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[Preprint]. 2023 Sep 23:2023.07.15.549149.
doi: 10.1101/2023.07.15.549149.

De novo purine metabolism is a metabolic vulnerability of cancers with low p16 expression

Naveen Kumar Tangudu  1 , Raquel Buj  1 , Hui Wang  1 , Jiefei Wang  2 , Aidan R Cole  1 , Apoorva Uboveja  1 , Richard Fang  1 , Amandine Amalric  1 , Peter Sajjakulnukit  3 , Maureen A Lyons  4 , Kristine Cooper  5 , Nadine Hempel  6 , Nathaniel W Snyder  7 , Costas A Lyssiotis  3 , Uma R Chandran  2 , Katherine M Aird  1
Affiliations

De novo purine metabolism is a metabolic vulnerability of cancers with low p16 expression

Naveen Kumar Tangudu et al. bioRxiv. .

Update in

Abstract

p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in ~50% of all human cancers. In its canonical role, p16 inhibits the G1-S phase cell cycle progression through suppression of cyclin dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. Whether other nucleotide metabolic genes and pathways are affected by p16/CDKN2A loss and if these can be specifically targeted in p16/CDKN2A-low tumors has not been previously explored. Using CRISPR KO libraries in multiple isogenic human and mouse melanoma cell lines, we determined that many nucleotide metabolism genes are negatively enriched in p16/CDKN2A knockdown cells compared to controls. Indeed, many of the genes that are required for survival in the context of low p16/CDKN2A expression based on our CRISPR screens are upregulated in p16 knockdown melanoma cells and those with endogenously low CDKN2A expression. We determined that cells with low p16/Cdkn2a expression are sensitive to multiple inhibitors of de novo purine synthesis, including anti-folates. Tumors with p16 knockdown were more sensitive to the anti-folate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2A-low tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents.

Keywords: anti-folates; anti-metabolites; folate metabolism; melanoma; one carbon metabolism.

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Conflict of interest statement

Declaration of Interests In the past three years, C.A.L. has consulted for Astellas Pharmaceuticals, Odyssey Therapeutics, Third Rock Ventures, and T-Knife Therapeutics, and is an inventor on patents pertaining to Kras regulated metabolic pathways, redox control pathways in pancreatic cancer, and targeting the GOT1-ME1 pathway as a therapeutic approach (US Patent No: 2015126580-A1, 05/07/2015; US Patent No: 20190136238, 05/09/2019; International Patent No: WO2013177426-A2, 04/23/2015). All other authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Multiple CRISPR knockout screens identify nucleotide metabolism genes that are selectively depleted in shp16/shCdkn2a cells.
(A) Schematic of our CRISPR screens. Isogenic p16/Cdkn2a wildtype cells were infected with lentiviruses expressing shGFP control (shCont), shp16 (human), or shCdkn2a (mouse). These cells were infected with the CRISPR gRNA libraries at an MOI of 0.2–0.3. After 14 days in culture, gDNA was harvested and sequenced. (B-C) Analysis of 128 genes related to nucleotide synthesis, salvage, and catabolism identified multiple genes that are negatively selected in shp16/shCdkn2a vs. shControl in (B) human SKMEL28 and (C) mouse Yumm5.2 melanoma cells.
Figure 2.
Figure 2.. p16/CDKN2A negatively correlates with mRNA and protein expression of multiple nucleotide metabolism genes.
(A-B) SKMEL28 human melanoma cells were infected with lentivirus expressing a short hairpin RNA (shRNA) targeting p16 (shp16). shGFP was used as a control (shCont). (A) Expression of the 128 nucleotide metabolism genes from RNA-Seq. (B) Polysome fractionation was performed and both heavy (>2 ribosomes) and total mRNA were sequenced, and the ratio of Heavy to Total was used to assess transcripts with increased translation. (C) Genes that are transcriptionally or translationally upregulated in shp16 SKMEL28 cells. (D-E) DepMap data of cutaneous melanoma cell lines. (D) mRNA expression of 31 genes identified in the CRISPR screens. (E) Protein expression of 31 genes identified in the CRISPR screens.
Figure 3.
Figure 3.. p16/CDKN2A-low cells are more sensitive to inhibitors of nucleotide metabolism.
(A) Table of inhibitors used in in vitro cell line studies. 1C metabolism = one carbon metabolism. (B) SKMEL28 human melanoma cells were infected with lentivirus expressing a short hairpin RNA (shRNA) targeting p16 (shp16). shGFP was used as a control (shCont). Cells were treated with the indicated inhibitors and proliferation was assessed by crystal violet staining. One of 2–3 independent experimental replicates is shown (n=6). (C) Increased drug sensitivity from DepMap data of cutaneous melanoma cell lines with high or low CDKN2A expression. Data are mean ± SD. T-test. *p<0.05, **p<0.01
Figure 4.
Figure 4.. Multiple anti-folates induce apoptosis in p16 knockdown cells.
(A-B) SKMEL28 human melanoma cells were infected with lentivirus expressing a short hairpin RNA (shRNA) targeting p16 (shp16). shGFP was used as a control (shCont). (A) Cells were treated with the indicated inhibitors and cytotoxicity was assessed using IncuCyte Cytotox Green reagent. One of 3 independent experimental replicates is shown (n=6). Data are mean ± SD. One-way ANOVA at endpoint. ****p<0.0001 (B) Cells were treated with the indicated inhibitors, and apoptosis was assessed using Annexin V/PI staining by flow cytometry. One of 3 independent experimental replicates is shown (n=3). Data are mean ± SD. One-way ANOVA of live cell population. ****p<0.0001
Figure 5.
Figure 5.. In vivo shp16 tumors are sensitive to the anti-folate methotrexate.
(A-B) SKMEL28 human melanoma cells were infected with lentivirus expressing a short hairpin RNA (shRNA) targeting GFP (shCont) or p16 (shp16). 107 cells were subcutaneously implanted into athymic nude mice. Individual tumor growth curves in the indicated groups. Shown are growth rates ± SE. Linear mixed-model group comparisons: shp16 vs. shCont p<0.001; shCont: MTX vs. Vehicle p=0.734; shp16: MTX vs. Vehicle p=0.031; (shp16: MTX vs. Vehicle) vs. (Control: Vehicle vs MTX) p=0.002.
Figure 6.
Figure 6.. De novo purine synthesis and one carbon metabolism genes are associated with worse overall survival in metastatic melanomas.
Data from The Cancer Genome Atlas (TCGA) Skin Cutaneous Melanoma PanCancer Atlas (367 metastatic melanomas). (A) De novo purine synthesis and one carbon metabolism genes in TCGA metastatic melanoma samples. Red indicates increased mRNA expression. Blue indicates decreased mRNA expression. Overall survival probability of patients with alterations in de novo purine synthesis and one carbon metabolism genes (B), de novo pyrimidine genes (C), overall nucleotide biosynthesis genes (D), and nucleotide salvage genes (E). Log rank p value and 95% confidence interval.
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