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.

CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny.

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

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.

Integrative phosphoproteomics defines two biologically distinct groups of KMT2A rearranged acute myeloid leukaemia with different drug response phenotypes.

Acute myeloid leukaemia (AML) patients harbouring certain chromosome abnormalities have particularly adverse prognosis. For these patients, targeted therapies have not yet made a significant clinical impact. To understand the molecular landscape of poor prognosis AML we profiled 74 patients from two different centres (in UK and Finland) at the proteomic, phosphoproteomic and drug response phenotypic levels. These data were complemented with transcriptomics analysis for 39 cases. Data integration highlighted a phosphoproteomics signature that define two biologically distinct groups of KMT2A rearranged leukaemia, which we term MLLGA and MLLGB. MLLGA presented increased DOT1L phosphorylation, HOXA gene expression, CDK1 activity and phosphorylation of proteins involved in RNA metabolism, replication and DNA damage when compared to MLLGB and no KMT2A rearranged samples. MLLGA was particularly sensitive to 15 compounds including genotoxic drugs and inhibitors of mitotic kinases and inosine-5-monosphosphate dehydrogenase (IMPDH) relative to other cases. Intermediate-risk KMT2A-MLLT3 cases were mainly represented in a third group closer to MLLGA than to MLLGB. The expression of IMPDH2 and multiple nucleolar proteins was higher in MLLGA and correlated with the response to IMPDH inhibition in KMT2A rearranged leukaemia, suggesting a role of the nucleolar activity in sensitivity to treatment. In summary, our multilayer molecular profiling of AML with poor prognosis and KMT2A-MLLT3 karyotypes identified a phosphoproteomics signature that defines two biologically and phenotypically distinct groups of KMT2A rearranged leukaemia. These data provide a rationale for the potential development of specific therapies for AML patients characterised by the MLLGA phosphoproteomics signature identified in this study.

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.

A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation.

The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using cross-linking immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial myelodysplasia (MDS)/acute myeloid leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease.

Targeting the lysine-specific demethylase 1 rewires kinase networks and primes leukemia cells for kinase inhibitor treatment.

Most tumor types either fail to respond or become resistant to kinase inhibitors, often because of compensatory prosurvival pathways in the cancer cell's broader signaling circuitry. Here, we found that intrinsic resistance to kinase inhibitors in cultured primary acute myeloid leukemia (AML) cells may be overcome by reshaping kinase networks into topologies that confer drug sensitivity. We identified several antagonists of chromatin-modifying enzymes that sensitized AML cell lines to kinase inhibitors. Of these, we confirmed that inhibitors of the lysine-specific demethylase (LSD1; also known as KDM1A) rewired kinase signaling in AML cells in a way that increased the activity of the kinase MEK and that broadly suppressed the activity of other kinases and feedback loops. As a result, AML cell lines and about half of primary human AML samples were primed for sensitivity to the MEK inhibitor trametinib. Primary human cells with KRAS mutations and those with high MEK pathway activity were the best responders to sequential treatment with LSD1 inhibitors then trametinib, whereas those with NRAS mutations and high mTOR activity were poor responders. Overall, our study reveals the MEK pathway as a mechanism of resistance to LSD1 inhibitors in AML and shows a way to modulate kinase network circuitry to potentially overcome therapeutic resistance to kinase inhibitors.

Ectopic humanized mesenchymal niche in mice enables robust engraftment of myelodysplastic stem cells.

Myelodysplastic syndrome (MDS) are clonal stem cell diseases characterized mainly by ineffective hematopoiesis. Here, we present an approach that enables robust long-term engraftment of primary MDS stem cells (MDS-SCs) in mice by implantation of human mesenchymal cell-seeded scaffolds. Critically for modelling MDS, where patient sample material is limiting, mononuclear bone marrow cells containing as few as 104 CD34+ cells can be engrafted and expanded by this approach with the maintenance of the genetic make-up seen in the patients. Non-invasive high-resolution ultrasound imaging shows that these scaffolds are fully perfused. Our data shows that human microenvironment but not mouse is essential to MDS-SCs homing and engraftment. Notably, the alternative niche provided by healthy donor MSCs enhanced engraftment of MDS-SCs. This study characterizes a new tool to model MDS human disease with the level of engraftment previously unattainable in mice, and offers insights into human-specific determinants of MDS-SC 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.

Loss of tRNA-modifying enzyme Elp3 activates a p53-dependent antitumor checkpoint in hematopoiesis.

The hematopoietic system is highly sensitive to perturbations in the translational machinery, of which an emerging level of regulation lies in the epitranscriptomic modification of transfer RNAs (tRNAs). Here, we interrogate the role of tRNA anticodon modifications in hematopoiesis by using mouse models of conditional inactivation of Elp3, the catalytic subunit of Elongator that modifies wobble uridine in specific tRNAs. Loss of Elp3 causes bone marrow failure by inducing death in committing progenitors and compromises the grafting activity of hematopoietic stem cells. Mechanistically, Elp3 deficiency activates a p53-dependent checkpoint in what resembles a misguided amino acid deprivation response that is accompanied by Atf4 overactivation and increased protein synthesis. While deletion of p53 rescues hematopoiesis, loss of Elp3 prompts the development of p53-mutated leukemia/lymphoma, and inactivation of p53 and Elongator cooperatively promotes tumorigenesis. Specific tRNA-modifying enzymes thus condition differentiation and antitumor fate decisions in hematopoietic stem cells and progenitors.

Mesenchymal niche remodeling impairs hematopoiesis via stanniocalcin 1 in acute myeloid leukemia.

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

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.

Correction: Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

c-Fos induces chondrogenic tumor formation in immortalized human mesenchymal progenitor cells.

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation.