Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

The DMT1 isoform lacking the iron-response element regulates normal and malignant hematopoiesis via NOTCH pathway activation.

Natural resistance-associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron-response element (nonIRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non-canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non-canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T-cell leukemia homeobox protein 1 (TLX1)-defective leukemia. This work sets the stage for future investigation using a multiple-hit T-cell acute lymphoblastic leukemia (T-ALL) model to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression.

Targeting serine/glycine metabolism improves radiotherapy response in non-small cell lung cancer.

BACKGROUND: Lung cancer is the most lethal cancer, and 85% of cases are classified as non-small cell lung cancer (NSCLC). Metabolic rewiring is a cancer hallmark that causes treatment resistance, and lacks insights into serine/glycine pathway adaptations upon radiotherapy. METHODS: We analyzed radiotherapy responses using mass-spectrometry-based metabolomics in NSCLC patient's plasma and cell lines. Efficacy of serine/glycine conversion inhibitor sertraline with radiotherapy was investigated by proliferation, clonogenic and spheroid assays, and in vivo using a serine/glycine dependent NSCLC mouse model by assessment of tumor growth, metabolite and cytokine levels, and immune signatures. RESULTS: Serine/glycine pathway metabolites were significantly consumed in response to radiotherapy in NSCLC patients and cell models. Combining sertraline with radiotherapy impaired NSCLC proliferation, clonogenicity and stem cell self-renewal capacity. In vivo, NSCLC tumor growth was reduced solely in the sertraline plus radiotherapy combination treatment group. Tumor weights linked to systemic serine/glycine pathway metabolite levels, and were inhibited in the combination therapy group. Interestingly, combination therapy reshaped the tumor microenvironment via cytokines associated with natural killer cells, supported by eradication of immune checkpoint galectin-1 and elevated granzyme B levels. CONCLUSION: Our findings highlight that targeting serine/glycine metabolism using sertraline restricts cancer cell recovery from radiotherapy and provides tumor control through immunomodulation in NSCLC.

Patient-derived glioblastoma organoids reflect tumor heterogeneity and treatment sensitivity.

Background: Treatment resistance and tumor relapse are the primary causes of mortality in glioblastoma (GBM), with intratumoral heterogeneity playing a significant role. Patient-derived cancer organoids have emerged as a promising model capable of recapitulating tumor heterogeneity. Our objective was to develop patient-derived GBM organoids (PGO) to investigate treatment response and resistance. Methods: GBM samples were used to generate PGOs and analyzed using whole-exome sequencing (WES) and single-cell karyotype sequencing. PGOs were subjected to temozolomide (TMZ) to assess viability. Bulk RNA sequencing was performed before and after TMZ. Results: WES analysis on individual PGOs cultured for 3 time points (1-3 months) showed a high inter-organoid correlation and retention of genetic variants (range 92.3%-97.7%). Most variants were retained in the PGO compared to the tumor (range 58%-90%) and exhibited similar copy number variations. Single-cell karyotype sequencing demonstrated preservation of genetic heterogeneity. Single-cell multiplex immunofluorescence showed maintenance of cellular states. TMZ treatment of PGOs showed a differential response, which largely corresponded with MGMT promoter methylation. Differentially expressed genes before and after TMZ revealed an upregulation of the JNK kinase pathway. Notably, the combination treatment of a JNK kinase inhibitor and TMZ demonstrated a synergistic effect. Conclusions: Overall, these findings demonstrate the robustness of PGOs in retaining the genetic and phenotypic heterogeneity in culture and the application of measuring clinically relevant drug responses. These data show that PGOs have the potential to be further developed into avatars for personalized adaptive treatment selection and actionable drug target discovery and as a platform to study GBM biology.

Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. While γ-secretase cleavage is necessary, we demonstrate it is insufficient for Notch activation and requires vesicular trafficking. Here, we report Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Dmt1-deficient cells are defective in Notch signalling and have perturbed endolysosomal trafficking and function. Dmt1 encodes for two isoforms, with and without an iron response element (ire). We show that isoform-specific silencing of Dmt1-ire and Dmt1+ire has opposite consequences on Notch-dependent cell fates in cell lines and intestinal organoids. Loss of Dmt1-ire suppresses Notch activation and promotes differentiation, whereas loss of Dmt1+ire causes Notch activation and maintains stem-progenitor cell fates. Dmt1 isoform expression correlates with Notch and Wnt signalling in Apc-deficient intestinal organoids and human colorectal cancers. Consistently, Dmt1-ire silencing induces Notch-dependent differentiation in colorectal cancer cells. These data identify Dmt1 isoforms as binary switches controlling Notch cell fate decisions in normal and tumour cells.

Drivers of de novo Serine/Glycine synthesis in acute leukemia.

Cancer cells hijack metabolic pathways in order to provide themselves with building blocks to support their proliferation and survival. Upregulation and addiction to de novo serine/glycine synthesis is an example of metabolic rewiring in cancer cells whereby serine and glycine are synthesised via a side branch of glycolysis. In this review, we focus on upregulation of endogenous serine/glycine production in acute leukemia, namely T-cell acute leukemia (T-ALL) and acute myeloid leukemia (AML). Several genetic lesions directly driving the serine/glycine addiction in acute leukemia have been established. Additionally, indirect regulation of de novo serine/glycine synthesis is observed in acute leukemia.

Transcription factor NKX2-1 drives serine and glycine synthesis addiction in cancer.

BACKGROUND: One-third of cancers activate endogenous synthesis of serine/glycine, and can become addicted to this pathway to sustain proliferation and survival. Mechanisms driving this metabolic rewiring remain largely unknown. METHODS: NKX2-1 overexpressing and NKX2-1 knockdown/knockout T-cell leukaemia and lung cancer cell line models were established to study metabolic rewiring using ChIP-qPCR, immunoblotting, mass spectrometry, and proliferation and invasion assays. Findings and therapeutic relevance were validated in mouse models and confirmed in patient datasets. RESULTS: Exploring T-cell leukaemia, lung cancer and neuroendocrine prostate cancer patient datasets highlighted the transcription factor NKX2-1 as putative driver of serine/glycine metabolism. We demonstrate that transcription factor NKX2-1 binds and transcriptionally upregulates serine/glycine synthesis enzyme genes, enabling NKX2-1 expressing cells to proliferate and invade in serine/glycine-depleted conditions. NKX2-1 driven serine/glycine synthesis generates nucleotides and redox molecules, and is associated with an altered cellular lipidome and methylome. Accordingly, NKX2-1 tumour-bearing mice display enhanced tumour aggressiveness associated with systemic metabolic rewiring. Therapeutically, NKX2-1-expressing cancer cells are more sensitive to serine/glycine conversion inhibition by repurposed anti-depressant sertraline, and to etoposide chemotherapy. CONCLUSION: Collectively, we identify NKX2-1 as a novel transcriptional regulator of serine/glycine synthesis addiction across cancers, revealing a therapeutic vulnerability of NKX2-1-driven cancers. Transcription factor NKX2-1 fuels cancer cell proliferation and survival by hyperactivating serine/glycine synthesis, highlighting this pathway as a novel therapeutic target in NKX2-1-positive cancers.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

Linking Serine/Glycine Metabolism to Radiotherapy Resistance.

The activation of de novo serine/glycine biosynthesis in a subset of tumors has been described as a major contributor to tumor pathogenesis, poor outcome, and treatment resistance. Amplifications and mutations of de novo serine/glycine biosynthesis enzymes can trigger pathway activation; however, a large group of cancers displays serine/glycine pathway overexpression induced by oncogenic drivers and unknown regulatory mechanisms. A better understanding of the regulatory network of de novo serine/glycine biosynthesis activation in cancer might be essential to unveil opportunities to target tumor heterogeneity and therapy resistance. In the current review, we describe how the activation of de novo serine/glycine biosynthesis in cancer is linked to treatment resistance and its implications in the clinic. To our knowledge, only a few studies have identified this pathway as metabolic reprogramming of cancer cells in response to radiation therapy. We propose an important contribution of de novo serine/glycine biosynthesis pathway activation to radioresistance by being involved in cancer cell viability and proliferation, maintenance of cancer stem cells (CSCs), and redox homeostasis under hypoxia and nutrient-deprived conditions. Current approaches for inhibition of the de novo serine/glycine biosynthesis pathway provide new opportunities for therapeutic intervention, which in combination with radiotherapy might be a promising strategy for tumor control and ultimately eradication. Further research is needed to gain molecular and mechanistic insight into the activation of this pathway in response to radiation therapy and to design sophisticated stratification methods to select patients that might benefit from serine/glycine metabolism-targeted therapies in combination with radiotherapy.

The ins and outs of serine and glycine metabolism in cancer.

Cancer cells reprogramme their metabolism to support unrestrained proliferation and survival in nutrient-poor conditions. Whereas non-transformed cells often have lower demands for serine and glycine, several cancer subtypes hyperactivate intracellular serine and glycine synthesis and become addicted to de novo production. Copy-number amplifications of serine- and glycine-synthesis genes and genetic alterations in common oncogenes and tumour-suppressor genes enhance serine and glycine synthesis, resulting in high production and secretion of these oncogenesis-supportive metabolites. In this Review, we discuss the contribution of serine and glycine synthesis to cancer progression. By relying on de novo synthesis pathways, cancer cells are able to enhance macromolecule synthesis, neutralize high levels of oxidative stress and regulate methylation and tRNA formylation. Furthermore, we discuss the immunosuppressive potential of serine and glycine, and the essentiality of both amino acids to promoting survival of non-transformed neighbouring cells. Finally, we point to the emerging data proposing moonlighting functions of serine- and glycine-synthesis enzymes and examine promising small molecules targeting serine and glycine synthesis.

Repurposing the Antidepressant Sertraline as SHMT Inhibitor to Suppress Serine/Glycine Synthesis-Addicted Breast Tumor Growth.

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival, and chemotherapy resistance. Although normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis-addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking, enzymatic assays, and drug-target interaction studies revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline's antiproliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1-S cell-cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis-addicted cancers.

Hallmarks of ribosomopathies.

Ribosomopathies are diseases caused by defects in ribosomal constituents or in factors with a role in ribosome assembly. Intriguingly, congenital ribosomopathies display a paradoxical transition from early symptoms due to cellular hypo-proliferation to an elevated cancer risk later in life. Another association between ribosome defects and cancer came into view after the recent discovery of somatic mutations in ribosomal proteins and rDNA copy number changes in a variety of tumor types, giving rise to somatic ribosomopathies. Despite these clear connections between ribosome defects and cancer, the molecular mechanisms by which defects in this essential cellular machinery are oncogenic only start to emerge. In this review, the impact of ribosomal defects on the cellular function and their mechanisms of promoting oncogenesis are described. In particular, we discuss the emerging hallmarks of ribosomopathies such as the appearance of 'onco-ribosomes' that are specialized in translating oncoproteins, dysregulation of translation-independent extra-ribosomal functions of ribosomal proteins, rewired cellular protein and energy metabolism, and extensive oxidative stress leading to DNA damage. We end by integrating these findings in a model that can provide an explanation how ribosomopathies could lead to the transition from hypo- to hyper-proliferation in bone marrow failure syndromes with elevated cancer risk.

Peptide microarray of pediatric acute myeloid leukemia is related to relapse and reveals involvement of DNA damage response and repair.

The majority of acute myeloid leukemia (AML) patients suffer from relapse and the exact etiology of AML remains unclear. The aim of this study was to gain comprehensive insights into the activity of signaling pathways in AML. In this study, using a high-throughput PepChip™ Kinomics microarray system, pediatric AML samples were analyzed to gain insights of active signal transduction pathway. Unsupervised hierarchical cluster analysis separated the AML blast profiles into two clusters. These two clusters were independent of patient characteristics, whereas the cumulative incidence of relapse (CIR) was significantly higher in the patients belonging to cluster-2. In addition, cluster-2 samples showed to be significantly less sensitive to various chemotherapeutic drugs. The activated peptides in cluster-1 and cluster-2 reflected the activity of cell cycle regulation, cell proliferation, cell differentiation, apoptosis, PI3K/AKT, MAPK, metabolism regulation, transcription factors and GPCRs signaling pathways. The difference between two clusters might be explained by the higher cell cycle arrest response in cluster-1 patients and higher DNA repair mechanism in cluster-2 patients. In conclusion, our study identifies different signaling profiles in pediatric AML in relation with CIR involving DNA damage response and repair.

Translatome analysis reveals altered serine and glycine metabolism in T-cell acute lymphoblastic leukemia cells.

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.

Cancer Biogenesis in Ribosomopathies.

Ribosomopathies are congenital diseases with defects in ribosome assembly and are characterized by elevated cancer risks. Additionally, somatic mutations in ribosomal proteins have recently been linked to a variety of cancers. Despite a clear correlation between ribosome defects and cancer, the molecular mechanisms by which these defects promote tumorigenesis are unclear. In this review, we focus on the emerging mechanisms that link ribosomal defects in ribosomopathies to cancer progression. This includes functional "onco-specialization" of mutant ribosomes, extra-ribosomal consequences of mutations in ribosomal proteins and ribosome assembly factors, and effects of ribosomal mutations on cellular stress and metabolism. We integrate some of these recent findings in a single model that can partially explain the paradoxical transition from hypo- to hyperproliferation phenotypes, as observed in ribosomopathies. Finally, we discuss the current and potential strategies, and the associated challenges for therapeutic intervention in ribosome-mutant diseases.

Correction: The ribosomal RPL10 R98S mutation drives IRES-dependent BCL-2 translation in T-ALL.

Following the publication of this article, the authors noted that Dr Laura Fancello was not listed among the authors. The corrected author list is given below. Additionally, the following was not included in the author contribution statement: 'L.F. analyzed RNA sequencing data'.

The ribosomal RPL10 R98S mutation drives IRES-dependent BCL-2 translation in T-ALL.

The R98S mutation in ribosomal protein L10 (RPL10 R98S) affects 8% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases, and was previously described to impair cellular proliferation. The current study reveals that RPL10 R98S cells accumulate reactive oxygen species which promotes mitochondrial dysfunction and reduced ATP levels, causing the proliferation defect. RPL10 R98S mutant leukemia cells can survive high oxidative stress levels via a specific increase of IRES-mediated translation of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2), mediating BCL-2 protein overexpression. RPL10 R98S selective sensitivity to the clinically available Bcl-2 inhibitor Venetoclax (ABT-199) was supported by suppression of splenomegaly and the absence of human leukemia cells in the blood of T-ALL xenografted mice. These results shed new light on the oncogenic function of ribosomal mutations in cancer, provide a novel mechanism for BCL-2 upregulation in leukemia, and highlight BCL-2 inhibition as a novel therapeutic opportunity in RPL10 R98S defective T-ALL.

Ribosomal Lesions Promote Oncogenic Mutagenesis.

Ribosomopathies are congenital disorders caused by mutations in ribosomal proteins (RP) or assembly factors and are characterized by cellular hypoproliferation at an early stage. Paradoxically, many of these disorders have an elevated risk to progress to hyperproliferative cancer at a later stage. In addition, somatic RP mutations have recently been identified in various cancer types, for example, the recurrent RPL10-R98S mutation in T-cell acute lymphoblastic leukemia (T-ALL) and RPS15 mutations in chronic lymphocytic leukemia (CLL). We previously showed that RPL10-R98S promotes expression of oncogenes, but also induces a proliferative defect due to elevated oxidative stress. In this study, we demonstrate that this proliferation defect is eventually rescued by RPL10-R98S mouse lymphoid cells that acquire 5-fold more secondary mutations than RPL10-WT cells. The presence of RPL10-R98S and other RP mutations also correlated with a higher mutational load in patients with T-ALL, with an enrichment in NOTCH1-activating lesions. RPL10-R98S-associated cellular oxidative stress promoted DNA damage and impaired cell growth. Expression of NOTCH1 eliminated these phenotypes in RPL10-R98S cells, in part via downregulation of PKC-θ, with no effect on RPL10-WT cells. Patients with RP-mutant CLL also demonstrated a higher mutational burden, enriched for mutations that may diminish oxidative stress. We propose that oxidative stress due to ribosome dysfunction causes hypoproliferation and cellular insufficiency in ribosomopathies and RP-mutant cancer. This drives surviving cells, potentiated by genomic instability, to acquire rescuing mutations, which ultimately promote transition to hyperproliferation. SIGNIFICANCE: Ribosomal lesions cause oxidative stress and increase mutagenesis, promoting acquisition of rescuing mutations that stimulate proliferation.