Slow TCA flux and ATP production in primary solid tumours but not metastases.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

Branching NOTCH1 to DNA damage in T-cell acute lymphoblastic leukemia.

Not available.

A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate-activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47-driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.

Sirt1 protects from K-Ras-driven lung carcinogenesis.

The NAD+-dependent deacetylase SIRT1 can be oncogenic or tumor suppressive depending on the tissue. Little is known about the role of SIRT1 in non-small cell lung carcinoma (NSCLC), one of the deadliest cancers, that is frequently associated with mutated K-RAS Therefore, we investigated the effect of SIRT1 on K-RAS-driven lung carcinogenesis. We report that SIRT1 protein levels are downregulated by oncogenic K-RAS in a MEK and PI3K-dependent manner in mouse embryo fibroblasts (MEFs), and in human lung adenocarcinoma cell lines. Furthermore, Sirt1 overexpression in mice delays the appearance of K-RasG12V-driven lung adenocarcinomas, reducing the number and size of carcinomas at the time of death and extending survival. Consistently, lower levels of SIRT1 are associated with worse prognosis in human NSCLCs. Mechanistically, analysis of mouse Sirt1-Tg pneumocytes, isolated shortly after K-RasG12V activation, reveals that Sirt1 overexpression alters pathways involved in tumor development: proliferation, apoptosis, or extracellular matrix organization. Our work demonstrates a tumor suppressive role of SIRT1 in the development of K-RAS-driven lung adenocarcinomas in mice and humans, suggesting that the SIRT1-K-RAS axis could be a therapeutic target for NSCLCs.

Synergistic antileukemic therapies in NOTCH1-induced T-ALL.

The Notch1 gene is a major oncogenic driver and therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL). However, inhibition of NOTCH signaling with γ-secretase inhibitors (GSIs) has shown limited antileukemic activity in clinical trials. Here we performed an expression-based virtual screening to identify highly active antileukemic drugs that synergize with NOTCH1 inhibition in T-ALL. Among these, withaferin A demonstrated the strongest cytotoxic and GSI-synergistic antileukemic effects in vitro and in vivo. Mechanistically, network perturbation analyses showed eIF2A-phosphorylation-mediated inhibition of protein translation as a critical mediator of the antileukemic effects of withaferin A and its interaction with NOTCH1 inhibition. Overall, these results support a role for anti-NOTCH1 therapies and protein translation inhibitor combinations in the treatment of T-ALL.

Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia.

Activating mutations of NOTCH1 (a well-known oncogene in T-cell acute lymphoblastic leukemia) are present in ∼4-13% of chronic lymphocytic leukemia (CLL) cases, where they are associated with disease progression and chemorefractoriness. However, the specific role of NOTCH1 in leukemogenesis remains to be established. Here, we report that the active intracellular portion of NOTCH1 (ICN1) is detectable in ∼50% of peripheral blood CLL cases lacking gene mutations. We identify a "NOTCH1 gene-expression signature" in CLL cells, and show that this signature is significantly enriched in primary CLL cases expressing ICN1, independent of NOTCH1 mutation. NOTCH1 target genes include key regulators of B-cell proliferation, survival, and signal transduction. In particular, we show that NOTCH1 transactivates MYC via binding to B-cell-specific regulatory elements, thus implicating this oncogene in CLL development. These results significantly extend the role of NOTCH1 in CLL pathogenesis, and have direct implications for specific therapeutic targeting.

cuRRBS: simple and robust evaluation of enzyme combinations for reduced representation approaches.

DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs. There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing (cuRRBS), a novel and easy-to-use computational method that solves this problem. By computing the optimal enzymatic digestions and size selection steps required, cuRRBS generalises the traditional MspI-based Reduced Representation Bisulfite Sequencing (RRBS) protocol to all restriction enzyme combinations. In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Moreover, we show in silico that cuRRBS-defined restriction enzymes consistently out-perform MspI digestion in many biological systems, considering both CpG and CHG contexts. Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets.

SIRT1 controls liver regeneration by regulating bile acid metabolism through farnesoid X receptor and mammalian target of rapamycin signaling.

UNLABELLED: Sirtuin1 (SIRT1) regulates central metabolic functions such as lipogenesis, protein synthesis, gluconeogenesis, and bile acid (BA) homeostasis through deacetylation. Here we describe that SIRT1 tightly controls the regenerative response of the liver. We performed partial hepatectomy (PH) to transgenic mice that overexpress SIRT1 (SIRT). SIRT mice showed increased mortality, impaired hepatocyte proliferation, BA accumulation, and profuse liver injury after surgery. The damaging phenotype in SIRT mice correlated with impaired farnesoid X receptor (FXR) activity due to persistent deacetylation and lower protein expression that led to decreased FXR-target gene expression; small heterodimer partner (SHP), bile salt export pump (BSEP), and increased Cyp7A1. Next, we show that 24-norUrsodeoxycholic acid (NorUDCA) attenuates SIRT protein expression, increases the acetylation of FXR and neighboring histones, restores trimethylation of H3K4 and H3K9, and increases miR34a expression, thus reestablishing BA homeostasis. Consequently, NorUDCA restored liver regeneration in SIRT mice, which showed increased survival and hepatocyte proliferation. Furthermore, a leucine-enriched diet restored mammalian target of rapamycin (mTOR) activation, acetylation of FXR and histones, leading to an overall lower BA production through SHP-inhibition of Cyp7A1 and higher transport (BSEP) and detoxification (Sult2a1) leading to an improved liver regeneration. Finally, we found that human hepatocellular carcinoma (HCC) samples have increased presence of SIRT1, which correlated with the absence of FXR, suggesting its oncogenic potential. CONCLUSION: We define SIRT1 as a key regulator of the regenerative response in the liver through posttranscriptional modifications that regulate the activity of FXR, histones, and mTOR. Moreover, our data suggest that SIRT1 contributes to liver tumorigenesis through dysregulation of BA homeostasis by persistent FXR deacetylation.

Acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is a haematological malignancy characterized by the uncontrolled proliferation of immature lymphoid cells. Over past decades, significant progress has been made in understanding the biology of ALL, resulting in remarkable improvements in its diagnosis, treatment and monitoring. Since the advent of chemotherapy, ALL has been the platform to test for innovative approaches applicable to cancer in general. For example, the advent of omics medicine has led to a deeper understanding of the molecular and genetic features that underpin ALL. Innovations in genomic profiling techniques have identified specific genetic alterations and mutations that drive ALL, inspiring new therapies. Targeted agents, such as tyrosine kinase inhibitors and immunotherapies, have shown promising results in subgroups of patients while minimizing adverse effects. Furthermore, the development of chimeric antigen receptor T cell therapy represents a breakthrough in ALL treatment, resulting in remarkable responses and potential long-term remissions. Advances are not limited to treatment modalities alone. Measurable residual disease monitoring and ex vivo drug response profiling screening have provided earlier detection of disease relapse and identification of exceptional responders, enabling clinicians to adjust treatment strategies for individual patients. Decades of supportive and prophylactic care have improved the management of treatment-related complications, enhancing the quality of life for patients with ALL.

BMP receptor 2 inhibition regulates mitochondrial bioenergetics to induce synergistic cell death with BCL-2 inhibitors in leukemia and NSLC cells.

Background: Bone morphogenetic protein (BMP) signaling cascade is a phylogenetically conserved stem cell regulator that is aberrantly expressed in non-small cell lung cancer (NSLC) and leukemias. BMP signaling negatively regulates mitochondrial bioenergetics in lung cancer cells. The impact of inhibiting BMP signaling on mitochondrial bioenergetics and the effect this has on the survival of NSLC and leukemia cells are not known. Methods: Utilizing the BMP type 2 receptor (BMPR2) JL189, BMPR2 knockout (KO) in cancer cells, and BMP loss of function mutants in C elegans, we determined the effects of BMPR2 inhibition (BMPR2i) on TCA cycle metabolic intermediates, mitochondrial respiration, and the regulation of mitochondrial superoxide anion (SOA) and Ca++ levels. We also examined whether BMPR2i altered the threshold cancer therapeutics induce cell death in NSLC and leukemia cell lines. KO of the mitochondria uniporter (MCU) was used to determine the mechanism BMPR2i regulates the uptake of Ca++ into the mitochondria, mitochondrial bioenergetics, and cell death. Results: BMPR2i increases mtCa++ levels and enhances mitochondrial bioenergetics in both NSLC and leukemia cell lines that is conserved in C elegans. BMPR2i induced increase in mtCa++ levels is regulated through the MCU, effecting mitochondria mass and cell survival. BMPR2i synergistically induced cell death when combined with BCL-2 inhibitors or microtubule targeting agents in both NSLC and leukemia cells. Cell death is caused by synergistic increase in mitochondrial ROS and Ca++ levels. BMPR2i enhances Ca++ uptake into the mitochondria induced by reactive oxygen species (ROS) produced by cancer therapeutics. Both acute myeloid leukemia (AML) and T-cell lymphoblastic leukemia cells lines were more responsive to the JL189 alone and when combined with venetoclax or navitoclax compared to NSLC.

A combination nutritional supplement reduces DNA methylation age only in older adults with a raised epigenetic age.

An increase in systemic inflammation (inflammaging) is one of the hallmarks of aging. Epigenetic (DNA methylation) clocks can quantify the degree of biological aging and this can be reversed by lifestyle and pharmacological intervention. We aimed to investigate whether a multi-component nutritional supplement could reduce systemic inflammation and epigenetic age in healthy older adults.We recruited 80 healthy older participants (mean age ± SD: 71.85 ± 6.23; males = 31, females = 49). Blood and saliva were obtained pre and post a 12-week course of a multi-component supplement, containing: Vitamin B3, Vitamin C, Vitamin D, Omega 3 fish oils, Resveratrol, Olive fruit phenols and Astaxanthin. Plasma GDF-15 and C-reactive protein (CRP) concentrations were quantified as markers of biological aging and inflammation respectively. DNA methylation was assessed in whole blood and saliva and used to derive epigenetic age using various clock algorithms.No difference between the epigenetic and chronological ages of participants was observed pre- and post-treatment by the blood-based Horvath or Hannum clocks, or the saliva-based InflammAge clock. However, in those with epigenetic age acceleration of ≥ 2 years at baseline, a significant reduction in epigenetic age (p = 0.015) and epigenetic age acceleration (p = 0.0058) was observed post-treatment using the saliva-based InflammAge clock. No differences were observed pre- and post-treatment in plasma GDF-15 and CRP, though participants with CRP indicative of an elevated cardiovascular disease risk (hsCRP ≥ 3µg/ml), had a reduction in CRP post-supplementation (p = 0.0195).Our data suggest a possible benefit of combined nutritional supplementation in individuals with an accelerated epigenetic age and inflammaging.

Sirtuin 1 regulation of developmental genes during differentiation of stem cells.

The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.

Therapeutic targeting of ACLY in T-ALL in vivo.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is a hematological malignancy in need of novel therapeutic approaches. Here, we identify the ATP-citrate lyase ACLY as a novel therapeutic target in T-ALL. Our results show that ACLY is overexpressed in T-ALL, and its expression correlates with NOTCH1 activity. To test the effects of ACLY in leukemia progression and the response to NOTCH1 inhibition, we developed an isogenic model of NOTCH1-induced Acly conditional knockout leukemia. Importantly, we observed intrinsic antileukemic effects upon loss of ACLY, which further synergized with NOTCH1 inhibition in vivo . Gene expression profiling analyses showed that the transcriptional signature of ACLY loss very significantly correlates with the signature of NOTCH1 inhibition in vivo , with significantly downregulated pathways related to oxidative phosphorylation, electron transport chain, ribosomal biogenesis and nucleosome biology. Consistently, metabolomic profiling upon ACLY loss revealed a metabolic crisis with accumulation of nucleotide intermediates and reduced levels of several amino acids. Overall, our results identify a link between NOTCH1 and ACLY and unveil ACLY as a novel promising target for T-ALL treatment.

A Therapeutically Targetable NOTCH1-SIRT1-KAT7 Axis in T-cell Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a NOTCH1-driven disease in need of novel therapies. Here, we identify a NOTCH1-SIRT1-KAT7 link as a therapeutic vulnerability in T-ALL, in which the histone deacetylase SIRT1 is overexpressed downstream of a NOTCH1-bound enhancer. SIRT1 loss impaired leukemia generation, whereas SIRT1 overexpression accelerated leukemia and conferred resistance to NOTCH1 inhibition in a deacetylase-dependent manner. Moreover, pharmacologic or genetic inhibition of SIRT1 resulted in significant antileukemic effects. Global acetyl proteomics upon SIRT1 loss uncovered hyperacetylation of KAT7 and BRD1, subunits of a histone acetyltransferase complex targeting H4K12. Metabolic and gene-expression profiling revealed metabolic changes together with a transcriptional signature resembling KAT7 deletion. Consistently, SIRT1 loss resulted in reduced H4K12ac, and overexpression of a nonacetylatable KAT7-mutant partly rescued SIRT1 loss-induced proliferation defects. Overall, our results uncover therapeutic targets in T-ALL and reveal a circular feedback mechanism balancing deacetylase/acetyltransferase activation with potentially broad relevance in cancer. SIGNIFICANCE: We identify a T-ALL axis whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results reveal T-ALL therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance. This article is highlighted in the In This Issue feature, p. 1.

Dietary glucosamine overcomes the defects in αß-T cell ontogeny caused by the loss of de novo hexosamine biosynthesis.

T cell development requires the coordinated rearrangement of T cell receptor (TCR) gene segments and the expression of either αß or γδ TCR. However, whether and how de novo synthesis of nutrients contributes to thymocyte commitment to either lineage remains unclear. Here, we find that T cell-specific deficiency in glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1), the rate-limiting enzyme of the de novo hexosamine biosynthesis pathway (dn-HBP), attenuates hexosamine levels, blunts N-glycosylation of TCRß chains, reduces surface expression of key developmental receptors, thus impairing αß-T cell ontogeny. GFAT1 deficiency triggers defects in N-glycans, increases the unfolded protein response, and elevates  γδ-T cell numbers despite reducing γδ-TCR diversity. Enhancing TCR expression or PI3K/Akt signaling does not reverse developmental defects. Instead, dietary supplementation with the salvage metabolite, glucosamine, and an α-ketoglutarate analogue partially restores αß-T cell development in GFAT1T-/- mice, while fully rescuing it in ex vivo fetal thymic organ cultures. Thus, dn-HBP fulfils, while salvage nutrients partially satisfy, the elevated demand for hexosamines during early T cell development.

Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming.

The expression of the pluripotency factors OCT4, SOX2, KLF4, and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, partial and reversible reprogramming does not change cell identity but can reverse markers of aging in cells, improve the capacity of aged mice to repair tissue injuries, and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome, and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen, and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites and biomarkers altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic, and metabolomic changes toward a younger configuration in multiple tissues and in the serum.

Inhibition of mitochondrial complex I reverses NOTCH1-driven metabolic reprogramming in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is commonly driven by activating mutations in NOTCH1 that facilitate glutamine oxidation. Here we identify oxidative phosphorylation (OxPhos) as a critical pathway for leukemia cell survival and demonstrate a direct relationship between NOTCH1, elevated OxPhos gene expression, and acquired chemoresistance in pre-leukemic and leukemic models. Disrupting OxPhos with IACS-010759, an inhibitor of mitochondrial complex I, causes potent growth inhibition through induction of metabolic shut-down and redox imbalance in NOTCH1-mutated and less so in NOTCH1-wt T-ALL cells. Mechanistically, inhibition of OxPhos induces a metabolic reprogramming into glutaminolysis. We show that pharmacological blockade of OxPhos combined with inducible knock-down of glutaminase, the key glutamine enzyme, confers synthetic lethality in mice harboring NOTCH1-mutated T-ALL. We leverage on this synthetic lethal interaction to demonstrate that IACS-010759 in combination with chemotherapy containing L-asparaginase, an enzyme that uncovers the glutamine dependency of leukemic cells, causes reduced glutaminolysis and profound tumor reduction in pre-clinical models of human T-ALL. In summary, this metabolic dependency of T-ALL on OxPhos provides a rational therapeutic target.