Suppression of p16 induces mTORC1-mediated nucleotide metabolic reprogramming

Buj, Raquel; Chen, Chi‐Wei; Dahl, Erika S.; Leon, Kelly E.; Kuskovsky, Ross; Maglakelidze, Natella; Navaratnarajah, Maithili; Zhang, Gao; Doan, Mary T.; Jiang, Helen; Zaleski, Michael; Kutzler, Lydia; Lacko, Holly A.; Lu, Yiling; Mills, G; Gowda, Raghavendra; Robertson, Gavin P.; Warrick, Joshua I.; Herlyn, Meenhard; Imamura, Yuka; Kimball, Scot R.; DeGraff, David J.; Snyder, Nathaniel W.; Aird, Katherine M.

doi:10.1101/393876

Cited by 10 publications

(17 citation statements)

References 91 publications

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“…1A-E), a classical model of OIS [5,61]. The shRNA hairpin specificity targeting p16 was previously validated [8]. Similar results were observed in normal skin fibroblasts Hs 895.Sk (Fig.…”

Section: Knockdown Of P16 Abrogates Oncogene-induced Il6 and Cxcl8 Exsupporting

confidence: 77%

“…Canonically, elevated p16 represses hyperphosphorylation of the retinoblastoma protein (RB), which inhibits E2F transcription factor-mediated expression of proliferative genes [7]. Loss of p16 is a common event in human cancer that has been linked to senescence bypass, increased proliferation, and malignant transformation though both canonical and non-canonical (RB-independent) pathways [8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

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Suppression of p16 alleviates the senescence-associated secretory phenotype

Buj

Leon

Aird

2020

Preprint

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Oncogene induced senescence (OIS) is characterized by increased expression of the cell cycle inhibitor p16, leading to a hallmark cell cycle arrest. Suppression of p16 in this context drives proliferation, senescence bypass, and contributes to tumorigenesis. OIS cells are also characterized by the expression and secretion of a widely variable group of factors collectively termed the senescence-associated secretory phenotype (SASP). The SASP can be both beneficial and detrimental and affects the microenvironment in a highly context-dependent manner. The relationship between p16 suppression and the SASP remains unclear. Here, we show that knockdown of p16 decreases expression of the SASP factors and pro-inflammatory cytokines IL6 and CXCL8 in both RASG12V- and BRAFV600E-induced senescence. Notably, this is likely not due to suppression of senescence as LMNB1 and cyclin A expression remain low and p21 remains high. Moreover, low p16 expression in both cancer cell lines and patient samples correspond to decreased SASP gene expression, suggesting this is a universal effect of loss of p16 expression. Together, our data suggest that p16 transcriptionally regulates SASP gene expression, which has implications for understanding how p16 modulates both the senescent and tumor microenvironment.

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“…1A-E), a classical model of OIS [5,61]. The shRNA hairpin specificity targeting p16 was previously validated [8]. Similar results were observed in normal skin fibroblasts Hs 895.Sk (Fig.…”

Section: Knockdown Of P16 Abrogates Oncogene-induced Il6 and Cxcl8 Exsupporting

confidence: 77%

Section: Introductionmentioning

confidence: 99%

Suppression of p16 alleviates the senescence-associated secretory phenotype

Buj

Leon

Aird

2020

Preprint

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“…To achieve sustainable long-term growth, oncogene-driven malignancies need to maintain sufficient dNTP pools, and this can be achieved through metabolic rewiring. This has been demonstrated in the case of oncogenic BRAF (19) and KRAS-driven PAAD (20), which upregulate de novo dNTP synthesis via the pentose phosphate pathway. Our analysis of clinical data suggests downregulation of dNTP catabolism via SAMHD1 could be an important component of this rewiring.…”

Section: Figure 1 Samhd1 Limits the Proliferation Of Cells Harbourinmentioning

confidence: 90%

SAMHD1 promotes oncogene-induced replication stress

Zhang

Calderón-Montaño

Rudd

2020

Preprint

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Oncogenes induce DNA replication stress in cancer cells. Although this was established more than a decade ago, we are still unravelling the molecular underpinnings of this phenomenon, which will be critical if we are to exploit this knowledge to improve cancer treatment. A key mediator of oncogene-induced replication stress is the availability of DNA precursors, which will limit ongoing DNA synthesis by cellular replicases. In this study, we identify a potential role for nucleotide catabolism in promoting replication stress induced by oncogenes. Specifically, we establish that the dNTPase SAMHD1 slows DNA replication fork speeds in human fibroblasts harbouring an oncogenic RAS allele, elevating levels of endogenous DNA damage, and ultimately limiting cell proliferation. We then show that oncogenic RAS-driven tumours express reduced SAMHD1 levels, suggesting they have overcome this tumour suppressor barrier, and that this correlates with worse overall survival for these patients.Abstract Figure

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“…Cells were routinely tested for mycoplasma as described in (27). Experiments targeting Ribonucleotide Reductase Regulatory Subunit M2 (RRM2), p16, mammalian target of rapamycin (mTOR), and ATR serine/threonine kinase (ATR) were previously included in Buj, et al (28). Lentiviral constructs were transfected into 293FT cells using Lipofectamine 2000 (Thermo Fisher, Cat# 11668019).…”

Section: Cell Culturementioning

confidence: 99%

“…To examine the effect of isotope dilution versus label free quantification we analyzed data from IMR90 cells with various short hairpin knockdown of genes found to disrupt nucleotide metabolism (28). This provided a bioanalytical meaningful way to modulate dNTP metabolites within similar cellular backgrounds.…”

Section: Stable Isotope Dilution Improves Analytical Performance Of Lmentioning

confidence: 99%

Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry

Kuskovsky

Buj

et al. 2018

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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.

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Suppression of p16 induces mTORC1-mediated nucleotide metabolic reprogramming

Cited by 10 publications

References 91 publications

Suppression of p16 alleviates the senescence-associated secretory phenotype

Suppression of p16 alleviates the senescence-associated secretory phenotype

SAMHD1 promotes oncogene-induced replication stress

Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry

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