De Novo Purine Metabolism is a Metabolic Vulnerability of Cancers with Low p16 Expression.

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. SIGNIFICANCE: 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.

αKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation.

Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.

Mass Spectrometry-Based Pipeline for Identifying RNA Modifications Involved in a Functional Process: Application to Cancer Cell Adaptation.

Cancer onset and progression are known to be regulated by genetic and epigenetic events, including RNA modifications (a.k.a. epitranscriptomics). So far, more than 150 chemical modifications have been described in all RNA subtypes, including messenger, ribosomal, and transfer RNAs. RNA modifications and their regulators are known to be implicated in all steps of post-transcriptional regulation. The dysregulation of this complex yet delicate balance can contribute to disease evolution, particularly in the context of carcinogenesis, where cells are subjected to various stresses. We sought to discover RNA modifications involved in cancer cell adaptation to inhospitable environments, a peculiar feature of cancer stem cells (CSCs). We were particularly interested in the RNA marks that help the adaptation of cancer cells to suspension culture, which is often used as a surrogate to evaluate the tumorigenic potential. For this purpose, we designed an experimental pipeline consisting of four steps: (1) cell culture in different growth conditions to favor CSC survival; (2) simultaneous RNA subtype (mRNA, rRNA, tRNA) enrichment and RNA hydrolysis; (3) the multiplex analysis of nucleosides by LC-MS/MS followed by statistical/bioinformatic analysis; and (4) the functional validation of identified RNA marks. This study demonstrates that the RNA modification landscape evolves along with the cancer cell phenotype under growth constraints. Remarkably, we discovered a short epitranscriptomic signature, conserved across colorectal cancer cell lines and associated with enrichment in CSCs. Functional tests confirmed the importance of selected marks in the process of adaptation to suspension culture, confirming the validity of our approach and opening up interesting prospects in the field.

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

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.

Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology.

Despite significant progress in targeted therapies, cancer recurrence remains a major cause of mortality worldwide. Identification of accurate biomarkers, through molecular profiling in healthy and cancer patient samples, will improve diagnosis and promote personalized medicine. While genetic and epigenetic alterations of DNA are currently exploited as cancer biomarkers, their robustness is limited by tumor heterogeneity. Recently, cancer-associated changes in RNA marks have emerged as a promising source of diagnostic and prognostic biomarkers. RNA epigenetics (also known as epitranscriptomics) is an emerging field in which at least 150 chemical modifications in all types of RNA (mRNA, tRNA, lncRNA, rRNA, and microRNA) have been detected. These modifications fine-tune gene expression in both physiological and pathological processes. A growing number of studies have established links between specific modified nucleoside levels in solid/liquid biopsies, and cancer onset and progression. In this review, we highlight the potential role of epitranscriptomic markers in refining cancer diagnosis and/or prognosis. RNA modification patterns may contain important information for establishing an initial diagnosis, monitoring disease evolution, and predicting response to treatment. Furthermore, recent developments in mass spectrometry allow reliable quantification of RNA marks in solid biopsies and biological fluids. We discuss the great potential of mass spectrometry for identifying epitranscriptomic biomarker signatures in cancer diagnosis. While there are various methods to quantify modified nucleosides, most are unable to detect and quantify more than one type of RNA modification at a time. Mass spectrometry analyses, especially GC-MS/MS and LC-MS/MS, overcome this limitation and simultaneously detect modified nucleosides by multiple reaction monitoring. Indeed, several groups are currently validating mass spectrometry methods that quantify several nucleosides at one time in liquid biopsies. The challenge now is to exploit these powerful analytical tools to establish epitranscriptomic signatures that should open new perspectives in personalized medicine. This review summarizes the growing clinical field of analysis of RNA modifications and discusses pre-analytical and analytical approaches, focusing in particular on the development of new mass spectrometry tools and their clinical applications.

FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell.

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2'-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.