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posted on 2024-05-02, 14:20 authored by Naveen Kumar Tangudu, Raquel Buj, Hui Wang, Jiefei Wang, Aidan R. Cole, Apoorva Uboveja, Richard Fang, Amandine Amalric, Baixue Yang, Adam Chatoff, Claudia V. Crispim, Peter Sajjakulnukit, Maureen A. Lyons, Kristine Cooper, Nadine Hempel, Costas A. Lyssiotis, Uma R. Chandran, Nathaniel W. Snyder, Katherine M. Aird Multiple antifolates induce apoptosis in p16 knockdown cells. A–D, SKMEL28 human melanoma cells were infected with lentivirus expressing a shRNA targeting p16 (shp16). shGFP was used as a control (shCont). A, Cells were treated with the indicated inhibitors (MTX – 0.17 µmol/L; LTX – 0.12 µmol/L) and cytotoxicity was assessed using IncuCyte Cytotox Green reagent. Data from one of three independent experimental replicates are shown (n = 6). Data are mean ± SD. One-way ANOVA at endpoint. ****, P < 0.0001. B, Cells were treated with the indicated inhibitors (MTX – 0.17 µmol/L; LTX – 0.12 µmol/L) for 72 hours, and apoptosis was assessed using Annexin V/PI (propidium iodide) staining by flow cytometry. Data from one of three independent experimental replicates are shown (n = 6). One-way ANOVA of live cells. ****, P < 0.0001. C, Purine metabolite abundance by mass spectrometry. Cells were treated with methotrexate (MTX; 0.17 µmol/L, 72 hours). Data represent one independent experimental replicate (n = 8). Controls are the same data as shown in Fig. 2G. D, Cells were treated with lometrexol (LTX; 0.17 µmol/L) or methotrexate (MTX; 0.17 µmol/L) for 72 hours, and immunofluorescence analysis for γH2AX foci was performed. Data from one of two independent experimental replicates are shown (n = 3). Data are mean ± SD. One-way ANOVA. ****, P < 0.0001; ns = not significant.
Funding
HHS | NIH | National Cancer Institute (NCI)
HHS | NIH | National Institute of General Medical Sciences (NIGMS)
Melanoma Research Foundation (MRF)
Ovarian Cancer Research Alliance (OCRA)
History
ARTICLE ABSTRACT
p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in approximately 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. However, the broader impact of p16/CDKN2A 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/CDKN2A. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low CDKN2A expression. We determined that cells with low p16/CDKN2A expression are sensitive to multiple inhibitors of de novo purine synthesis, including antifolates. Finally, tumors with p16 knockdown were more sensitive to the antifolate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2Alow tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents.
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.
