression using the SCTransform function.
Principal Component Analysis (PCA) was conducted using the Seurat RunPCA function, and the top 40 principal components (PCs) were chosen for further analysis. Subsequently, the Seurat FindNeighbors function established a Shared Nearest Neighbor (SNN) Graph, followed by the application of the RunUMAP function to visualize the selected significant PCs. Cell clustering was accomplished using the FindClusters function, with a parameter of “resolution = 1”.
Marker genes for distinct clusters or cell clusters or cell types were identified using the Seurat FindMarkers function, which compared the gene expression values of cells within a particular cluster against those from other clusters. Cell clusters were annotated using two approaches: canonical marker genes were utilized to assign known biological cell types, while predicted marker genes helped confirm annotated cell types and name clusters lacking canonical marker genes.
The access number of scRNA-seq dataset generated from colon tissues is GSE271836.
For analysis of small intestinal scRNAseq, a dataset GSE261216 from a separate unpublished study was used.

Enzymatic assays for NAD and ATP
Cells or organoids were seeded in clear-bottom white plates obtained from Corning (#3610). The determination of total NAD using enzymatic methods was conducted utilizing the NAD/NADH-Glo™ kit sourced from Promega. Additionally, CellTiter-Glo® or CellTiter-Glo® 3D from Promega were employed for determination of ATP levels and served as a surrogate measure for number of viable cells.

Sample preparation for LC-MS
Cells were cultured in 6-well plates and subjected to various treatments. Metabolites were extracted following a brief saline wash. This extraction process involved scraping the cells on dry ice into a solution comprising 80% methanol and 20% water. Subsequently, the plates were incubated at -80 degrees for 15 minutes, and the resulting extracts were transferred to microcentrifuge tubes. Samples were incubated at 4 degrees with 800 rpm mixing followed by centrifugation at 14000 rpm for 10 minutes. Supernatants were collected and dried using a vacuum concentrator at room temperature. The resultant dry pellets were reconstituted in 30 μl of sample solvent (a mixture of water, methanol, and acetonitrile in a 2:1:1 ratio, v/v), and 3 μl was subjected to further analysis by liquid chromatography-mass spectrometry (LC-MS). The final results were normalized to the total protein content.
In the case of tissue analysis, frozen tissues were pulverized using a mortar in the presence of liquid nitrogen and maintained on dry ice. Approximately 10-20 mg of tissue powder was accurately weighed and subjected to extraction using cold 80% methanol and 20% water, following the same procedure as described for the cells. LC-MS signals for different metabolites were normalized to tissue weight.

Targeted LC-MS methods for NAD pathway metabolites
The methods for sample preparation and relative and absolute LC-MS measurements of unlabeled and isotopically labeled NAD pathway metabolites NAD, NAAD, NA, NAM, NAMN, NMN were as described under Targeted Analyses of Compounds from the NAD Pathway (NIEHS LC-MS) in 21. Similarly, NADP and ADPR measurements were made using the identical C18-reversed-phase method described previously21 with the additional narrow MS1 channels for NADP (MS acquisition range of m/z 735 – m/z 760 with an EIC drawn for the precursor m/z 744.083 and retention time of 2.3 minutes) and ADPR (MS acquisition range of m/z 555 – m/z 580 with an EIC draw for the precursor m/z 560.080 and a retention time of 2.0 minutes). The representative spectral peaks of these targeted measurements are provided in Supplementary Data 8.
Relative quantification of nicotinuric acid (NUA) (Supplementary Data 9) was performed separately using an Agilent 1200 Series HPLC coupled to a Thermo Q Exactive Plus mass spectrometer. An autosampler was used to inject the samples onto an Agilent Zorbax 300SB-C18 reversed-phase column (2.1 × 150 mm, 5-micron) using a 1.0 µL injection volume for each. Solvent A consisted of 10 mM ammonium acetate in H2O + 0.1% formic acid, and Solvent B consisted of methanol + 0.1% formic Acid. A flow rate of 50 µL per minute was used for the first 6 minutes of the run at 2.0% B solvent, followed by a gradient from 2.0–95.0% B from 6 to 20 minutes along with an increased flow rate of 150 µl per minute. The solvent was held at 95.0% B from 20 to 30 min and returned to 2.0% B for the remainder of the run. A HESI (heated electrospray ionization) source was used with positive polarity, a capillary temperature of 320 °C, the source voltage of 3.2 kV, S-lens RF level of 60, and a sheath gas flow rate of 16.0. One full scan from 100–900 m/z was performed at 17,500 resolution, followed by targeted MS2 scans at 35,000 resolution with an isolation width of 2.0 m/z. Normalized CID collision energy was set to 25. The total run time was 38 min. A blank was run in-between each sample to minimize and monitor for carryover.

Targeted LC-MS methods for NAD pathway metabolites in mouse plasma
5 µL mouse plasma was mixed with 5 µL 13C yeast extract, followed by the addition of 40 µL ice-cold methanol. The mixture was vortexed for 1 min, kept on ice for 10 min with occasional vortexing, and then centrifuged for 10 min at 20,000 g at 4 °C. The supernatant was transferred to an LC vial, and 2 µL was injected.
A Vanquish Horizon UHPLC system equipped with ACQUITY UPLC BEH Amide column (1 mm x 100 mm, 1.7 μm, Waters) was used for LC separation, coupled to Exploris 480 mass spectrometer (Thermo Fisher Scientific). LC mobile phase A was water with 5 mM ammonium acetate (pH 6.8), and mobile phase B was 100% Acetonitrile. The LC gradient was as follows: 0-1.5 min, 85% B; 5.5 min, 35% B; 10.5 min, 35% B; 10.6 min, 10% B; 12.5 min, 10% B; 13.5-20 min, 85% B. The flow rate was 40 µL/min, except at 13.5 min when it was increased to 70 µL/min to accelerate column re-equilibration. The column temperature was maintained at 25 °C.
For all metabolites, the following MS parameters were used: spray voltage, 3000 V; sheath gas, 18; auxiliary gas, 5; vaporizer temperature, 80 °C; ion transfer tube temperature, 300 °C; and S-lens, 30%. For Nicotinic acid (NA) and Nicotinamide (NAM) analysis, full-scan acquisition in positive mode was performed over an m/z range of 100–400 with a mass resolution of 120,000. Extracted ion chromatograms corresponding to m/z values of metabolites with 0, 3, or 4 deuterium incorporations (M+0, M+3, and M+4, respectively) were used for quantification. 13C-labeled NAM from 13C yeast extract was used as an internal standard for both NA and NAM due to structural and retention time similarity. To determine the concentration of 13C-labeled NAM in the yeast extract and to correct for differential ion responses between NA and NAM, standards of NA and NAM (0.16, 0.8, 4, and 20 ng/µL) were spiked into 5 µL 13C yeast extract, followed by methanol extraction and LC–MS analysis. Peak areas of extracted ions corresponding to NA, NAM, and 13C-labeled NAM were used to determine internal standard concentration and ion response factors. For nicotinic acid riboside (NAR) analysis, due to interference in the D4-NAR (M+4) channel at MS1 level, characteristic fragment ions (m/z 124.0392, 127.0581, 128.0643, and 130.0594) from MS2 spectra of NAR, D3-NAR, D4-NAR, and 13C-NAR precursors were used for quantification. MS2 data acquisition was performed with the following parameters: isolation window, 0.8 m/z; HCD collision energy, 30%; resolution, 30,000; and a targeted mass list of m/z 256.0816, 259.1004, 260.1067, and 267.1185. To determine the concentration of 13C-labeled NAR in the yeast extract, standards of NAR (0.16 and 0.8 ng/µL) were spiked into 5 µL 13C yeast extract, followed by methanol extraction and LC–MS analysis. Skyline was used for MS2-based quantification, while the FrameSeed function in SIEVE was applied for MS1-based quantification. LC-MS data and representative spectral peaks from this measurement are provided in Supplementary Data 10.

Untargeted Metabolomics
Pre-Analytical Materials and Methods
Sample Preparation
Pre-extracted, dried colon samples were removed from storage at -80°C, allowed to warm to room temperature, and resuspended via the addition of a range of 200 µL - 334 µL of water-acetonitrile 98%:2% v/v, normalized based on extract weights (ranging from 10.0 mg – 16.7 mg). Resuspended extracts were briefly vortexed (3 s) and then centrifuged for 10 minutes at 14000 rcf at 4°C (Eppendorf Centrifuge 5425R). 100 µL of supernatant of the resuspended extract, the soluble fraction of the extract, was transferred to 2 mL autosampler vial (12 mm x 32 mm height vial, 12 mm screw cap, and PTFE/silicone septa, Agilent) containing a glass microvolume insert (Agilent). The pellet (insoluble extract) was discarded in accordance with chemical and biological safety procedures. 30 µL of supernatant from each sample was pooled into a 2 mL autosampler vial for a quality control standard.
Analytical Materials and Methods
Untargeted Metabolomics: Vanquish – Tribrid Fusion
Instrumentation
Samples were analyzed using an ultra-high performance liquid chromatograph (VanquishTM Horizon UHPLC, Thermo Scientific) coupled to a high-resolution mass spectrometer (Orbitrap FusionTM Tribrid, Thermo Scientific). EASY-Max NGTM was used as the ionization source, operated in the heated-electrospray ionization (H-ESI) configuration. The source parameters in positive ionization mode were as follows: spray voltage of +4000 V, sheath gas of 50 arbitrary units (arb), auxiliary gas of 10 arb, sweep gas of 1 arb, ion transfer tube at 325°C, vaporizer at 350 °C. The source parameters used in negative ionization mode were identical, with the exception of the spray voltage of -3000 V. Prior to measurement, the mass spectrometer was calibrated using FlexMix (Thermo Scientific) following manufacture directions. EASY-IC™ (Thermo Scientific) was used during data collection; this is a secondary reagent ion source, yielding fluoranthene ions that are used as a lock mass to improve m/z accuracy by correcting for mass errors that result from variation in m/z measurement (e.g. scan-to-scan variation) and environmental changes (e.g. ambient temperature).
Chromatographic separation was carried out on a Kinetex F5 analytical column (2.1 inner diameter, 100 mm length, 100 Å, 2.6 µm particle size, Phenomenex) with corresponding guard cartridge. The column was maintained at 30°C during separation. The Vanquish solvent pre-heater was maintained at 30°C. Gradient elution was performed after an initial period of isocratic elution using water with 0.1% acetic acid v/v (A) and acetonitrile with 0.1% acetic acid v/v (B). Separation was performed as follows: 0% B from 0 - 2.0 min, 0% to 100% B from 2.0 to 10.5 min, 100% B from 10.5 to 12.0 min, 100% to 0% B from 12.0 to 13.0 min, 0% B from 13.0 to 20.0 min. The flow rate was 500 µL min-1. A 10 µL static mixer was used. The post column flow path consisted of a ViperTM connection (0.1 mm ID, 550 mm length, Thermo Scientific) from the column to a six-port value and PEEK tubing from the six-port value to the ionization source.
Data Acquisition: Liquid chromatography – mass spectrometry (LC-MS)
LC-MS data were collected from individual samples, system blanks, and a pooled quality control. System blanks consist of the same solvent used to resolubilize samples without biological material and are used to evaluate the chemical background of the results from the analysis system; system blanks were analyzed after every seven samples in the data acquisition order. The pooled quality control (QC) was generated by combining 30 µL aliquots from every sample in the data set into one pooled sample. Samples and system blanks were each injected at 4 µL; the QC pool was injected 9 times at different volumes (3 injections each at 2 µL, 4 µL, and 6 µL). MS data were collected with an anticipated LC peak width of 8 s and a default charge of 1. MS data were acquired at 120,000 resolution from m/z 100-1000 with an RF lens of 60% and maximum injection time of 50 ms.
Data Acquisition: Liquid chromatography – tandem mass spectrometry (LC-MS/MS)
Prior to acquiring LC-MS data for samples, liquid chromatography – tandem mass spectrometry (LC-MS/MS) data were acquired using the AcquireX (Thermo Scientific) deep scan methodology. The acquisition order for AcquireX was the following: system blank (n=1 injection) for exclusion list generation, QC (n=1 injection) for inclusion list generation, and n=7 injections of QC. IMS and MS/MS data were collected with an anticipated LC peak width of 8 s and a default charge of 1. MS data were acquired at 120,000 resolution from m/z 100-1000 with an RF lens of 60% and maximum injection time of 50 ms. MS/MS data were acquired at 30,000 resolution using an isolation width of 1.5 (m/z), stepped assisted higher-energy collision induced dissociation was used with energy steps of 20, 35, and 60 normalized collision energy, and a maximum injection time of 54 ms. The inclusion list was generated and updated via AcquireX with a low and high mass tolerance of 5 part-per-million (ppm) mass error. An intensity filter was applied with an intensity threshold of 2.0 x 104. Dynamic exclusion was used with the following parameters: exclude after n = 3 times; if occurs within 15 s; exclusion duration of 6 s; a low mass tolerance of 5 ppm mass error; a high mass tolerance of 5 ppm mass error; and excluding isotopes.
Data Processing
Processing of Raw Data – Feature Finding
Data files (.raw) were processed with Compound Discoverer 3.3.0.550 (ThermoFisher Scientific) to identify unique molecular features and, where possible, annotate them with chemical names. Features with distinct measured accurate mass, unique retention time, and MS/MS data were tabulated after removal of isotope peaks, blank contaminants, and noise artifacts from the data (workflow and parameters displayed in Supplementary Data 11). The table contained feature descriptors (e.g. m/z and retention time), annotation information (e.g. MS/MS database match), and peak area. Features were then processed using R via Jupyter Notebook. Processing steps included formatting of the table output from Compound Discoverer, comparison of m/z and retention time of annotated features versus an in-house generated list of m/z and retention time based on authentic chemical standards, assessment of signal response in pooled QC samples, assessment of signal variance in pooled QC samples versus samples (i.e. dispersion ratio), and multi- and univariate statistics.
Data Quality and Data Filtering
Signal Response Evaluation
We evaluated the signal response of the pooled sample with the intention of evaluating the fundamental principle of LC-MS/MS that if more of a given feature is present, a corresponding increase in the signal should be obtained. To do this, the pooled QC sample was injected and analyzed at three volumes in technical triplicate: 2 µL, 4 µL, and 6 µL, where 4 µL is the amount of material injected during the untargeted metabolomics assay in the present experiment. For any given feature, the peak area of the feature (i.e. the integration of a signal of unique m/z over a specific retention time) in the 4 µL sample is assumed to represent the mean value of that feature in the dataset; the peak area of that feature in the 2 µL sample and 6 µL sample should therefore represent 50% and 150% of the mean value, respectively. The metric used to evaluate the signal response was coefficient of determination (R2, evaluates fit to a linear model). For every feature in the dataset, a value of these metrics was calculated to evaluate the signal response for that feature over the QC range. Any feature for which the value does not meet the filtering parameter is filtered out of the dataset, ensuring that only features displaying positive correlations within the R2 = 0.7 parameter are retained for further evaluation and interpretation.
Assessment of Dispersion Ratio
The pooled QC was utilized to calculate the dispersion ratio for the sample set. The dispersion ratio is a metric for describing the measurement precision of a detected metabolite; the metric focuses on statistical dispersion of the pooled QC samples in relation to the dispersion of the biological test samples. If the distribution of both the biological test sample measurements and the QC random error are Gaussian, then the dispersion ratio (D-ratio) equal to 50 is the ratio of the sample standard deviation for the pooled QC samples to the sample standard deviation for the biological test samples. (If the data distribution is not Gaussian, then the raw data must be mathematically transformed.) D-ratio is a measurement of technical variance: a D-ratio of 0% means that the technical variance is zero (a “perfect” measurement), and all observed variances can therefore be attributed to a non-measurement (putatively biological) cause, whereas a D-ratio of 100% indicates that all variances can be attributed to the measurement.
Annotation
Features were annotated based on MS/MS spectral matching, in alignment with the Metabolomics Standards Initiative guidelines (Level 2). Prior to annotation, the data acquisition strategy AcquireX (Thermo Fisher Scientific) was used to improve coverage compared to similar data acquisition strategies (i.e. data-dependent acquisition). The public, commercial, and in-house MS/MS spectral libraries NIST2020, GNPS (accessed 04-01-2022), mzCloud (offline, endogenous metabolites) were used to evaluate MS/MS spectral matches, as well as an in-house MS/MS spectral library acquired from authentic chemical standards purchased and analyzed by the MCFS. The MS/MS spectral matching was performed in Compound Discoverer (Thermo RFisher Scientific).
During processing in the Jupyter Notebook, each feature was assigned an MSI level of confidence based on the measured data (without manual review). MSI Level 2 features have an MS/MS match to either a database entry or an in-house library reference match. MSI Level 4 and Level 5 features have either an MS/MS match but no database match, or no MS/MS match. MSI Level 2 features were promoted to MSI Level 1 after matching in-house m/z and retention time lists acquired on the same analytical platform using identical analytical conditions. The MSI Level 1 annotations are referred to as identified.
Abundance matrices of 277 annotated metabolites from positive and negative modes were combined (Supplementary Data 1) and used to generate PCA plot in MetaboAnalyst 6.0 without any filtering or normalization. For heatmap generation, 50 top differential annotated metabolites were used.

Flow cytometry analysis
Blood samples were collected in EDTA coated tubes. Red blood cells were lysed with ACK lysis buffer at room temperature for 10 min. The collected lymphocytes were incubated with anti-mouse CD16/32 at room temperature for 10 min to block the IgG Fc receptors. Expression of surface markers was detected by simultaneously staining with the following antibodies (eFluor 450 anti-mouse CD45, AF 700 anti-mouse CD4, FITC anti-mouse CD3, PE-Cy7 anti-mouse MHCII, BV711 anti-mouse CD8, PerCP-Cy™5.5 anti-mouse CD11b, APC anti-mouse CD19, BV510 anti-mouse CD11c, PE anti-mouse Ly6G, PE-Cyanine5 anti-mouse F4/80, APC/Cyanine7 anti-mouse Ly6C, PE-Dazzle 594 anti-mouse CD115) on ice for 30 min followed by wash and flow cytometry.
For flow cytometry analysis of colonic immune cells, colons were flushed with ice-cold PBS (Mg²⁺/Ca²⁺-free) and surrounding fat and mesentery were removed. Colons were then opened longitudinally and transferred to a 50 mL Falcon tube containing 20 mL ice-cold PBS. The tissue was shaken vigorously five times in fresh PBS until the intestines appeared clean and pale (white/pink). Colons were cut into 0.5–1 cm pieces and incubated in 10 mL of pre-digestion buffer (Mg²⁺/Ca²⁺-free HBSS with 2% FBS, 10 mM HEPES, 2 mM EDTA, and 1 mM DTT) at 37°C in a shaking incubator (250 rpm) for 15 minutes followed by three washes with cold PBS. Tissue was digested in 10 mL RPMI-1640 containing 5% FBS, 10 mM HEPES, 0.6 mg/mL Collagenase IV, 0.4 mg/mL Collagenase I, 1 mg/mL Dispase II, and 50 µg/mL DNase I at 37°C with shaking at 250 rpm for 15 minutes. After vortexing for 10 seconds, cells were filtered through a 70 µm strainer and collected in RPMI-1640 with 10% FBS and 10 mM HEPES on ice. The cell suspension was centrifuged at 500 × g for 5 minutes. Before flow cytometry, cells were diluted to 10⁶ cells per 100 μL and incubated with a blocking cocktail containing anti-mouse CD16/CD32 to prevent non-specific binding. Following blocking, cells were stained for surface markers described above for 10 minutes at room temperature.
Flow cytometric analysis was performed on BD LSRFortessa instrument (BD Biosciences) and analyzed using FACSDiva (BD Biosciences) software. All antibodies are listed in Supplementary Data 12. The FACS gating strategy is shown in Supplementary Fig. 10.

Analysis of publicly available expression and clinical data
To analyze the expression of NAD metabolic enzymes in IBD samples, RNA-seq dataset GSE111889 was utilized 42.
To analyze the expression of NAMPT and NAPRT in different human solid tumors in comparison with their respective normal tissues, TCGA data were downloaded from UCSC Xena web portal. The expression values of NAMPT and NAPRT are HTSeq FPKM-UQ normalized values. Normal tissue samples taken from tissue adjacent the tumor were used as normal tissue controls, and only a limited number of normal samples have been sequenced. Primary tumors matching adjacent normal tissues (some normal tissues might have two or more matched tumor samples) as well as all primary tumor samples with expression values were used in separate analyses. Based on the expression values in normal tissue samples, the expression of NAMPT or NAPRT in tumors were stratified into three categories: Below, in which tumor values were below the 25th percentile of the corresponding normal tissue data; Normal, in which tumor values were within the 25th to 75th percentile of the corresponding normal data; and Above, in which tumor values were above the 75th percentile of the corresponding normal data.
Similar results were obtained in matched primary tumors and all primary tumors (Supplementary Data 6).
To analyze the expression of NAMPT and NAPRT in colonic polyps in comparison with normal colonic tissues, single-cell RNA-seq datasets from Becker et al. 60 and Chen et al. 61 were downloaded from public resources. Chen’s dataset was obtained from the CELLxGENE collection (HTAN VUMC: a48f5033-3438-4550-8574-cdff3263fdfd) and further processed using the Scanpy Python package. Becker’s dataset was downloaded from GEO (GSE201348) and processed using the Seurat R package. For both single-cell datasets, mean-based, sample-level pseudobulk counts were aggregated from the raw cell counts and then normalized to CPM (counts per million). The CPM expression values for the genes of interest were retrieved and compared between polyp and normal samples.
For association of NAPRT expression and cancer patients’ survival, RNAseq expression and clinical data for TCGA PanCancer datasets of colon, liver and kidney cancers were downloaded from https://www.cbioportal.org/ 62, 63. Survival analysis was performed in GraphPad Prism v 9.5.1 using cutoffs of NAPRT expression that resulted in best separation of NAPRT-low and NAPRT-high samples for each dataset.

Statistics and Reproducibility
Values are presented as mean with either standard deviation (SD) or standard error of mean (SEM) from a minimum of three independent experiments or biological replicates, unless specified differently in the figure legend. To evaluate significant differences between the means, two-tailed, unpaired, non-parametric Mann-Whitney test was employed when sample size 4, while two-tailed, unpaired, Student’s t-test was utilized when sample size < 4 64. Statistical significance was set at p<0.05. Differences between the means with more than two comparison groups were analyzed by either Kruskal-Wallis test or two-way ANOVA with correction for multiple comparisons by controlling the false discovery rate and adjusted p-values (q-values) were reported. For all figures, *p (or q)<0.05, **p (or q)<0.01, *** p (or q)<0.001, **** p (or q)<0.0001, n.s., not significant. The exact p values are included in the Source Data file. Data analyses were performed using Prism Software 10.0 (GraphPad) or Microsoft Office Excel (Version 16.79.1). No methods were applied to ascertain whether the data met the assumptions of the statistical approach (e.g., normal distribution test).
For animal studies, the sample size in each independent experiment was determined based on our prior experience and mouse availability. The combined sample size for DSS-colitis study was estimated to achieve 2-fold difference of colitis phenotypes (tissue damage scores or rectal bleeding scores) with 80% of power. The sample size for other phenotypes (metabolic changes or tumor burden) was estimated to achieve 30% difference with 80% of power. For gene expression analysis, the sample size was estimated to achieve 2-fold difference of gene expression level with 80% of power. In vitro cell culture experiments were independently performed at least three times and similar results were observed. Each independent experiment was performed with at least three biological replicates, no explicit calculations were done to determine the sample size.

Data availability
The RNA-seq and scRNA-seq datasets of this study have been deposited to Gene Expression Omnibus under the following accession numbers:
RNA-seq dataset of the AOM experiment:
GSE271834 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE271834]
RNA-seq dataset of the DSS-colitis experiment:
GSE271250 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE271250]
scRNA-seq datasets of colon tissues:
GSE271836 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE271836]
scRNA-seq datasets of small intestinal tissues:
GSE261216 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE261216]

Metabolomics data of this study have been deposited to massive.ucsd.edu with the accession number MSV000100103, https://doi.org/doi:10.25345/C56M33H4M. The detailed data of 277 identified or annotated metabolites are provided in Supplementary Data 1.
LC-MS data for targeted measurement of mouse tissue NAD metabolites have been deposited to massive.ucsd.edu with the accession number MSV000100196, https://doi.org/doi:10.25345/C5639KJ8W. The representative spectral peaks are provided in Supplementary Data 8.
LC-MS parameters and representative spectral peaks from targeted measurement of NUA in mouse kidney are provided in Supplementary Data 9.
The LC-MS raw data files for NAD metabolites in mouse plasma have been uploaded to metabolomicsworkbench.org under tracking ID 6868 (NA and NAM) and 6875 (NAR), http://dx.doi.org/10.21228/M8BN9Z. The LC-MS data and representative spectral peaks are provided in Supplementary Data 10.
Public datasets used in this study are:
GSE111889 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111889]
GSE201348 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE201348]
HTAN VUMC [https://cellxgene.cziscience.com/collections/a48f5033-3438-4550-8574-cdff3263fdfd]
TCGA PanCancer datasets: https://www.cbioportal.org/
Source Data containing the exact p-values for all statistical tests are provided with this paper.

Code availability
Scripts used for scRNA-seq analyses in this study are available with no restriction at github: https:github.com/JohnXu24/NAPRTKO_scRNA. Published software packages and pipelines are used for RNA-seq and scRNA-seq analyses. Detailed description of these data analyses is included in Methods.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, Wu X et al. have revealed that Naprt deficiency promotes two major processes in colon tumorigenesis: tumor initiation (DNA damage) and promotion (inflammation). They propose that the increased DNA damage in the Naprt-KO mice is likely due to a reduced PARP activity associated with lower NAD levels in epithelial cells. Given these results, a phenotype that Naprt deficiency leads to an increased incidence of colon tumors is convincing. These are novel and interesting findings. However, there are two major concerns regarding this paper that will require many additional efforts, as outlined below;\nThe first concern relates to the inverse correlation between Naprt expression and prognosis in several cancers (Fig 6J-L). As noted above, data regarding increased tumor formation in Naprt-KO mice is convincing. However, this does not imply that tumors with Naprt deficiency exhibit higher malignancy or more therapeutic resistance. After diagnosis, the prognosis of cancer patients typically worsens with increasing malignancy and/or treatment resistance of tumor cells. Importantly, tumor cells with DNA repair defects often show higher sensitivity to chemotherapy and radiation therapy, which can extend patient survival. Thus, even if Naprt-low cancers show worse prognoses compared to Naprt-high cancers, it does not seem reasonable to attribute this to an impaired DNA repair ability associated with Naprt deficiency.\nAnother major issue is that this study overlooks recent important findings on systemic NA metabolism. In brief, NA absorbed from the intestine is actively converted to nicotinic acid riboside (NAR) NAPRT-dependently, then released into the circulation (Nomura, Nat Commun ’23. PMID: 38092728). Their paper reports that (1) the blood concentration of NA is much lower than that of NAR, and (2) Naprt deficiency significantly decreases the blood NAR levels in mice. These findings are critical to interpret the data presented in this paper. For example, in the culture experiments of this study, the authors added NA at a concentration of 100 µM, which is likely supraphysiological by a factor of 1000 or more in most organs. Direct fueling form the dietary NA or NA converted from Nam by gut microbiota may result in higher NA concentrations in the colon epithelium compared to the circulation. However, there is little direct evidence that NAD synthesis using NA as a substrate is active in other organs. Instead, it is questionable whether the proposed mechanism in this paper applies beyond the intestinal epithelium. Given the NA to NAR conversion, many of author’s claim in this paper remain speculative.\nBesides the two major issues mentioned above, the following points require significant attention:\nFigs 1-3: The authors added NA at a final concentration of 100 µM in many experiments in vitro. A theoretical basis for this concentration setting must be provided. Additionally, It would be better to present results with lower concentrations of NA for comparison.\nFig 2: The differences in PARylation and DNA damage between the parental and NAPRT-KO cells are relatively small. Therefore, add-back experiments should be conducted. Alternatively, experiments with multiple KO clones should be performed. Additionally, gH2AX levels should be examined by WB for a clearer comparison.\nFig 3: The NAD levels in various organs of Naprt-KO mice under steady-state conditions (without Nam load) should be reported.\nFig 4: The reason for administering additional NA through drinking water, given that NA is already provided in the dietary chow, should be clarified. Panels C-E are underwhelming, and most of them could be moved to supplementary items.\nFig 6G: The experiment appears to be very important, but the method section lacks essential details. Were comparisons made using littermates? Were events (e.g., mortality) monitored up to the time of inspection at 24 months? These are critical details for interpreting the data. Moreover, the results should be presented separately for male and female mice.\nThe following statements appear overly speculative or exaggerated:\nLine 160-1: “suggesting that NAD pool maintained by … … DNA repair.”\nLine 185: “in gut epithelial cells”\nLine 186-8: “despite accumulation of more severe DNA … … the surviving cells.”\nLine 213: “NAPRT-mediated deamidated NAD … … all tested tissues.”\nLine 218-20: “deamidated biosynthesis is a major route of … … and lung.”\nLine 296: “NAPRT deficiency is associated with an increased sensitivity to IBD.”\nLine 377: “NAPRT is believed to be oncogenic.”\nLine 394-5: “These results indicate that … … in both mice and humans.”\nThe reasoning behind the statements in lines 381-8 and lines 401-3 is difficult to follow.", "reviewer_2": "The study by Wu et al. explores how the Preiss-Handler pathway in NAD biosynthesis, mediated by NAPRT, impacts NAD metabolism, DNA repair, and tumorigenesis in the gut. Key findings highlight the role of NAPRT in sustaining NAD levels across tissues such as the liver, colon, and pancreas, and the consequences of NAPRT deficiency on NAD synthesis and tissue vulnerability to NAD-depleting stress.\nIn NAPRT KO mice, NAD synthesis is significantly compromised under DNA damage conditions, like those induced by AOM treatment, leading to inadequate metabolic and DNA repair responses, heightened inflammation, and increased gut cell damage. This dysfunction predisposes NAPRT KO mice to more severe colitis and tumorigenesis, resulting in a higher tumor burden, advanced adenomas, and upregulated inflammatory genes, underscoring the critical function of NAPRT-mediated NAD synthesis in safeguarding gut tissue from inflammation and cancer progression.\nThis is a very well written paper, that provides important insights into the role of the Preiss-Handler pathway in NAD biosynthesis, particularly in the context of NAPRT and its impact on DNA repair and tumorigenesis. However, it lacks novelty in certain aspects, as highlighted in major concerns, particularly point 5. The pioneering study on the role of NAD+ in tumorigenesis by Tummala et al. (2014) as well as the controversial work by Lee et al. (2022) should be cited, discussed, and integrated into the context of the manuscript, supported by experimental evidence. Additionally, several key issues outlined below need to be addressed before this manuscript is considered suitable for publication.Once these issues are addressed, this manuscript could make a valuable contribution to the field.\nMajor concerns\n1. The connection between NAD+ depletion and DNA damage is well-established, as shown in pivotal studies like Tummala et al. (2014) and Gomes et al. (2016), whose papers have not been cited but should. However, in their study, authors should mechanistically provide more insights into how NAPRT KO leads to DNA damage. This could result from decreased PARP activity, inhibition of Sirtuins, reduced dNTP synthesis, or perturbation in alkylation routes, all pathways where NAD+ plays a crucial role. To clarify this, the authors should measure the activities of PARP, Sirtuins, and dNTP synthesis in the NAPRT KO context. These assays would provide insight into the specific mechanisms by which NAD+ depletion leads to genotoxic stress and tumorigenesis in their model. Furthermore, performing rescue experiments, such as supplementing precursors to restore NAD+ levels or enhancing activity in the affected pathways, would significantly strengthen the study’s conclusions regarding the role of NAD+ metabolism in DNA damage and cancer progression. Rescue experiments with dNTPs, or using PARP inhibitors are also requested.\n2. A key concern is that while NAMPT depletion results in cell death, NAPRT KO does not induce such effects, in particular because the medium is supplemented with NAM. Likewise, NAPRT KO mice develop normally without obvious phenotypes, whereas NAMPT KO mice are embryonically lethal. The authors conclude that the deamidated NAD biosynthesis pathway is non-essential for survival, development, or normal cellular function. However, the underlying reason for this disparity remains unclear. Are NAD+ levels significantly more reduced to be completely abolished in NAMPT KO compared to NAPRT KO, thereby leading to extensive DNA damage, cell death, and catastrophic outcomes in NAMPT KO but not in NAPRT KO? Could it be that NAD+ depletion is less severe in NAPRT KO, allowing partial compensation through NAD+ synthesis from tryptophan catabolism? Notably QPRT is expressed in human colons and therefore this pathway might still be active despite the enzymes are not highly expressed in mice. Resolving these questions is crucial, and the study would benefit from comparative NAD+ quantification between NAMPT and NAPRT KO models, along with an analysis of compensatory metabolic pathways.\n3. The authors claim that their scRNA-seq analysis reveals distinct cell-type expression patterns for NAMPT and NAPRT in the small intestine and colon, with NAMPT being ubiquitously expressed across all cell types, particularly in gut immune cells, while NAPRT is relatively enriched in the epithelial compartments (Figure 1A and S1B). However, this is not entirely accurate, as NAPRT also appears to be expressed in immune cells. Thus, a complete NAPRT knockout could indeed affect NAD+ levels in immune cells, potentially impacting their functional role in reducing tumorigenesis. A conditional knockout mouse model would have been a more suitable approach to target specific cell types for this study. By restricting the NAPRT knockout to particular tissues or cell types, the authors could better assess the cell-type-specific effects of NAD+ depletion on tumorigenesis. Alternatively, the authors could utilize a xenograft or patient-derived xenograft model (that will give some clinicl relevance to the study), comparing NAPRT knockout with NAMPT knockout, to assess the tumor-inducing potential in immunodeficient mice.\n4. In line with the previous comment, the authors should provide a more comprehensive explanation of their results addressing inflammation, in particular when using the DSS-induced colitis models. Reduction of NAD+ levels in their Naprt KO model induces DNA damage and this is well-known to be linked to tumour formation, especially in epithelial cells, but the connection between the reduction of NAD+ levels with inflammation is not clear. Why the reduction of NAD+ levels leads to more inflammation? Which cells contribute to this enhanced sensitivity in their KO mice? Which are the cellular pathways important for this process? Perhaps an immune cell characterization in the presence of DSS in NAPRT KO mice should be provided via scRNA seq of isolated CD45+ cells. The conditional KO mouse model would have solved these questions. We do understand that this is not possible at this stage, but authors could explored alternative experiments such as PDX or/and xenografts studies in immunodeficient mice to circumvent this.\n5. A rescue experiment using nicotinamide riboside (NR) both in vitro (Figure 1) and in vivo to counteract the effects of NAPRT depletion on tumorigenesis would be highly valuable. For tumorigenesis, NR should be implemented in prevention and in therapy once tumors are formed to better understand the pro- or anti-tumorigenic effects of NAPRT KO. This experiment could provide direct evidence for the potential therapeutic role of NR in restoring NAD+ levels and mitigating the negative effects of NAD+ depletion on DNA repair, inflammation, and tumorigenesis. Including such data would significantly strengthen the manuscript by demonstrating the feasibility of NR as a potential intervention for restoring NAD+ homeostasis and reducing cancer progression in the context of NAPRT deficiency.\n6. While this study introduces the first NAPRT KO mouse model to explore the role of the Preiss-Handler pathway in tumorigenesis, the concept of NAD+ depletion in DNA damage and cancer is not novel. The authors overlook the foundational work by Tummala et al., published in Cancer Cell in 2014, which first demonstrated the significance of NAD+ depletion in tumorigenesis. Additionally, the study by Lee et al., published in Cancer Discovery in 2022, has been ignored. In their work, the authors showed that APC-deficient colorectal cancer models were susceptible to TDO2 depletion or pharmacological inhibition, leading to NAD+ depletion, impaired cancer cell proliferation, and enhanced antitumor immune responses. These important studies should be cited, discussed, and integrated into the context of the current manuscript through experimental evidence. One possible explanation to the study of Lee et al. is that since APC deletion drives cell proliferation, cells might rely on increased NAD+ from de novo synthesis to initiate DNA synthesis and repair pathways. Without the support of the NAPRT-NAD+ arm, APC-deleted cells may not survive. Therefore, it is crucial to measure NAD+ levels under different conditions in order to determine how the depletion of NAPRT affects cellular NAD+ metabolism, especially in the context of APC deletion. This would help clarify whether NAPRT-mediated NAD+ synthesis is essential for the survival and function of APC-deleted cells and further elucidate the role of NAD+ in DNA repair and cell survival under genotoxic stress.\n7. Figure 2D is difficult to understand, and the data interpretation lacks clarity. The authors claim that STF treatment sensitizes cells to MMS-induced cell death by comparing WT MMS+STF to WT MMS alone. However, when discussing the inactivation of deamidated synthesis in KO cells, the authors compare KO MMS to WT MMS instead, which seems inconsistent. According to Figure 2C, KO cells appear to have more DNA damage; therefore, in Figure 2D, MMS treatment alone would be expected to result in fewer colonies in KO cells compared to WT, but this outcome is not shown. Furthermore, if the comparison in KO cells were done similarly to WT cells—that is, MMS+STF vs. MMS alone—a similar observation (fewer colonies with STF treatment) would likely emerge. The precise methodology of this experiment, particularly the comparisons drawn between groups, needs more explanation.\nMinor points\n1. Some data described in the text is not shown in the figures (e.g. Figure 1 is missing to show cell morphology and proliferation rates).\n2. Some panels of the figures are not even mentioned in the text (e.g. Figure 2A). All data shown in the figures must be discussed.\n3. Figure legends contain explanations already present in the results section. They should be more descriptive and summarized.\n4. Full uncut immunoblots should not be added in the supplementary figures (Fig. S1D, Fig. S2B).\n5. In lines 141-142, the authors claim that ‘’ NAPRT deficiency impairs cellular metabolism and increases the sensitivity of gut epithelial cells to NAMPT inhibition induced NAD depletion and subsequence cell death’’. However, the authors should provide an experiment that shows that cell death occurs such as Brdu incorporation or stainings for apoptotic markers.\n6. The authors should consider changing the title of the manuscript since it does not align with the findings shown.\n7. Figure 3D, quantify the number of organoids\n8. The authors explain the observed phenotypes due to the accumulation of DNA damage due to NAD+ depletion. Therefore, DNA damage measures are key to support their conclusions. In this regard, authors rely on yH2AX staining quantified by intensity/cell. The use of additional DNA markers for DNA damage, beyond yH2AX, should be also included to further corroborate their results. In addition, measure of DNA damage in their NARPT KO aged mice is also requested.\n9. The authors should show the activation of DNA repair pathways in gut epithelial cells upon DNA damage.\n10. Microbiome or diet are the source of NA is their study? The relevance of the microbiome could be explored via antibiotics treatment to get insights into its role.", "reviewer_3": "The manuscript by Xiaoyue and colleagues “NAPRT-mediated deamidated NAD biosynthesis enhances tissue resiliency and suppresses tumorigenesis,” aims to investigate the tumour suppressing roles of the deamidated arm of NAD biosynthesis. To achieve that, they constructed NAPRT gene knock-out models and assess their effects using both in vitro (CRC119 cell lines and colonic organoids) and in vivo (mouse) systems. Here they found that removal of NAPRT gene altered metabolism such as glycolysis, increased DNA damage, impaired PARP activity, heightened sensitivity to inflammatory stress, and susceptibility to tumorigenesis in mouse models. Furthermore, reduced NAPRT expression correlates with poor prognosis in stage 1, not stage 3 and 4, CRC patients.\nThe manuscript is well-written, and the analyses are thorough and comprehensive. In my view, the findings presented in this manuscript are valuable and provide some insights into the mechanisms underlying colorectal cancer (CRC) development. Additionally, these insight may also extend to understanding precancerous conditions, such as polyps, offering a foundation for further research in this area.\nHowever, I have a few comments that I hope the authors will address to strengthen the manuscript further:\n1. The authors focus their study on gut, but occasionally deviate and discuss NAPRT expression in other organs without clear purpose or sufficient discussion, which may confuse the reader. For example, it is not clear why the author show NAD metabolites in liver instead of colon in figure 3A?\n2. For NAPRT/NAPRT expression in humans (Fig. 1A or Fig. 6I), it would be helpful to stratify the data based on previously published transcriptomic subtypes, such as CMS (https://doi.org/10.1038/nm.3967), IMF (https://doi.org/10.1038/s41588-022-01100-4), or MSI/MSS, to provide a more detailed understanding.\n3. It would also be interesting to investigate whether there is any dysregulation of NAPRT in the polyps. The authors could consider utilizing previously published polyp datasets, such as those from Chen et al. (Cell, Volume 184, Issue 26, 6262 - 6280.e26) or Becker et al. (Nat Genet 54, 985–995 (2022)), to explore this further.\n4. The mechanism behind the increase in NA following NAM feeding in the mouse model is unclear. The authors suggest that gut bacteria may convert dietary NAM to NA, but a similar increase in NA was also observed in other organs (Fig. 3C). Further explanation are needed.\n5. It is unclear why NA was used as the substrate in the in vitro experiments, while NAM was used in the in vivo studies. The authors may consider using NAM as the substrate in the in vitro system as well, which could also help address my previous question (point 4).\n6. Figure 4A, why NA level was decreased in KO mice after AOM treatment?\n7. Figure 4C:\na. Why was the group highlighted in the black box below are not discussed? This group appears to include genes that are highly expressed in both WT and KO at 12 hours but downregulated in WT at 48 hours.\nb. It would also be interesting to compare genes within the group across different time points (e.g., WT0 vs. WT12 vs. WT48) and then examine which pathways are altered in the KO\n8. Figure 5H: The author write in line 330-333 that NAMPT, NAPRT and NADSYN1 are differentially altered in IBD. However, figure 5H clearly shown only NAMPT are differentially altered. This results actually contradict their conclusion that \"NAPRT deficiency is associate with increased sensitivity to IBD\". Better explanation and support are needed.\na. The author may also be interested in checking the NAPRT expression in colon instead of intestine (https://doi.org/10.1038/s41586-019-0992-y).\n9. Figure 6I: The authors state in lines 387-388 that “TCGA datasets showed that the mRNA levels of NAPRT are reduced in a significant proportion of tumors,” which is inaccurate. Figure 6I shows that, in COAD, only 40% of patients show a reduction in NAPRT, while another 40% actually show an increase. This discrepancy is even more pronounced in the LUAD and LUSC datasets, where a larger proportion of patients exhibit increased NAPRT expression. This contradicts the authors' claim that reduced deamidated NAD biosynthesis is associated with tumorigenesis in both mice and humans (lines 394-395). A better explanation and additional support for this discrepancy are needed.\n10. The figure legends could be improved. Ex: it is unclear what is M+2, M+3, and M+4 in figure 3A and what is 0.5,1,1.5, and 2 in figure 5A means."}, "author_response": "Thanks for this insightful comment. We agree with the reviewer that we don’t have a clear mechanistic understanding why cancer patients with low NAPRT-low tumors have worse prognosis than patients with NAPRT-high tumors. To address the reviewer’s concern, we deleted the sentence “This observation in CRC supports the notion that NAPRT-mediated deamidated NAD biosynthesis is important for DNA repair, thereby suppressing tumor initiation” from the last paragraph of the Results section. We also modified the Discussion section and toned down the link of NAPRT and DNA repair in human patients.\nAnother major issue is that this study overlooks recent important findings on systemic NA metabolism. In brief, NA absorbed from the intestine is actively converted to nicotinic acid riboside (NAR) NAPRT-dependently, then released into the circulation (Nomura, Nat Commun ’23. PMID: 38092728). Their paper reports that (1) the blood concentration of NA is much lower than that of NAR, and (2) Naprt deficiency significantly decreases the blood NAR levels in mice. These findings are critical to interpret the data presented in this paper. For example, in the culture experiments of this study, the authors added NA at a concentration of 100 µM, which is likely supraphysiological by a factor of 1000 or more in most organs. Direct fueling form the dietary NA or NA converted from Nam by gut microbiota may result in higher NA concentrations in the colon epithelium compared to the circulation. However, there is little direct evidence that NAD synthesis using NA as a substrate is active in other organs. Instead, it is questionable whether the proposed mechanism in this paper applies beyond the intestinal epithelium. Given the NA to NAR conversion, many of author’s claim in this paper remain speculative.\nIf we understood the argument here correctly, the reviewer believed that based on the referenced study, NAR but NA is the major circulating deamidated NAD precursor after dietary supplementation of NA/NAM. Therefore, the reduced NAD synthesis we observed in organs other than the colon epithelium in NAPRT KO mice was due to reduced circulating NAR released from the colon, rather than deletion of NAPRT in these organs. Thereby, the reviewer concluded that “there is little direct evidence that NAD synthesis using NA as a substrate is active in other organs”.\nThis argument would hold true if only the data from the referenced paper were considered.\nHowever, our findings from the current study, along with those from a previous study, contradict this interpretation. First, our finding that NA was massively accumulated in all tested NAPRT KO mouse tissues (Figure 3C, S3F, and S3G) provided direct evidence that NAD synthesis using NA as a substrate is active in all these tissues. Moreover, the observation that robust NAAD and NAD biosynthesis in WT mice occurred without significant accumulation of NA, even at the earliest time point examined (except for the colon) (Figure 3C, NA), indicating that NAPRT-mediated NA assimilation is swift and highly efficient. Second, in our prior study 1, we analyzed systemic NAM/NR/NA/NAR metabolism. As shown in Figure 6D and Figure S5G of that paper, following oral NAM gavage, NAMN was the most abundant deamidated NAD precursor in both portal blood (12 pmol/mg) and inferior vena cava blood (8 pmol/mg), compared to 1.5 pmol/mg NA and 0.3 pmol/mg NR in inferior vena cava blood (Figure R1). Thus, in our experimental system, NAR is not the major circulating deamidated precursor. Yet despite the presence of various circulating deamidated precursors, NA accumulated in KO tissues beyond the intestinal epithelium, strongly indicating that NA serves as a main source for deamidated NAD biosynthesis in these tissues.\nFor the criticism on the concentration of NA we used in the cell culture system, please see our response below.\nBesides the two major issues mentioned above, the following points require significant attention:\nFigs 1-3: The authors added NA at a final concentration of 100 µM in many experiments in vitro. A theoretical basis for this concentration setting must be provided. Additionally, It would be better to present results with lower concentrations of NA for comparison.\nIndeed, the concentration of NA in the serum of mice on regular chow diet is around 1 µM NA 1. However, it has been reported that this concentration goes up to 50-100 µM 8 hours after oral gavage with 500 mg/kg NR 2. Please note that we and others have shown that NR is degraded in the gut to NAM, which is then deamidated by the gut microbiota to NA 1, 2. Moreover, we have previously showed that in the colon, the NA concentration reaches 400 µM after gavage with 80 mg/kg NAM 1. Thus, 100 µM of NA in our culture system is well within the physiological range after supplementation with NAD boosting supplements.\nTo further address the reviewer’s concern, we performed a dose-response experiment, which revealed a biphasic response of NAD levels to increasing NA concentrations with 12.5-25 µM NA resulting in maximal increase in cellular NAD (Figure S2D). We then repeated the DNA damage experiment shown in Figure 2C, but now using 20 µM instead of 100 uM NA. We confirmed that NAPRT KO cells also have higher basal and MMS-induced DNA damage in the presence of this lower NA concentration (Figure S2E and new Figure 2B, see below).\nFig 2: The differences in PARylation and DNA damage between the parental and NAPRT-KO cells are relatively small. Therefore, add-back experiments should be conducted.\nAs suggested by the reviewer, we performed add-back experiments by transiently overexpressing NAPRT in two independent NAPRT KO clones. Cells were then treated with MMS for 1 hour and co-stained for NAPRT and gH2AX. As shown in Figure S2F and 2D, in both clones DNA damage was significantly lower in NAPRT-positive than in NAPRT-negative cells, confirming the genuine role of NAPRT in DNA repair.\nAlternatively, experiments with multiple KO clones should be performed. Additionally, gH2AX levels should be examined by WB for a clearer comparison.\nThe WT and NAPRT KO cells we used in the original Figure 2 are mixture of two independent clones each. We apologize that we forgot to mention this in the Methods section. Additionally, the PARylation results in Figure S2B are from two independent clones.\nTo further address the reviewer’s concern, we optimized the timeframe of MMS treatment and analyzed Mono-ADP-ribosylation (MARylation) and gH2AX by western blotting in two independent KO clones separately. Cells were supplemented with 20 µM NA to address reviewer’s request to repeat some experiments with a lower NA concentration. Similar to our results on PARylation (Figure S2B and S2C), auto MARylation of PARP1 (upper band) and MARylation of histones (lower band), which was recently shown to be important for efficient DNA repair 3, were less induced in both KO clones (Figure 2B), demonstrating reduced induction of PARP activity. The results further confirmed that NAPRT KO cells have increased gH2AX after MMS treatment in both KO clones, providing an additional piece of clear evidence that NAPRT KO cells have defective DNA repair thereby accumulating DNA damage after MMS treatment.\nPlease note that in the revision, the new MARylation and gH2AX results are shown in new Figure 2B, and the original PARylation data are moved into supplementary figures as Figure S2C.\nFig 3: The NAD levels in various organs of Naprt-KO mice under steady-state conditions (without Nam load) should be reported.\nWe added a new Figure S3D showing that without dietary supplementation of NAD precursors, the steady-state tissue NAD levels were normal in all tested tissues of NAPRT KO mice. As stated in our response to the next point, we believe this is due to compensation from amidated and/or de novo pathways.\nFig 4: The reason for administering additional NA through drinking water, given that NA is already provided in the dietary chow, should be clarified.\nThe reason why we supplemented experimental mice with NA in this experiment is because the chow diet (NIH-31) in our animal facility contains a very low level of NA (0.0083%) but a high level of Trp (0.20%), the precursor for de novo NAD biosynthesis. Given that hepatocytes, which have high expression of Qprt (Figure S1B), are the main producers of circulating NAM from NAD (>95% of total circulating NAM) and that circulating NAM serves as the primary NAD precursor for most tissues in vivo 4, it is likely that hepatic de novo NAD biosynthesis from Trp compensated for the defective deamidated NAD synthesis in NAPRT KO mice fed the chow diet. Additionally, although the expression of Qprt in mouse intestinal epithelial cells is very low under normal conditions (Figure 1A), we were concerned that it might be activated by increased NAD consumption upon AOM-induced DNA damage, potentially masking the deficiency caused by NAPRT deletion. We therefore decided to supplement NA to increase the dependence of experimental mice on the deamidated pathway.\nPanels C-E are underwhelming, and most of them could be moved to supplementary items.\nWe agree with the reviewer, therefore moved Figure 4E into Figure S5D.\nFig 6G: The experiment appears to be very important, but the method section lacks essential details. Were comparisons made using littermates? Were events (e.g., mortality) monitored up to the time of inspection at 24 months? These are critical details for interpreting the data.\nThe WT and NAPRT KO mice were originally littermates. After being backcrossed into the C57BL/6J background for four generations, they were bred separately within each genotype to produce sufficient experimental animals within the available space for this study.\nFor two cohorts of mice analyzed in this specific experiment, their health status and mortality were regularly monitored by the institute veterinary medicine staff during the two-year period of the study. But unfortunately, due to the renovation of the animal facility during this period, they were transferred multiple times between rooms, and we were not able to locate their health status and mortality records. The only data we have for these two cohorts is that at the time of inspection at 24 months, 6 out of 17 WT and 12 out 17 KO female mice were removed/lost, and 11 out 23 WT and 8 out of 27 KO male mice were removed/lost due to health issues/mortality. This result suggests that female NAPRT KO mice may have increased mortality. Indeed, analyzing another two ongoing independent aging cohorts confirmed that female but not male NAPRT KO mice had significantly reduced symptom-free survival compared with age-matched WT females (new Figure 7G). Among those females removed/lost from the aging cohort, 5 out 16 KO were due to visible large abdominal mass or rectal prolapse, whereas none of 6 removed/lost WT displayed these tumor-related symptoms.\nMoreover, the results should be presented separately for male and female mice.\nThanks for the suggestion. We re-graphed the original tumor incidence data and presented male and female mice separately and showed them in new Figure 7H.\nThe following statements appear overly speculative or exaggerated:\nLine 160-1: “suggesting that NAD pool maintained by … … DNA repair.”\nThis statement was deleted in the revision.\nLine 185: “in gut epithelial cells”\nDeleted in the revision.\nLine 186-8: “despite accumulation of more severe DNA … … the surviving cells.”\nWe hope that our new data will convince the reviewer that DNA damage is indeed more severe in NAPRT KO cells. We also now provide additional evidence that a “comparable fraction of NAPRT-deficient gut epithelial cells survives the stress” using Cell-titer GLO assay after 72 hours of continuous MMS treatment (Figure 2F, ATP (72 hours)). We believe that we were careful enough at this early point in the paper to qualify our hypothesis that increased DNA damage coupled with equal cell survival may be “potentially leading to increased mutagenesis and tumorigenesis within the surviving cells.” We use this hypothesis as a setup for the following cancer-related experiments and analyses. We are glad that the Reviewer found our results on increased tumorigenesis in NAPRT KO mice in 3 different models (AOM-DSS, APC, spontaneous aging) convincing. Since we do not provide direct evidence on increased mutagenesis, we deleted “mutagenesis” from this sentence.\nLine 213: “NAPRT-mediated deamidated NAD … … all tested tissues.”\nPlease see our response to the second major concern. This is an accurate statement.\nLine 218-20: “deamidated biosynthesis is a major route of … … and lung.”\nPlease see our response to the second major concern. This is an accurate statement.\nLine 296: “NAPRT deficiency is associated with an increased sensitivity to IBD.”\nWe changed the title of this section to “NAPRT deficiency is associated with an increased sensitivity to DSS-induced colitis.”\nLine 377: “NAPRT is believed to be oncogenic.”\nThis statement was changed into “NAPRT is found to be frequently amplified and/or overexpressed in various human cancer types and its knockdown slowed growth of xenograft tumors, suggesting tumor-promoting role of this gene 5, 6.”\nLine 394-5: “These results indicate that … … in both mice and humans.”\nThis statement was deleted in the revision.\nThe reasoning behind the statements in lines 381-8 and lines 401-3 is difficult to follow.\nWe modified the statement in lines 381-8 into “In support of this hypothesis, AOM/DSS-induced tumorigenesis in WT mice was coupled with reduced expression of key deamidated NAD biosynthesis genes (Figure 8A). Compared to normal colon tissues from age- and gender-matched untreated mice, tumor adjacent colon tissues from AOM/DSS treated WT mice had significantly reduced expression of both Naprt and Nadsyn but not Nampt (Figure 8A, Adjacent vs Normal). The mRNA levels of these two key deamidated NAD biosynthesis enzymes were further reduced in AOM/DSS-induced tumors (Figure 8A, Tumor vs Adjacent).”\nThe statement in lines 401-3 was deleted."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This manuscript is a revised version of a paper I previously reviewed. In this revision, several issues have been addressed and improved. However, I believe there remain critical concerns, particularly regarding aspects of systemic nicotinic acid (NA) metabolism, that require further revision.\nMajor points\n1. In my previous review, I pointed out that there is a Nat Commun paper (Nomura M, PMID: 38092728) demonstrating that NA is converted to NAR in a NAPRT-dependent manner. During this revision period, an additional study reported NA-to-NAR conversion in mice (Song WS, Cell Metab ’25, PMID: 40315855). Thus, two independent groups have now shown that NA can be converted to NAR. Nevertheless, the present manuscript does not mention or consider this conversion pathway at all. I believe this omission constitutes a significant limitation.\n2. The authors state that the NA concentration used in the in vitro experiments (100 uM) is comparable to the peak serum concentration following NAD booster administration, as reported in the previous study (Chellappa K, Cell Metab ’22, PMID: 36476934). While this is factually correct, the study also reported that steady-state levels of NA (in the absence of NR administration) are very low. Omitting this point could lead readers to overestimate physiological NA concentrations.\n3. To allow for a more accurate and reliable interpretation of the metabolic data presented in this study, I strongly recommend that the authors include, at a minimum, plasma or serum (not whole blood) concentrations of NAD-related metabolites in WT and Naprt-KO mice, both at both at baseline (prior to NA administration) and at multiple time points following NA administration. Blood sampling should preferably be performed from the systemic circulation, rather than the portal vein, to better reflect whole-body metabolism. In this context, blood collection from the right ventricle is considered the most appropriate.\nMinor points.\n4. In Figures 1 and 2, the in vitro experiments were conducted using only one colorectal cancer cell line, CRC119. The authors should be more cautious in generalizing the results to normal “gut epithelial cells” as a whole. In addition, more information about CRC119 should be provided in the Methods section; at present, even the species is not indicated.\n5. In Figure 2D, the results of the add-back experiment are not clear. Such experiments should be performed using a stable expression system, rather than transient transfection.", "reviewer_2": "At the outset, I was indeed very positive about this manuscript, under the condition that my initial comments were thoroughly addressed. While the paper remains a very well-written and insightful contribution, providing important advances on the role of the Preiss–Handler pathway in NAD biosynthesis, particularly the function of NAPRT in DNA repair and tumorigenesis, it is concerning that some of my key points appear to have been overlooked.\nSeveral other key issues outlined below remain unaddressed. I strongly recommend that these be carefully revised and clarified before the manuscript can be considered suitable for publication. If adequately addressed, this work could indeed represent a valuable contribution to the field.\nPoint 1\nIn particular, as highlighted in my Major Concern, the pioneering study by Tummala et al. (2014) on the role of NAD⁺ in tumorigenesis, along with the more recent and controversial work by Lee et al. (2022), should be not only cited—which the authors have done—but also properly discussed and integrated into the conceptual framework of the manuscript. These citations currently lack critical engagement.\nI respectfully disagree with the response provided to Comment 1. The authors argue that their study is mechanistically distinct from the work of Tummala et al. and Gomes et al., both of which demonstrated that NAD⁺ depletion leads to DNA damage, likely through PARP inactivation or reduced dNTP synthesis. While the authors emphasize that their study centers on NAPRT and its role in DNA repair, they also conclude that NAPRT knockout cells, which have compromised NAD⁺ levels, exhibit defective DNA repair and accumulate DNA damage upon MMS treatment.\nIn my view, the underlying message of these studies remains fundamentally aligned. Therefore, a proper and critical discussion of these prior findings is necessary. Without it, the current manuscript falls short in demonstrating sufficient novelty.\nMechanistic insights into how NAPRT deficiency leads to DNA damage remain insufficiently resolved. Although the authors now demonstrate reduced PARylation and MARylation in NAPRT KO cells, this evidence essentially confirms the role of PARP in their phenotype, thereby aligning their study more closely with previously published findings. Similarly, while SIRT1's role remains unclear in their system, and dNTP synthesis appears largely unaffected. As established in seminal studies such as Tummala et al. (2014) and Gomes et al. (2016), NAD⁺ depletion can lead to DNA damage through several converging pathways, including PARP inactivation, Sirtuin inhibition, and reduced dNTP synthesis, all of which are potentially relevant in this context. These studies should be properly cited and integrated into the manuscript’s conceptual framework.\nTherefore, I respectfully maintain that the manuscript would benefit from a more rigorous and integrative discussion acknowledging how their findings intersect with existing knowledge and litteratire. The notion that NAPRT-derived NAD⁺ is essential for DNA repair in gut epithelial cells is interesting; however, the message conveyed converges with previously published observations about NAD⁺ depletion and genome instability. This convergence must be transparently acknowledged and discussed to better contextualize the contribution of this work and to address concerns around novelty.\nIt is unclear why the authors have chosen to omit citation and discussion of previous pioneering studies that have significantly shaped the understanding of NAD⁺ metabolism and its role in DNA damage and tumorigenesis.\nPoint 2\nThe authors fairly replied to this point: Orally administered NR and NMN are largely converted by gut microbiota into nicotinic acid (NA), which then enters the Preiss-Handler pathway requiring NAPRT for NAD⁺ synthesis. In NAPRT knockout (KO) models, this pathway is disrupted, preventing NAD⁺ restoration from NR/NMN, as seen in both in vitro (e.g., CRC119 cells) and in vivo studies.\nDespite this, NR and NMN remain widely used because, in organisms with functional NAPRT and normal gut microbiota, they effectively boost NAD⁺ levels and provide physiological benefits. Thus, the efficacy of NR/NMN depends on the presence of NAPRT and highlights the critical role of the Preiss-Handler pathway in their metabolism.\nYet, dietary levels of tryptophan may be sufficient to compensate for NAPRT deficiency in many tissues. This has not been tested. A rescue experiment can still be important.\nPoint 3\nThis is justified by the authors stating, “We agree that conditional NAPRT KO in different cell types would be more informative. However, as the reviewer noted here, such studies are beyond the scope of the current revision.”\nThis is inaccurate; the reviewer never claimed it was beyond the scope but explicitly requested this experiment. Nevertheless, the xenograft studies presented do not fully support the paper’s conclusions, and conditional KO mouse models would be important to address this gap.\nOther important points\nI regret to note that γH2AX, the standard marker for DNA damage, has not been successfully demonstrated in this study, despite its widespread and well-established use in numerous publications. It is unclear why evidence of DNA damage is not provided or visible here.", "reviewer_3": "The authors have addressed all my concern"}, "author_response": "To answer the reviewer’s question about whether NA or NAR is the main circulating deamidated NAD precursor beyond the intestinal epithelium during the first revision, we provided evidence that NA is the main source for deamidated NAD biosynthesis in various tested mouse tissues. However, we did not realize that the reviewer would also like to see our measurement on the conversion of NA to NAR. We apologize for the oversight.\nTo address this concern as well as Point #3, we orally gavaged D4-labeled NA into WT and NAPRT KO mice, then followed the kinetics of NA, NAR, and NAM in systemic plasma. As shown in the new Figure S3I, the baseline concentration of NA (M+0) in plasma of mice under the regular chow diet, which was reported to be around 1 µM by Chellappa et al. 1, was below the detection limit of our platform (1-2 µM). One hour after oral gavage of D4-NA at 80 mg/Kg, plasma NA levels rose to 278±73 µM in WT and 824±217 µM in KO mice, indicating reduced NA utilization in NAPRT KO mice.\nNA levels dropped below the detection limit by three hours after gavage (Figure S3I, NA).\nSupporting the reviewer’s note that NA can be converted into NAR in an NAPRT-dependent manner 2, we observed a significant increase in plasma NAR levels (from 0.028± 0.007 µM to 0.202± 0.007 µM) in WT mice one hour post-gavage, and this increase was almost completely abolished in KO mice (Figure S3I, NAR). Additionally, D4-NA gavage elevated plasma levels of both M+0 and M+3 NAM, peaking between 1- 3 hours in WT but not in KO mice (Figure S3I, NAM), suggesting increased NAD consumption following NA-induced NAD boosting in WT mice.\nBecause the peak NAR concentration we observed in WT mice (~0.2 µM) is much lower than levels (1-2 µM) reported by recent studies 2, 3, we repeated the experiment using 400 mg/Kg D4-NA as in Song et al. 3. Even then, plasma NAR levels remained below 0.3 µM three hours after gavage, in contrast to 540 µM of NA (Figure S3I, right panels).\n2. The authors state that the NA concentration used in the in vitro experiments (100 uM) is comparable to the peak serum concentration following NAD booster administration, as reported in the previous study (Chellappa K, Cell Metab ’22, PMID: 36476934). While this is factually correct, the study also reported that steady-state levels of NA (in the absence of NR administration) are very low. Omitting this point could lead readers to overestimate physiological NA concentrations.\nThe reviewer is correct that the baseline concentration of NA in plasma of mice under the regular chow diet without supplementation was reported to be around 1 µM by Chellappa et al. 1. We agree with the reviewer that even the reduced concentration of 20 µM NA, which we used in the first revision to address this criticism, is still significantly higher than 1 µM. In response to this point, we used 1 µM NA in the new NAPRT add-back MMS experiment requested in point 5.\nAs shown in new Figure 2E, the higher DNA damage in NAPRT KO cells was also observed with this physiologically relevant NA concentration. Stable re-expression of HA-mNAPRT rescued this defect, confirming the genuine role of NAPRT in DNA repair. Our results further suggest that NAPRT-mediated NAD synthesis may be important for maintenance of homeostatic DNA repair.\n3. To allow for a more accurate and reliable interpretation of the metabolic data presented in this study, I strongly recommend that the authors include, at a minimum, plasma or serum (not whole blood) concentrations of NAD-related metabolites in WT and Naprt-KO mice, both at both at baseline (prior to NA administration) and at multiple time points following NA administration. Blood sampling should preferably be performed from the systemic circulation, rather than the portal vein, to better reflect whole-body metabolism. In this context, blood collection from the right ventricle is considered the most appropriate.\nWe performed the experiment requested by the reviewer and the new data are included in new Figure S3I (please see our response to Point #1).\nMinor points.\n4. In Figures 1 and 2, the in vitro experiments were conducted using only one colorectal cancer cell line, CRC119. The authors should be more cautious in generalizing the results to normal “gut epithelial cells” as a whole.\nIn addition to CRC119 cancer line data, the conclusion about the increased sensitivity of NAPRT KO to DNA damage is based on increased gH2Ax staining of normal gut epithelial cells before and after in-vivo AOM treatment of mice (Figure 4D). However, as requested by the reviewer, since our mechanistic studies were performed in CRC119 cells and presented on a separate figure than the in vivo data, we changed “NAPRT-deficient gut epithelial cells” to “NAPRT-deficient cells“ when describing the results on Figure 2.\nIn addition, more information about CRC119 should be provided in the Methods section; at present, even the species is not indicated.\nWe did mention in the main text (page 7) that CRC119 is a human colon cancer cell line. We provided a reference to the paper describing the derivation of this cell line 4 and added this information also in the method section in this revision.\n5. In Figure 2D, the results of the add-back experiment are not clear. Such experiments should be performed using a stable expression system, rather than transient transfection.\nPer the reviewer’s request, we stably expressed HA-mNAPRT in KO cells and performed MMS experiment using a more physiological 1 µM NA concentration in response to Point #2. The new data are shown in new Figure 2E (please see our response to Point #2).\nReviewer #2 (Remarks to the Author):\nPoint 1\nWe apologize that we did not provide a rigorous and integrative discussion to acknowledge previous pioneering studies on NAD, DNA damage, and tumorigenesis in our first revision. In this revision, we added the following paragraph in the Discussion section:\n“The importance of NAD in supporting DNA repair and maintaining genome stability has been well-established in the literature (reviewed in 5, 6). Consistently, pathological conditions that lead to NAD depletion, such as aging, high-fat diet feeding, and oncogenic gene expression, have been reported to increase DNA damage and promote tumorigenesis through various NAD-dependent mechanisms, including PARP inactivation, sirtuin inhibition, and reduced dNTP synthesis. For instance, a seminal study by Tummala et al. showed that in hepatocytes, suppression of the de novo NAD biosynthesis pathway by expression of an oncogene unconventional prefoldin RPB5 interactor (URI) leads to reduced cellular NAD levels and increased DNA damage at early stages of tumorigenesis possibly by inhibition of PARP 7. Restoration of hepatic NAD pool with NR supplementation prevents DNA damage and tumor formation 7. In a following-up study, Gomes et al. reported that URI-induced inhibition of de novo NAD biosynthesis and subsequent DNA damage also trigger hepatic T cell infiltration and increase IL-17A production, which further promotes non-alcoholic steatohepatitis and hepatocellular carcinoma 8. These defects could also be abolished by dietary supplementation of NR 8. In the present study, by utilizing a genetic NAPRT KO model that enabled us to investigate the role of the Preiss-Handler arm of the deamidated NAD biosynthesis during tumor initiation, we demonstrated that in the gut epithelium, deficiency of this pathway impairs PARP activity and compromises DNA repair, thereby increasing susceptibility to inflammation and tumorigenesis. To our knowledge, this is the first study to identify endogenous NAPRT as a tumor suppressor rather than a tumor promoter.”\nWe hope this addition addressed the reviewer’s concern.\nPoint 2\nWe are sorry that we are not sure what type of “rescue” experiment the reviewer is requesting here.\nIf \"a rescue experiment” refers to tryptophan (Trp) supplementation, we are concerned that such an experiment may not yield meaningful outcomes. The conversion of Trp to NAD is only one of several Trp metabolic fates that requires eight metabolic reactions, and the ratio of conversion is low (e.g. ~ 60:1 in humans) 9, 10. This conversion is expected to be particularly limited in the gut, where QPRT expression is minimal (Figure 1A). Indeed, in a recent report published in Cell Metabolism, Song et al. showed that Trp only boosts NAD levels in the liver and kidney 3. Since our study is focused on the gut, a rescue experiment with Trp supplementation is not feasible.\nAlternatively, if the reviewer’s intention is to ask us to test whether “dietary Trp compensates for NAPRT deficiency in many tissues”, then we could potentially place NAPRT KO mice on a Trp-deficient diet so that any possible compensations from de novo NAD synthesis will be unmasked. However, the NAMPT-mediated amidated NAD salvage could very likely compensate loss of both NAPRT deficiency and inhibition of de novo pathway, masking any effects from Trp restriction. Moreover, Trp is an essential amino acid with broad roles beyond NAD synthesis, including protein biosynthesis and generation of multiple bioactive metabolites 11. It also has extensive interactions with gut microbiota and Trp-deficient diet feeding has been shown to induce intestinal inflammation and many other adverse effects 12. These NAD-independent effects would significantly complicate interpretation of any observed phenotypes.\nGiven these factors, we are unsure how Trp rescue and/or restriction experiments would yield interpretable or meaningful results.\nPoint 3\nWe apologize that we are not able to generate a conditional KO mouse model within a reasonable time-frame to meet this request.\nIn terms of the xenograft results, we respectfully disagree with the reviewer’s interpretation. As previously noted (in the first revision), using NAPRT KO mouse model, we found that impaired deamidated NAD biosynthesis in NAPRT deficient cells leads to diminished PARP activity and compromised DNA repair in response to carcinogen-induced DNA damage. This defect in DNA repair increases the rate of oncogenic mutations, thereby promoting cancer initiation. Established cancer cell lines used in the xenograft experiment, on the other hand, are not suitable to study oncogenic mutation-associated cancer initiation because they have already accumulated all the mutations needed for malignant growth. Further inhibition of DNA repair by deletion of NAPRT in these cell lines is not expected to further enhance their growth rate. It can instead decrease the growth rate of xenograft tumors due to excessive DNA damage and reduced metabolic fitness. This outcome was supported by results from our xenograft experiment as well as by previous published findings 13.\nWe would like to again emphasize that our study is the first to demonstrate that NAPRT can function as a tumor suppressor rather than a tumor promoter. This novel finding was made possible by our use of a newly developed genetic NAPRT KO model, which enabled us to investigate the effects of NAPRT deficiency during tumor initiation, in contrast to prior studies using xenograft models based on established cancer cell lines. In fact, our observations that NAPRT deficiency increased tumor initiation in mice but reduced tumor growth in established cancer cell lines reveal an intriguing stage-specific role of NAD in cancer: suppressing tumor initiation at early stages while supporting tumor growth and progression at later stages. We believe this finding offers valuable guidance for the development of future NAD-based strategies in cancer prevention and treatment.\nOther important points\nI regret to note that γH2AX, the standard marker for DNA damage, has not been successfully demonstrated in this study, despite its widespread and well-established use in numerous publications. It is unclear why evidence of DNA damage is not provided or visible here.\nWe are perplexed by the comment here because we believe that our data in Figures 2B, 2C, 2D, new 2E, 4D, S2E, and S2F clearly showed increased γH2AX in NAPRT KO cells and mice following genotoxic stress. Particularly, our new NAPRT add-back experiment (Figure 2E) further validates the authentic role of NAPRT in DNA repair at physiological levels of NA (1 µM) as demonstrated by higher γH2AX in KO cells and rescue of this defect by NAPRT add-back."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I appreciate that in this revision the authors have addressed the concerns I previously raised. However, there remains one important issue that should be resolved before publication. I apologize for not noticing it earlier, but I believe it is critical for the validity of the study.\nThis concerns the mutant mouse line referred to as “Naprt-KO” in the manuscript. Typically, genome-editing–based gene disruption involves a frameshift mutation, which leads to nonsense-mediated mRNA decay and thus a marked reduction in transcript levels. However, as the authors noted, the allele in this mouse carries an in-frame deletion of seven amino acid residues (Fig. S3A), which would not normally cause a severe decrease in mRNA levels as reported in Fig. S3B.\nAlthough several functional studies in the paper suggest a loss of Naprt activity, it remains essential to confirm that Naprt protein is indeed absent in this mutant line. This could be demonstrated, for example, by Western blotting of tissue lysates. Such verification would clarify whether the allele truly represents a null mutation.\nI am aware that the deletion may have disrupted an exon splicing enhancer (ESE), potentially leading to cryptic splicing; however, this possibility further underscores the need to verify the actual loss of Naprt protein.", "reviewer_2": "Although I was initially very critical of certain experiments and aspects of the discussion, the authors have addressed all my concerns. I am satisfied with their responses and the revisions provided.\nOne minor point is that the authors should discuss the limitation of the study arising from the use of xenograft models, rather than the use of conditional NAPRT knockout in different cell types.\nWith this minor suggestion, I recommend that this paper be accepted for publication."}, "author_response": "We are sorry that our data in Figure S3B confused the reviewer. We have noted in the figure legend that this qPCR analysis was done using WT specific primers, which were designed to specifically amplify the full length Naprt mRNA containing the 21-bp fragment that is present in the WT but not in the mutant allele. The aim of this verification qPCR is to confirm the deletion of the 21-bp in the Naprt KO cells and tissues. The sequences of these WT specific primers have already been included in Table S7 (mNAPRT genotyping-F and mNAPRT genotyping-R). We have another set of qPCR primers of mNAPRT, which are specific for Exon 1 of Naprt gene, for regular qPCR analysis. Their sequences are also included in Table S7 (mNAPRT-F and mNAPRT-R).\nTo present our figures and results more clearly to the reader, we have made the following modifications in this revision:\n(1) We labeled the positions of the aforementioned four primers in Figure S3A (new Supplementary Figure 3a) in green.\n(2) We conducted additional qPCR analysis in primary cells and tissues from WT and NAPRT KO mice using both primer sets to show that while the genotyping (GT) primer set failed to amplify the mutant Naprt mRNA in cells/tissues from NAPRT KO mice, the regular primer set revealed comparable levels of Naprt mRNA between WT and KO mice (new Supplementary Figure 3b).\n(3) We performed immunoblotting analysis of NAPRT in the liver and colon per the reviewer’s request (new Supplementary Figure 3c). Our results showed that in both liver and colon, the deletion of the 7 amino acids in NAPRT KO mice resulted in a slight down shift of NAPRT protein bands. Moreover, this deletion was coupled with significant protein degradation, particularly in the liver.\nWe are glad to know that we have addressed all concerns of the reviewer.\nFor the minor point of the reviewer, we modified the Discussion section to include the following language:\nThe first paragraph on Page 28: “In fact, our observations that NAPRT deficiency increased tumor initiation in mice together with previous reports on the anti-tumor growth effects of NAPRT inhibition in established cancer cell lines and xenografts reveal an intriguing stage-specific role of NAD in cancer: suppressing tumor initiation at early stages while supporting tumor growth and progression at later stages. These findings could offer valuable guidance for the development of future NAD-based strategies in cancer prevention and treatment.”\nAt the end of the first paragraph on Page 29: “Future studies employing tissue-specific NAPRT knockout models are needed to further elucidate the role of NAPRT in different cell types and at different stages along the carcinogenesis process.”\nHope these are sufficient to address this minor point."}]

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