Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy.

Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell's viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and a lack of the favorable survival seen with other SF3B1 mutations. Moreover, compared to other hotspot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.

Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death.

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukaemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.

Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase (COASY), induced by mutations in SF3B1, affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.

The transcription factor DDIT3 is a potential driver of dyserythropoiesis in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.

Germline ERCC excision repair 6 like 2 (ERCC6L2) mutations lead to impaired erythropoiesis and reshaping of the bone marrow microenvironment.

Despite the inclusion of inherited myeloid malignancies as a separate entity in the World Health Organization Classification, many established predisposing loci continue to lack functional characterization. While germline mutations in the DNA repair factor ERCC excision repair 6 like 2 (ERCC6L2) give rise to bone marrow failure and acute myeloid leukaemia, their consequences on normal haematopoiesis remain unclear. To functionally characterise the dual impact of germline ERCC6L2 loss on human primary haematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs), we challenged ERCC6L2-silenced and patient-derived cells ex vivo. Here, we show for the first time that ERCC6L2-deficiency in HSPCs significantly impedes their clonogenic potential and leads to delayed erythroid differentiation. This observation was confirmed by CIBERSORTx RNA-sequencing deconvolution performed on ERCC6L2-silenced erythroid-committed cells, which demonstrated higher proportions of polychromatic erythroblasts and reduced orthochromatic erythroblasts versus controls. In parallel, we demonstrate that the consequences of ERCC6L2-deficiency are not limited to HSPCs, as we observe a striking phenotype in patient-derived and ERCC6L2-silenced MSCs, which exhibit enhanced osteogenesis and suppressed adipogenesis. Altogether, our study introduces a valuable surrogate model to study the impact of inherited myeloid mutations and highlights the importance of accounting for the influence of germline mutations in HSPCs and their microenvironment.

ER Stress and Unfolded Protein Response in Leukemia: Friend, Foe, or Both?

The unfolded protein response (UPR) is an evolutionarily conserved adaptive signaling pathway triggered by a stress of the endoplasmic reticulum (ER) lumen compartment, which is initiated by the accumulation of unfolded proteins. This response, mediated by three sensors-Inositol Requiring Enzyme 1 (IRE1), Activating Transcription Factor 6 (ATF6), and Protein Kinase RNA-Like Endoplasmic Reticulum Kinase (PERK)-allows restoring protein homeostasis and maintaining cell survival. UPR represents a major cytoprotective signaling network for cancer cells, which frequently experience disturbed proteostasis owing to their rapid proliferation in an usually unfavorable microenvironment. Increased basal UPR also participates in the resistance of tumor cells against chemotherapy. UPR activation also occurs during hematopoiesis, and growing evidence supports the critical cytoprotective role played by ER stress in the emergence and proliferation of leukemic cells. In case of severe or prolonged stress, pro-survival UPR may however evolve into a cell death program called terminal UPR. Interestingly, a large number of studies have revealed that the induction of proapoptotic UPR can also strongly contribute to the sensitization of leukemic cells to chemotherapy. Here, we review the current knowledge on the consequences of the deregulation of UPR signaling in leukemias and their implications for the treatment of these diseases.

Translational Regulations in Response to Endoplasmic Reticulum Stress in Cancers.

During carcinogenesis, almost all the biological processes are modified in one way or another. Among these biological processes affected, anomalies in protein synthesis are common in cancers. Indeed, cancer cells are subjected to a wide range of stresses, which include physical injuries, hypoxia, nutrient starvation, as well as mitotic, oxidative or genotoxic stresses. All of these stresses will cause the accumulation of unfolded proteins in the Endoplasmic Reticulum (ER), which is a major organelle that is involved in protein synthesis, preservation of cellular homeostasis, and adaptation to unfavourable environment. The accumulation of unfolded proteins in the endoplasmic reticulum causes stress triggering an unfolded protein response in order to promote cell survival or to induce apoptosis in case of chronic stress. Transcription and also translational reprogramming are tightly controlled during the unfolded protein response to ensure selective gene expression. The majority of stresses, including ER stress, induce firstly a decrease in global protein synthesis accompanied by the induction of alternative mechanisms for initiating the translation of mRNA, later followed by a translational recovery. After a presentation of ER stress and the UPR response, we will briefly present the different modes of translation initiation, then address the specific translational regulatory mechanisms acting during reticulum stress in cancers and highlight the importance of translational control by ER stress in tumours.

Improving Outcome in Infantile Autism with Folate Receptor Autoimmunity and Nutritional Derangements: A Self-Controlled Trial.

BACKGROUND: In contrast to multiple rare monogenetic abnormalities, a common biomarker among children with infantile autism and their parents is the discovery of serum autoantibodies directed to the folate receptor alpha (FRα) localized at blood-brain and placental barriers, impairing physiologic folate transfer to the brain and fetus. Since outcome after behavioral intervention remains poor, a trial was designed to treat folate receptor alpha (FRα) autoimmunity combined with correction of deficient nutrients due to abnormal feeding habits. METHODS: All participants with nonsyndromic infantile autism underwent a routine protocol measuring CBC, iron, vitamins, coenzyme Q10, metals, and trace elements. Serum FRα autoantibodies were assessed in patients, their parents, and healthy controls. A self-controlled therapeutic trial treated nutritional derangements with addition of high-dose folinic acid if FRα autoantibodies tested positive. The Childhood Autism Rating Scale (CARS) monitored at baseline and following 2 years of treatment was compared to the CARS of untreated autistic children serving as a reference. RESULTS: In this self-controlled trial (82 children; mean age ± SD: 4.4 ± 2.3 years; male:female ratio: 4.8:1), FRα autoantibodies were found in 75.6 % of the children, 34.1 % of mothers, and 29.4 % of fathers versus 3.3 % in healthy controls. Compared to untreated patients with autism (n=84) whose CARS score remained unchanged, a 2-year treatment decreased the initial CARS score from severe (mean ± SD: 41.34 ± 6.47) to moderate or mild autism (mean ± SD: 34.35 ± 6.25; paired t-test p<0.0001), achieving complete recovery in 17/82 children (20.7 %). Prognosis became less favorable with the finding of higher FRα autoantibody titers, positive maternal FRα autoantibodies, or FRα antibodies in both parents. CONCLUSIONS: Correction of nutritional deficiencies combined with high-dose folinic acid improved outcome for autism, although the trend of a poor prognosis due to maternal FRα antibodies or FRα antibodies in both parents may warrant folinic acid intervention before conception and during pregnancy.

The PERK Branch of the Unfolded Protein Response Promotes DLL4 Expression by Activating an Alternative Translation Mechanism.

Delta-like 4 (DLL4) is a pivotal endothelium specific Notch ligand that has been shown to function as a regulating factor during physiological and pathological angiogenesis. DLL4 functions as a negative regulator of angiogenic branching and sprouting. Interestingly, Dll4 is with Vegf-a one of the few examples of haplo-insufficiency, resulting in obvious vascular abnormalities and in embryonic lethality. These striking phenotypes are a proof of concept of the crucial role played by the bioavailability of VEGF and DLL4 during vessel patterning and that there must be a very fine-tuning of DLL4 expression level. However, to date the expression regulation of this factor was poorly studied. In this study, we showed that the DLL4 5'-UTR harbors an Internal Ribosomal Entry Site (IRES) that, in contrast to cap-dependent translation, was efficiently utilized in cells subjected to several stresses including hypoxia and endoplasmic reticulum stress (ER stress). We identified PERK, a kinase activated by ER stress, as the driver of DLL4 IRES-mediated translation, and hnRNP-A1 as an IRES-Trans-Acting Factor (ITAF) participating in the IRES-dependent translation of DLL4 during endoplasmic reticulum stress. The presence of a stress responsive internal ribosome entry site in the DLL4 msRNA suggests that the process of alternative translation initiation, by controlling the expression of this factor, could have a crucial role in the control of endothelial tip cell function.

Hypoxia and ER stress promote Staufen1 expression through an alternative translation mechanism.

Under physiological stress conditions the cell protects itself through a global blockade on cap-dependent translation of mRNA. This allows cap-independent mechanisms such as internal ribosome entry site (IRES)-mediated translation to take over and initiate the translation of a specific pool of mRNAs that encode proteins involved in protecting the cell from stress. Staufen 1 (Stau1) is an RNA-binding protein that has been previously implicated in the regulation of stress granule formation and therefore could play a key role in protecting the cell against stress stimuli such as oxidative and endoplasmic reticulum (ER) stress. We hypothesized that Stau1 mRNA could, like many stress response genes, contain an IRES in its 5'UTR. Here we describe that a bona fide IRES element is present in the 5'UTR of Stau1 mRNA, which is activated under hypoxic and ER stress conditions. Further, we show that the activity of PERK kinase, a major effector of the ER stress response, is required for Stau1 IRES-mediated translation during ER stress. These results suggest that Stau1 is a stress response gene that remains efficiently translated during hypoxia and ER stress despite the substantial global inhibition of cap-dependent protein translation, promoting cell recovery following stress.

PERK mediates the IRES-dependent translational activation of mRNAs encoding angiogenic growth factors after ischemic stress.

Angiogenesis is induced by various conditions, including hypoxia. Although cap-dependent translation is globally inhibited during ischemia, the mRNAs encoding two important proangiogenic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2), are translated at early time points in ischemic muscle. The translation of these mRNAs can occur through internal ribosome entry sites (IRESs), rather than through cap-dependent translation. Hypoxic conditions also induce the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, leading us to assess the interplay between hypoxia, ER stress, and IRES-mediated translation of FGF-2 and VEGF We found that unlike cap-dependent translation, translation through FGF-2 and VEGF IRESs was efficient in cells and transgenic mice subjected to ER stress-inducing stimuli. We identified PERK, a kinase that is activated by ER stress, as the driver of VEGF and FGF-2 IRES-mediated translation in cells and in mice expressing IRES-driven reporter genes and exposed to hypoxic stress. These results demonstrate the role of IRES-dependent translation in the induction of the proangiogenic factors VEGF and FGF-2 in response to acute hypoxic stress. Furthermore, the PERK pathway could be a viable pharmacological target to improve physiological responses to ischemic situations.

Key contribution of eIF4H-mediated translational control in tumor promotion.

Dysregulated expression of translation initiation factors has been associated with carcinogenesis, but underlying mechanisms remains to be fully understood. Here we show that eIF4H (eukaryotic translation initiation factor 4H), an activator of the RNA helicase eIF4A, is overexpressed in lung carcinomas and predictive of response to chemotherapy. In lung cancer cells, depletion of eIF4H enhances sensitization to chemotherapy, decreases cell migration and inhibits tumor growth in vivo, in association with reduced translation of mRNA encoding cell-proliferation (c-Myc, cyclin D1) angiogenic (FGF-2) and anti-apoptotic factors (CIAP-1, BCL-xL). Conversely, each isoform of eIF4H acts as an oncogene in NIH3T3 cells by stimulating transformation, invasion, tumor growth and resistance to drug-induced apoptosis together with increased translation of IRES-containing or structured 5'UTR mRNAs. These results demonstrate that eIF4H plays a crucial role in translational control and can promote cellular transformation by preferentially regulating the translation of potent growth and survival factor mRNAs, indicating that eIF4H is a promising new molecular target for cancer therapy.

Anatomical and electrophysiological plasticity of locomotor networks following spinal transection in the salamander.

Recovery of locomotor behavior following spinal cord injury can occur spontaneously in some vertebrates, such as fish, urodele amphibians, and certain reptiles. This review provides an overview of the current status of our knowledge on the anatomical and electrophysiological changes occurring within the spinal cord that lead to, or are associated with the re-expression of locomotion in spinally-transected salamanders. A better understanding of these processes will help to devise strategies for restoring locomotor function in mammals, including humans.