SUMMARY Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A ( RPIA ), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo . These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.

Quantification of cellular deoxyribonucleoside mono-(dNMP), di-(dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13 C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13 C 15 N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13 C isotope tracing from major substrates including 13 C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.

Polyunsaturated fatty acids (PUFAs) are critical for brain development and have been linked with neurodevelopmental outcomes. We conducted a population-based case control study in California to examine the association between PUFAs measured in mid-pregnancy serum samples and child autism spectrum disorder (ASD). ASD cases (n = 499) were identified through the Department of Developmental Services and matched to live-birth population controls (n = 502) on birth month, year (2010–2011), and sex. Crude and adjusted logistic regression models were used to examine associations. Secondary analyses examined ASD with and without co-occurring intellectual disability (ID; n = 67 and 432 respectively), and effect modification by sex and ethnicity. No clear patterns emerged, though there was a modest inverse association with the top quartile of linoleic acid (highest versus lowest quartile, adjusted OR = 0.74, 95% CI: 0.49, 1.11; P for trend = 0.10). Lower levels of total and ω 3 PUFAs were associated with ASD with ID (lowest decile versus deciles 4–7, total PUFA adjusted OR = 2.78 95% CI: 1.13, 6.82) but not ASD without ID. We did not observe evidence of effect modification by factors examined. Findings do not suggest a strong association between mid-pregnancy PUFA levels and ASD. Further work should consider associations with ASD with ID and in other time windows.

Keywords dNTP, nucleotide, metabolism, high resolution mass spectrometry Acknowledgements This work was made possible by a NARSAD young investigator grant from the Brain and Behavior Foundation (NWS) as well as NIH grants K22ES026235 (NWS), R00CA194309 (KA), and P30ES013508. AbstractQuantification of cellular deoxyribonucleoside mono-(dNMP), di-(dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13 C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13 C 15 N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13 C isotope tracing from major substrates including 13 C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.