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.

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.

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.

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.

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.

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.

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.

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.

A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia.

Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the PTEN promoter in multiple hematopoietic populations, including T-cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced PTEN levels in T-ALL cells. Moreover, PE-null mice show reduced Pten levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by Pten loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased PTEN levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.

Freshwater monitoring by nanopore sequencing.

While traditional microbiological freshwater tests focus on the detection of specific bacterial indicator species, including pathogens, direct tracing of all aquatic DNA through metagenomics poses a profound alternative. Yet, in situ metagenomic water surveys face substantial challenges in cost and logistics. Here, we present a simple, fast, cost-effective and remotely accessible freshwater diagnostics workflow centred around the portable nanopore sequencing technology. Using defined compositions and spatiotemporal microbiota from surface water of an example river in Cambridge (UK), we provide optimised experimental and bioinformatics guidelines, including a benchmark with twelve taxonomic classification tools for nanopore sequences. We find that nanopore metagenomics can depict the hydrological core microbiome and fine temporal gradients in line with complementary physicochemical measurements. In a public health context, these data feature relevant sewage signals and pathogen maps at species level resolution. We anticipate that this framework will gather momentum for new environmental monitoring initiatives using portable devices.

Many water-dwelling bacteria can cause severe diseases such as cholera, typhoid or leptospirosis. One way to prevent outbreaks is to test water sources to find out which species of microbes they contain, and at which levels. Traditionally, this involves taking a water sample, followed by growing a few species of 'indicator bacteria' that help to estimate whether the water is safe. An alternative technique, called metagenomics, has been available since the mid-2000s. It consists in reviewing (or 'sequencing') the genetic information of most of the bacteria present in the water, which allows scientists to spot harmful species. Both methods, however, require well-equipped laboratories with highly trained staff, making them challenging to use in remote areas. The MinION is a pocket-sized device that ­ when paired with a laptop or mobile phone ­ can sequence genetic information 'on the go'. It has already been harnessed during Ebola, Zika or SARS-CoV-2 epidemics to track the genetic information of viruses in patients and environmental samples. However, it is still difficult to use the MinION and other sequencers to monitor bacteria in water sources, partly because the genetic information of the microbes is highly fragmented during DNA extraction. To address this challenge, Urban, Holzer et al. set out to optimise hardware and software protocols so the MinION could be used to detect bacterial species present in rivers. The tests focussed on the River Cam in Cambridge, UK, a waterway which faces regular public health problems: local rowers and swimmers often contract waterborne infections, sometimes leading to river closures. For six months, Urban, Holzer et al. used the MinION to map out the bacteria present across nine river sites, assessing the diversity of species and the presence of disease-causing microbes in the water. In particular, the results showed that optimising the protocols made it possible to tell the difference between closely related species ­ an important feature since harmful and inoffensive bacteria can sometimes be genetically close. The data also revealed that the levels of harmful bacteria were highest downstream of urban river sections, near a water treatment plant and river barge moorings. Together, these findings demonstrate that optimising MinION protocols can turn this device into a useful tool to easily monitor water quality. Around the world, climate change, rising urbanisation and the intensification of agriculture all threaten water quality. In fact, access to clean water is one of the United Nations sustainable development goals for 2030. Using the guidelines developed by Urban, Holzer et al., communities could harness the MinION to monitor water quality in remote areas, offering a cost-effective, portable DNA analysis tool to protect populations against deadly diseases.

Spleen plays a major role in DLL4-driven acute T-cell lymphoblastic leukemia.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4+CD8+ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL.

SHMT inhibition is effective and synergizes with methotrexate in T-cell acute lymphoblastic leukemia.

Folate metabolism enables cell growth by providing one-carbon (1C) units for nucleotide biosynthesis. The 1C units are carried by tetrahydrofolate, whose production by the enzyme dihydrofolate reductase is targeted by the important anticancer drug methotrexate. 1C units come largely from serine catabolism by the enzyme serine hydroxymethyltransferase (SHMT), whose mitochondrial isoform is strongly upregulated in cancer. Here we report the SHMT inhibitor SHIN2 and demonstrate its in vivo target engagement with 13C-serine tracing. As methotrexate is standard treatment for T-cell acute lymphoblastic leukemia (T-ALL), we explored the utility of SHIN2 in this disease. SHIN2 increases survival in NOTCH1-driven mouse primary T-ALL in vivo. Low dose methotrexate sensitizes Molt4 human T-ALL cells to SHIN2, and cells rendered methotrexate resistant in vitro show enhanced sensitivity to SHIN2. Finally, SHIN2 and methotrexate synergize in mouse primary T-ALL and in a human patient-derived xenograft in vivo, increasing survival. Thus, SHMT inhibition offers a complementary strategy in the treatment of T-ALL.

Application of Crosslinked Polybenzimidazole-Poly(Vinyl Benzyl Chloride) Anion Exchange Membranes in Direct Ethanol Fuel Cells.

Crosslinked membranes have been synthesized by a casting process using polybenzimidazole (PBI) and poly(vinyl benzyl chloride) (PVBC). The membranes were quaternized with 1,4-diazabicyclo[2.2.2]octane (DABCO) to obtain fixed positive quaternary ammonium groups. XPS analysis has showed insights into the changes from crosslinked to quaternized membranes, demonstrating that the crosslinking reaction and the incorporation of DABCO have occurred, while the 13C-NMR corroborates the reaction of DABCO with PVBC only by one nitrogen atom. Mechanical properties were evaluated, obtaining maximum stress values around 72 MPa and 40 MPa for crosslinked and quaternized membranes, respectively. Resistance to oxidative media was also satisfactory and the membranes were evaluated in single direct ethanol fuel cell. PBI-c-PVBC/OH 1:2 membrane obtained 66 mW cm-2 peak power density, 25% higher than commercial PBI membranes, using 0.5 bar backpressure of pure O2 in the cathode and 1 mL min-1 KOH 2M EtOH 2 M aqueous solution in the anode. When the pressure was increased, the best performance was obtained by the same membrane, reaching 70 mW cm-2 peak power density at 2 bar O2 backpressure. Based on the characterization and single cell performance, PBI-c-PVBC/OH membranes are considered promising candidates as anion exchange electrolytes for direct ethanol fuel cells.

GATA3-Controlled Nucleosome Eviction Drives MYC Enhancer Activity in T-cell Development and Leukemia.

Long-range enhancers govern the temporal and spatial control of gene expression; however, the mechanisms that regulate enhancer activity during normal and malignant development remain poorly understood. Here, we demonstrate a role for aberrant chromatin accessibility in the regulation of MYC expression in T-cell lymphoblastic leukemia (T-ALL). Central to this process, the NOTCH1-MYC enhancer (N-Me), a long-range T cell-specific MYC enhancer, shows dynamic changes in chromatin accessibility during T-cell specification and maturation and an aberrant high degree of chromatin accessibility in mouse and human T-ALL cells. Mechanistically, we demonstrate that GATA3-driven nucleosome eviction dynamically modulates N-Me enhancer activity and is strictly required for NOTCH1-induced T-ALL initiation and maintenance. These results directly implicate aberrant regulation of chromatin accessibility at oncogenic enhancers as a mechanism of leukemic transformation. SIGNIFICANCE: MYC is a major effector of NOTCH1 oncogenic programs in T-ALL. Here, we show a major role for GATA3-mediated enhancer nucleosome eviction as a driver of MYC expression and leukemic transformation. These results support the role of aberrant chromatin accessibility and consequent oncogenic MYC enhancer activation in NOTCH1-induced T-ALL.This article is highlighted in the In This Issue feature, p. 1631.

Screening for genes that accelerate the epigenetic aging clock in humans reveals a role for the H3K36 methyltransferase NSD1.

BACKGROUND: Epigenetic clocks are mathematical models that predict the biological age of an individual using DNA methylation data and have emerged in the last few years as the most accurate biomarkers of the aging process. However, little is known about the molecular mechanisms that control the rate of such clocks. Here, we have examined the human epigenetic clock in patients with a variety of developmental disorders, harboring mutations in proteins of the epigenetic machinery. RESULTS: Using the Horvath epigenetic clock, we perform an unbiased screen for epigenetic age acceleration in the blood of these patients. We demonstrate that loss-of-function mutations in the H3K36 histone methyltransferase NSD1, which cause Sotos syndrome, substantially accelerate epigenetic aging. Furthermore, we show that the normal aging process and Sotos syndrome share methylation changes and the genomic context in which they occur. Finally, we found that the Horvath clock CpG sites are characterized by a higher Shannon methylation entropy when compared with the rest of the genome, which is dramatically decreased in Sotos syndrome patients. CONCLUSIONS: These results suggest that the H3K36 methylation machinery is a key component of the epigenetic maintenance system in humans, which controls the rate of epigenetic aging, and this role seems to be conserved in model organisms. Our observations provide novel insights into the mechanisms behind the epigenetic aging clock and we expect will shed light on the different processes that erode the human epigenetic landscape during aging.

The MYC Enhancer-ome: Long-Range Transcriptional Regulation of MYC in Cancer.

MYC is one of the most important oncogenes in cancer. Indeed, MYC is upregulated in 50-60% of all tumors. MYC overexpression can be achieved through a variety of mechanisms, including gene duplications, chromosomal translocations, or somatic mutations leading to increased MYC stability. However, recent studies have identified numerous tissue-specific noncoding enhancers of MYC that play major roles in cancer, highlighting long-range transcriptional regulation of MYC as a critical novel mechanism leading to MYC hyperactivation and as a potential target for new therapeutic strategies in the near future. Here we summarize the regions and mechanisms involved in the long-range transcriptional regulation of MYC, underscoring the relevance of MYC enhancers both in normal physiological development and in MYC-driven cancer initiation and progression.