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Supplementary Methods

Supplementary Tables S1-S6_S17-S18

Supplementary Figures

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments.Significance:Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy.This article is highlighted in the In This Issue feature, p. 1

Supplementary Table S7-S16

Supplementary Tables and Figures

Several cytotoxic agents used in cancer therapy cause DNA damage and replication stress. Understanding the metabolic determinants of the cell response to replication stress-inducing agents could have relevant implications for cancer treatment. In a recent study, we showed that cell survival during replication stress is influenced by the availability of amino acids, as well as by TORC1 and Gcn2-mediated amino acid sensing pathways. Amino acid starvation, or TORC1 inhibition, sensitizes cells to replication stress conditions, whereas Gcn2 ablation promotes cell survival by stimulating protein synthesis. The Vps34-Vps15-Vps30/Atg6/BECN1-Vps38/UVRAG phosphatidylinositol-3-phosphate (PtdIns3P) complex at the endosomes sets the balance between survival and death signals during replication stress and amino acid starvation. The Vps34-Vps15-Vps30/Atg6/BECN1-Vps38/UVRAG axis promotes the degradation of amino acid transporters, thus sensitizing cells to amino acid starvation, while Vps34-Vps15-Vps30/Atg6/BECN1-Vps38/UVRAG inactivation promotes cell survival by enabling synthesis of stress response proteins mediating survival under replication stress conditions. Our study unravels an autophagy-independent mechanism through which Vps34-Vps30/Atg6/BECN1 promotes lethal events during replication stress.

Glutamine analogs are potent suppressors of general glutamine metabolism with anti-cancer activity. 6-diazo-5-oxo-L-norleucine (DON) is an orally available glutamine analog which has been recently improved by structural modification for cancer treatment. Here, we explored the chemogenomic landscape of DON sensitivity using budding yeast as model organism. We identify evolutionarily conserved proteins that mediate cell resistance to glutamine analogs, namely Ura8CTPS1/2, Hpt1HPRT1, Mec1ATR, Rad53CHK1/CHK2 and Rtg1. We describe a function of Ura8 as inducible CTP synthase responding to inhibition of glutamine metabolism and propose a model for its regulation by CTP levels and Nrd1-dependent transcription termination at a cryptic unstable transcript. Disruption of the inducible CTP synthase under DON exposure hyper-activates the Mec1-Rad53 DNA damage response (DDR) pathway, which prevents chromosome breakage. Simultaneous inhibition of CTP synthase and Mec1 kinase synergistically sensitizes cells to DON, whereas CTP synthase over-expression hampers DDR mutant sensitivity. Using genome-wide suppressor screening, we identify factors promoting DON-induced CTP depletion (TORC1, glutamine transporter) and DNA breakage in DDR mutants. Together, our results identify CTP regulation and the Mec1-Rad53 DDR axis as key glutamine analog response pathways, and provide a rationale for the combined targeting of glutamine and CTP metabolism in DDR-deficient cancers.

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. Significance: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy. This article is highlighted in the In This Issue feature, p. 1

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.

Purpose: The mTOR complex C1 (mTORC1) inhibitor everolimus in combination with the aromatase inhibitor exemestane is an effective treatment for patients with hormone receptor—positive (HR+), HER2-negative (HER2−), advanced breast cancer (HR+/HER2− aBC). However, everolimus can cause hyperglycemia and hyperinsulinemia, which could reactivate the PI3K/protein kinase B (AKT)/mTORC1 pathway and induce tumor resistance to everolimus. Experimental Design: We conducted a multicenter, retrospective, Italian study to investigate the impact of baseline and on-treatment (i.e., during first 3 months of therapy) blood glucose levels on progression-free survival (PFS) in patients with HR+/HER2− aBC treated with everolimus-exemestane. Results: We evaluated 809 patients with HR+/HER2− aBC treated with everolimus-exemestane as any line of therapy for advanced disease. When evaluated as dichotomous variables, baseline and on-treatment glycemia were not significantly associated with PFS. However, when blood glucose concentration was evaluated as a continuous variable, a multivariable model accounting for clinically relevant patient- and tumor-related variables revealed that both baseline and on-treatment glycemia are associated with PFS, and this association is largely attributable to their interaction. In particular, patients who are normoglycemic at baseline and experience on-treatment diabetes have lower PFS compared with patients who are already hyperglycemic at baseline and experience diabetes during everolimus-exemestane therapy (median PFS, 6.34 vs. 10.32 months; HR, 1.76; 95% confidence interval, 1.15–2.69; P = 0.008). Conclusions: The impact of on-treatment glycemia on the efficacy of everolimus-exemestane therapy in patients with HR+/HER2− aBC depends on baseline glycemia. This study lays the foundations for investigating novel therapeutic approaches to target the glucose/insulin axis in combination with PI3K/AKT/mTORC1 inhibitors in patients with HR+/HER2− aBC.

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1ATM and Rpd3HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes., The relationship between DNA damage response (DDR) and regulation of the tolerance to glucose restriction is currently unclear. Here the authors reveal that maintaining a physiological level of histones by Rad53-Spt21 is necessary for glucose tolerance via multiple parallel pathways, including derepression of subtelomeric genes and acetyl-coA regulation by histone acetylation.

The mTOR inhibitor everolimus is effective against advanced pancreatic neuroendocrine tumors (pNETs). However, it can cause metabolic adverse events, such as hyperglycemia, hypertriglyceridemia and hypercholesterolemia. In this work we aimed at evaluating the impact of systemic and tumor lipid metabolism on everolimus efficacy. We carried out a monocentric, retrospective study to correlate plasma triglyceride and cholesterol levels with the progression free survival (PFS) of advanced pNET patients treated with everolimus. In formalin fixed, paraffin embedded (FFPE) tumor specimens, we also assessed by mRNA quantification and immunohistochemistry the expression of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), two enzymes crucially involved in fatty acid biosynthesis, and we analyzed their impact on PFS. We evaluated 58 consecutive pNET patients who started everolimus between December 2006 and January 2015. Patients with higher plasma triglycerides during the first 3 months of treatment had an increased risk of disease progression (aHR 3.08, 95% CIs 1.15-8.21; p = 0.025). In 23 FFPE tumor specimens amenable for IHC evaluations, we found a positive correlation between ACC1 and FASN at both mRNA (r = 0.87, p = 0.00045) and protein (r = 0.68, p = 0.0004) level. Patients with higher ACC1 protein expression in metastatic lesions had significantly lower PFS when compared to patients with lower ACC1 levels (5.5 vs. 36 months; aHR 4.49, 95% CIs 1.08-18.72; p = 0.039). In conclusion, systemic and tumor lipid metabolism are associated with the PFS of everolimus-treated patients with advanced pNETs; based on these findings, dietary and pharmacological interventions targeting lipid metabolism could improve everolimus efficacy in this patient population.

Summary Replication stress activates the Mec1ATR and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1., Graphical Abstract, Highlights • Rrm3 and Pif1 promote fork reversal and ssDNA gaps at stalled forks in rad53 cells • Rrm3 and Pif1 associate with stalled DNA replication forks • Rad53 phosphorylates Rrm3 and Pif1 at stalled forks • Rrm3 and Pif1 promote chromosome fragility in hydroxyurea-treated rad53 cells, Rossi et al. show that the DNA helicases Rrm3 and Pif1 associate with stalled DNA replication forks and undergo Rad53-mediated phosphorylation. The authors suggest that Rrm3 and Pif1 promote fork reversal, fork stalling, and chromosome fragility in rad53 defective cells under replication stress.