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.This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the “Miguel Servet” program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees.S

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.Peer reviewe

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.Peer reviewe

Adaptive from Innate: Human IFN-γ+CD4+ T Cells Can Arise Directly from CXCL8-Producing Recent Thymic Emigrants in Babies and Adults.

We recently demonstrated that the major effector function of neonatal CD4+ T cells is to produce CXCL8, a prototypic cytokine of innate immune cells. In this article, we show that CXCL8 expression, prior to proliferation, is common in newly arising T cells (so-called "recent thymic emigrants") in adults, as well as in babies. This effector potential is acquired in the human thymus, prior to TCR signaling, but rather than describing end-stage differentiation, such cells, whether isolated from neonates or adults, can further differentiate into IFN-γ-producing CD4+ T cells. Thus, the temporal transition of host defense from innate to adaptive immunity is unexpectedly mirrored at the cellular level by the capacity of human innate-like CXCL8-producing CD4+ T cells to transition directly into Th1 cells

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

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

Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Disease Progression and Drug Response in Acute Myeloid Leukemia

D.P. was supported by a non-clinical research fellowship from EHA. This work was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001045), The UK Medical Research Council (FC001045), and the Welcome Trust (FC001045)

HIF-1α and HIF-2α knockdowns induce deficiency in the long-term reconstitution ability of human haematopoietic cells

Le taux physiologique d’oxygène est finement régulé dans les tissus des mammifères, l’oxygénation diffère d’un tissu à l’autre, ainsi qu’au sein même de ces derniers, ce phénomène est en grande partie dû à l’architecture vasculaire. L’oxygène est un stimulus clef dans la destinée des cellules durant l’embryogenèse et la progression tumorale. Il est actuellement reconnu que les cellules souches se distribuent en suivant un gradient d’oxygène, où les faibles concentrations en oxygène (Hypoxie) favorisent la conservation d’un état indifférencié. Les cellules souches hématopoïétiques (CSH) résident dans des niches osseuses où la disponibilité en oxygène est limitée voir nulle. Le modèle de l’hématopoïèse a été largement décrit au niveau cellulaire et moléculaire et est un des principaux modèles utilisé dans l’étude des mécanismes gouvernant le caractère souche d’une cellule. Des études récentes révèlent dans des modèles murins que les facteurs de transcriptions induits par l’hypoxie (HIF) affectent le comportement des cellules souches hématopoïétiques. Ici, nous étudions le rôle potentiel des facteurs HIF-1α et HIF-2α dans les CSH Humaines. Le knockdown des deux facteurs a été obtenu en combinant une stratégie de short-hairpin RNA et de vecteurs de transferts lentiviraux. La transduction de cellules de sang de cordon CD34-positive révèle que les deux knockdowns affectent à court terme la croissance des progéniteurs et CSH et à long terme leurs capacités de reconstitution dans des souris immuno-déficientes NOD/LtSz-scid IL2rgnull. Cependant, nous observons un effet plus délétère de HIF-2α sur le maintien des cellules souches en comparaison à HIF-1α.Physiological oxygen level is tightly regulated in mammalian tissues. Moreover, oxygenation differs from one tissue to the other, as well as within a single tissue, due to vasculature modeling. Oxygen levels have been shown to be a key stimulus of cell fate during embryogenesis and cancer progression. It is now widely admitted that stem cells fate followes oxygen gradients with low levels (hypoxia) promoting an undifferentiated state. Hematopoietic stem cells (HSC) reside in bone marrow niches in which oxygen availability is low, even absent. Hypoxia-inducible factors (HIF) are the main factors regulating the cell-response to oxygen variation. Studies on mouse models reveal that HIFs affect HSC activity. Here, we investigate the potential function of HIF-1α and HIF-2α within human HSCs. Knockdown of both factors was obtained by lentivirus-mediated short-hairpin RNA strategy. Transductions of CD34-positive cord blood cells reveal that HIF-2α knockdown affects myelo-erythroid differentiation. Both HIF-α are required for long-term reconstitution in immune-deficient NOD/LtSz-scid IL2rγnull mice, whereas a more pronounced deficiency was observed for HIF-2α. Overall, our data strongly suggest the existence of multiple but preponderant functions in human stem cell maintenance and differentiation

HIF-1α and HIF-2α knockdowns induce deficiency in the long-term reconstitution ability of human haematopoietic cells

Le taux physiologique d’oxygène est finement régulé dans les tissus des mammifères, l’oxygénation diffère d’un tissu à l’autre, ainsi qu’au sein même de ces derniers, ce phénomène est en grande partie dû à l’architecture vasculaire. L’oxygène est un stimulus clef dans la destinée des cellules durant l’embryogenèse et la progression tumorale. Il est actuellement reconnu que les cellules souches se distribuent en suivant un gradient d’oxygène, où les faibles concentrations en oxygène (Hypoxie) favorisent la conservation d’un état indifférencié. Les cellules souches hématopoïétiques (CSH) résident dans des niches osseuses où la disponibilité en oxygène est limitée voir nulle. Le modèle de l’hématopoïèse a été largement décrit au niveau cellulaire et moléculaire et est un des principaux modèles utilisé dans l’étude des mécanismes gouvernant le caractère souche d’une cellule. Des études récentes révèlent dans des modèles murins que les facteurs de transcriptions induits par l’hypoxie (HIF) affectent le comportement des cellules souches hématopoïétiques. Ici, nous étudions le rôle potentiel des facteurs HIF-1α et HIF-2α dans les CSH Humaines. Le knockdown des deux facteurs a été obtenu en combinant une stratégie de short-hairpin RNA et de vecteurs de transferts lentiviraux. La transduction de cellules de sang de cordon CD34-positive révèle que les deux knockdowns affectent à court terme la croissance des progéniteurs et CSH et à long terme leurs capacités de reconstitution dans des souris immuno-déficientes NOD/LtSz-scid IL2rgnull. Cependant, nous observons un effet plus délétère de HIF-2α sur le maintien des cellules souches en comparaison à HIF-1α.Physiological oxygen level is tightly regulated in mammalian tissues. Moreover, oxygenation differs from one tissue to the other, as well as within a single tissue, due to vasculature modeling. Oxygen levels have been shown to be a key stimulus of cell fate during embryogenesis and cancer progression. It is now widely admitted that stem cells fate followes oxygen gradients with low levels (hypoxia) promoting an undifferentiated state. Hematopoietic stem cells (HSC) reside in bone marrow niches in which oxygen availability is low, even absent. Hypoxia-inducible factors (HIF) are the main factors regulating the cell-response to oxygen variation. Studies on mouse models reveal that HIFs affect HSC activity. Here, we investigate the potential function of HIF-1α and HIF-2α within human HSCs. Knockdown of both factors was obtained by lentivirus-mediated short-hairpin RNA strategy. Transductions of CD34-positive cord blood cells reveal that HIF-2α knockdown affects myelo-erythroid differentiation. Both HIF-α are required for long-term reconstitution in immune-deficient NOD/LtSz-scid IL2rγnull mice, whereas a more pronounced deficiency was observed for HIF-2α. Overall, our data strongly suggest the existence of multiple but preponderant functions in human stem cell maintenance and differentiation