<div>Abstract<p>p16 is a tumor suppressor encoded by the <i>CDKN2A</i> gene whose expression is lost in approximately 50% of all human cancers. In its canonical role, p16 inhibits the G<sub>1</sub>–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. However, the broader impact of p16/<i>CDKN2A</i> loss on other nucleotide metabolic pathways and potential therapeutic targets remains unexplored. Using CRISPR knockout libraries in isogenic human and mouse melanoma cell lines, we determined several nucleotide metabolism genes essential for the survival of cells with loss of p16/<i>CDKN2A</i>. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low <i>CDKN2A</i> expression. We determined that cells with low p16/<i>CDKN2A</i> expression are sensitive to multiple inhibitors of <i>de novo</i> purine synthesis, including antifolates. Finally, tumors with p16 knockdown were more sensitive to the antifolate methotrexate <i>in vivo</i> than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/<i>CDKN2A</i><sup>low</sup> tumors as loss of p16/<i>CDKN2A</i> may provide a therapeutic window for these agents.</p>Significance:<p>Antimetabolites were the first chemotherapies, yet many have failed in the clinic due to toxicity and poor patient selection. Our data suggest that p16 loss provides a therapeutic window to kill cancer cells with widely-used antifolates with relatively little toxicity.</p></div>
<p>Multiple CRISPR KO screens identify nucleotide metabolism genes that are selectively depleted in p16/<i>CDKN2A</i><sup>low</sup> cells. <b>A,</b> Schematic of our CRISPR screens. p16/<i>Cdkn2a</i> wildtype cells were infected with lentiviruses expressing shGFP control (shCont), shp16 (human), or sh<i>Cdkn2a</i> (mouse). Human and mouse isogenic cell pairs were infected with nucleotide-focused or whole metabolism-focused CRISPR gRNA libraries, respectively, at an MOI of <0.3. After 14 days in culture, gDNA was harvested and sequenced. Analysis of genes included in the “nucleotide metabolism signature” (<a href="#SMT1" target="_blank">Supplementary Table S1</a>) identified multiple genes that are negatively enriched in shp16/sh<i>Cdkn2a</i> vs. shCont in human SKMEL28 (<b>B</b>) and mouse Yumm5.2 (<b>C</b>) melanoma cells. Raw data can be found in <a href="#SMT3" target="_blank">Supplementary Tables S3</a> and <a href="#SMT4" target="_blank">S4</a>. <b>D,</b> Comparison of datasets and list of 31 common genes negatively enriched (log<sub>2</sub> fold change <0) in the indicated analyses.</p>
<div>Abstract<p>p16 is a tumor suppressor encoded by the <i>CDKN2A</i> gene whose expression is lost in approximately 50% of all human cancers. In its canonical role, p16 inhibits the G<sub>1</sub>–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. However, the broader impact of p16/<i>CDKN2A</i> loss on other nucleotide metabolic pathways and potential therapeutic targets remains unexplored. Using CRISPR knockout libraries in isogenic human and mouse melanoma cell lines, we determined several nucleotide metabolism genes essential for the survival of cells with loss of p16/<i>CDKN2A</i>. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low <i>CDKN2A</i> expression. We determined that cells with low p16/<i>CDKN2A</i> expression are sensitive to multiple inhibitors of <i>de novo</i> purine synthesis, including antifolates. Finally, tumors with p16 knockdown were more sensitive to the antifolate methotrexate <i>in vivo</i> than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/<i>CDKN2A</i><sup>low</sup> tumors as loss of p16/<i>CDKN2A</i> may provide a therapeutic window for these agents.</p>Significance:<p>Antimetabolites were the first chemotherapies, yet many have failed in the clinic due to toxicity and poor patient selection. Our data suggest that p16 loss provides a therapeutic window to kill cancer cells with widely-used antifolates with relatively little toxicity.</p></div>
<p>p16/<i>CDKN2A</i> negatively correlates with multiple nucleotide metabolism genes, proteins, and metabolites. <b>A–D,</b> SKMEL28 human melanoma cells were infected with lentivirus expressing a shRNA targeting p16 (shp16). shGFP was used as a control (shCont). <b>A,</b> Expression of the 128 nucleotide metabolism gene signature from RNA-seq. Raw data can be found in <a href="#SMT6" target="_blank">Supplementary Table S6</a>. <b>B,</b> Polysome fractionation was performed and both the heavy fraction (>2 ribosomes) and total mRNA were sequenced. The ratio of heavy to total was used to assess transcripts with increased translation. Raw data can be found in <a href="#SMT7" target="_blank">Supplementary Table S7</a>. <b>C,</b> Genes that are transcriptionally or translationally upregulated in shp16 SKMEL28 cells. <b>D,</b> Expression of the indicated proteins by proteomics. <b>E</b> and <b>F,</b> DepMap data of cutaneous melanoma cell lines. <b>E,</b> mRNA expression of 23 genes identified in the CRISPR screens. <b>F,</b> Protein expression of genes identified in the CRISPR screens. Note only 20 proteins were found in the DepMap data. <b>G,</b> Steady-state metabolite profile of one carbon metabolites and purines.</p>
<p>shp16 tumors are more sensitive to the antifolate methotrexate. <b>A</b> and <b>B,</b> SKMEL28 human melanoma cells were infected with lentivirus expressing a shRNA targeting GFP (shCont) or p16 (shp16). A total of 10<sup>7</sup> cells were subcutaneously implanted into athymic nude mice. Mice were treated with vehicle controls or methotrexate (MTX). Individual tumor growth curves in shp16 tumors (<b>A</b>) and shCont tumors (<b>B</b>). Shown are growth rates ± SE. Linear mixed-model group comparisons: shp16 versus shCont <i>P</i> < 0.001; shCont: MTX versus vehicle <i>P</i> = 0.734; shp16: MTX versus vehicle <i>P</i> = 0.031; (shp16: MTX vs. vehicle) versus (Control: vehicle vs. MTX) <i>P</i> = 0.002.</p>
