DNA damage and cellular metabolism exhibit a complex interplay characterized by bidirectional feedback. Key mediators of these pathways include ATR and mTORC1, respectively. Previous studies established ATR as a regulatory upstream factor of mTORC1 during replication stress; however, the precise mechanisms remain poorly defined. Additionally, the activity of this signaling axis in unperturbed cells has not been extensively investigated. We demonstrate that ATR promotes mTORC1 activity across various human cancer cells and both human and mouse normal cells under basal conditions. This effect is enhanced in human cancer cells (SKMEL28, RPMI-7951, HeLa) following knockdown of p16, a cell cycle inhibitor that we have previously found increases mTORC1 activity and here found increases ATR activity. Mechanistically, ATR promotes de novo cholesterol synthesis and mTORC1 activation through the phosphorylation and upregulation of lanosterol synthase (LSS), independently of both CHK1 and the TSC complex. Interestingly, this pathway is distinct from the regulation of mTORC1 by ATM and may be specific to cancer cells. Finally, ATR-mediated increased cholesterol correlates with enhanced localization of mTOR to lysosomes. Collectively, our findings demonstrate a novel connection linking ATR and mTORC1 signaling through the modulation of cholesterol metabolism.

Anti-PD1 therapies are primarily thought to rely on functional T cell responses; yet tumors with limited T cell infiltration can still benefit, suggesting alternative mechanisms contribute to therapeutic efficacy. Indeed, we found that myeloid-rich, T cell-poor tumor models respond to anti-Pd1, and this is dependent on a cancer cell-macrophage crosstalk mediated by cancer cell Cdkn2a expression. Mechanistically, we found that cancer cells with decreased Cdkn2a expression (C dkn2a Low ), which occurs in ∼50% of all human cancers, reorganize zinc compartmentalization by upregulating the zinc importer Slc39a9 at the plasma membrane. Increased cancer cell plasma membrane Slc39a9 leads to intracellular zinc accumulation in cancer cells and depletion of zinc in the tumor microenvironment (TME), resulting in zinc-starved tumor-associated macrophages (TAMs) with reduced phagocytic activity. Restoring zinc availability in TAMs-via dietary supplementation or Slc39a9 knockdown in cancer cells-reprograms TAMs to a pro-phagocytic state and sensitizes Cdkn2a Low tumors to anti-Pd1 therapy. Remarkably, Slc39a9 knockdown tumors respond to anti-Pd1 in Rag1 -/- mice, and co-injection of zinc-replete macrophages is sufficient to drive an anti-Pd1 response in immunodeficient mice, demonstrating the T cell-independent nature of this response. Clinically, TAMs from CDKN2A Low cancer patients show reduced zinc and phagocytosis gene signatures. Moreover, patients with lower circulating zinc levels have significantly worse time-to-event outcomes than those with higher levels. Together, these findings uncover a previously unrecognized mechanism by which Cdkn2a Low cancer cells outcompete TAMs for zinc, impairing their function and limiting anti-Pd1 efficacy. They also provide evidence that macrophages alone, without T cells, can enhance anti-PD1 response through zinc-mediated reprogramming of phagocytosis.

CRISPR screen results for human SKMEL28 cells

De novo purine synthesis and one carbon metabolism genes are associated with worse overall survival in metastatic melanomas. Data from TCGA Skin Cutaneous Melanoma PanCancer Atlas (367 metastatic melanomas). Raw data can be found in Supplementary Table S8. 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.

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.

shp16 tumor bearing mice treated with methotrexate have a trend towards a survival advantage; and methotrexate does not affect body weight or blood cell counts. Related to Figure 5.

mRNA expression of nucleotide metabolism genes

p16/CDKN2Alow 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 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. IC50 and fold change (shp16 vs. shCont, FC) are indicated. Data from one of 2–3 independent experimental replicates are 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.

CRISPR screen results for mouse Yumm5.2 cells

Knockdown or knockout of Cdkn2a in mouse melanoma cell lines increases sensitivity to multiple anti-folates but not to de novo pyrimidine synthesis; Knockdown of RB1 does not recapitulate the anti-folate response exhibited by shp16 cells. Related to Figure 3.

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.

31 common genes identified in screens and publicly available data

Poly-Seq data of nucleotide metabolism genes

CRISPR KO library containing 128 genes comprising the nucleotide metabolism signature

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. 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.

Abstract Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. Contamination of dU in DNA is limited by the DNA damage response kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA replication. ATR inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell cycle checkpoints, ATRi induce more dU contamination than antimetabolites. ATRi-induced dU contamination in DNA is associated with an innate immune response. We showed this with the simple observation that ATRi-induced dU contamination and IFN-α/β expression is reversed by low doses of thymidine. Here we show that ATRi-induced IFN-α/β response is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA, ultimately generating cytoplasmic dsDNA that induces IFN-α/β. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell dependent anti-tumor responses in mouse models of cancer treated with radiation therapy. Citation Format: Pinakin Pandya, Frank P. Vendetti, Joseph A. Ghoubaira, Sudipta Pathak, Joshua J. Deppas, Reyna E. Jones, Yunqi Zhang, Daniel Ivanov, Raquel Buj, Katherine M. Aird, Jan H. Beumer, Robert W. Sobol, Christopher J. Bakkenist. Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA022.

The persistence of ovarian cancer stem-like cells (OvCSCs) after chemotherapy resistance has been implicated in relapse. However, the ability of these relatively quiescent cells to produce the robust tumor regrowth necessary for relapse remains an enigma. Since normal stem cells exist in a niche, and tumor-associated macrophages (TAMs) are the highest abundance immune cell within ovarian tumors, we hypothesized that TAMs may influence OvCSC proliferation. To test this, we optimized OvCSC enrichment by sphere culture and in vitro polarization of monocytes to a TAM-like M2 phenotype. Using cocultures that permitted the exchange of only soluble factors, we found that M2 macrophages increased the proliferation of sphere cells. Longer-term exposure (5-7 days) to soluble TAM factors led to retention of some stem cell features by OvCSCs but loss of others, suggesting that TAMs may support an intermediate stemness phenotype in OvCSCs. Although TAM coculture decreased the percentage of OvCSCs surviving chemotherapy, it increased the overall number. We therefore sought to determine the influence of this interaction on chemotherapy efficacy in vivo and found that inhibiting macrophages improved chemotherapy response. Comparing the gene expression changes in OvCSCs cocultured with TAMs to publicly available patient data identified 34 genes upregulated in OvCSCs by exposure to soluble TAM factors whose expression correlates with outcome. Overall, these data suggest that TAMs may influence OvCSC proliferation and impact therapeutic response.

Ovcar3 and Ovcar10 cells are wildtype for IDH1; RT-qPCR primers and shRNA binding sites.

ABSTRACT High grade serous ovarian cancer (HGSOC) is the most lethal gynecological cancer. Platinumbased therapies such as cisplatin are standard-of-care for HGSOC patients; however, the majority of HGSOCs initially treated with cisplatin will recur with widespread disseminated disease. Cisplatin induces cellular senescence, a stable cell cycle arrest. Although they are non-proliferative, senescent cells secrete a complex mix of cytokines and small molecules, named the senescence associated secretory phenotype (SASP), that have been shown to have pro-tumorigenic effects. To investigate how the SASP contributes to HGSOC progression, we used conditioned media from cisplatin therapy-induced senescent cells to culture naïve HGSOC spheroids. We report that while the SASP does not affect spheroid formation, the adhesion of cells within spheroids is altered, leading to cell detachment from spheroids. Interestingly, our data indicate that this occurs in an MMP-independent manner. Analysis of RNA-Seq samples indicates many adhesion-related genes and adhesion factors are transcriptionally downregulated by the SASP, particularly fibronectin and integrins, which was validated by immunofluorescence in spheroids. These data reveal that senescent cells contribute to a transcriptional program in nearby cancer cells in a paracrine fashion that decreases their adhesion, which may contribute to tumor dissemination.