Hypoxia regulates CD9 expression and dissemination of B lymphoblasts.

Acute lymphoblastic leukemias (ALL) are the most frequent cancer in children and derive most often from B-cell precursors. Current survival rates roughly reach 90% at 10 years from diagnosis. However, 15-20% of children still relapse with a significant risk of death. Our previous work showed that the transmembrane protein CD9 plays a major role in lymphoblasts migration into sanctuary sites, especially in testis, through the activation of RAC1 signaling upon blasts stimulation with C-X-C chemokine ligand 12 (CXCL12). Here, we identified common factors shared by the bone marrow and extramedullary niches which could upregulate CD9 expression and function. We found that low oxygen levels enhance CD9 expression both at mRNA and protein levels. We further determined that Hypoxia Inducible Factor 1α (HIF1α), the master transcription factor involved in hypoxia response, binds directly CD9 promoter and induce CD9 transcription. We also showed that CD9 protein is crucial for leukemic cell adhesion and migration at low oxygen levels, possibly through its action on RAC1 signaling. Mouse xenograft experiments indicate that HIF1α signaling pathway promotes ALL cells engraftment in a CD9-dependent manner. The present work increments our understanding of CD9 implication in ALL pathogenesis.

Autophagy Targeting and Hematological Mobilization in FLT3-ITD Acute Myeloid Leukemia Decrease Repopulating Capacity and Relapse by Inducing Apoptosis of Committed Leukemic Cells.

Targeting FLT3-ITD in AML using TKI against FLT3 cannot prevent relapse even in the presence of complete remission, suggesting the resistance and/or the persistence of leukemic-initiating cells in the hematopoietic niche. By mimicking the hematopoietic niche condition with cultures at low oxygen concentrations, we demonstrate in vitro that FLT3-ITD AML cells decrease their repopulating capacity when Vps34 is inhibited. Ex vivo, AML FLT3-ITD blasts treated with Vps34 inhibitors recovered proliferation more slowly due to an increase an apoptosis. In vivo, mice engrafted with FLT3-ITD AML MV4-11 cells have the invasion of the bone marrow and blood in 2 weeks. After 4 weeks of FLT3 TKI treatment with gilteritinib, the leukemic burden had strongly decreased and deep remission was observed. When treatment was discontinued, mice relapsed rapidly. In contrast, Vps34 inhibition strongly decreased the relapse rate, and even more so in association with mobilization by G-CSF and AMD3100. These results demonstrate that remission offers the therapeutic window for a regimen using Vps34 inhibition combined with mobilization to target persistent leukemic stem cells and thus decrease the relapse rate.

CITED2 coordinates key hematopoietic regulatory pathways to maintain the HSC pool in both steady-state hematopoiesis and transplantation.

Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic differentiation hierarchy and sustain multilineage hematopoiesis. Here, we show that the transcriptional regulator CITED2 is essential for life-long HSC maintenance. While hematopoietic-specific Cited2 deletion has a minor impact on steady-state hematopoiesis, Cited2-deficient HSCs are severely depleted in young mice and fail to expand upon aging. Moreover, although they home normally to the bone marrow, they fail to reconstitute hematopoiesis upon transplantation. Mechanistically, CITED2 is required for expression of key HSC regulators, including GATA2, MCL-1, and PTEN. Hematopoietic-specific expression of anti-apoptotic MCL-1 partially rescues the Cited2-deficient HSC pool and restores their reconstitution potential. To interrogate the Cited2→Pten pathway in HSCs, we generated Cited2;Pten compound heterozygous mice, which had a decreased number of HSCs that failed to reconstitute the HSC compartment. In addition, CITED2 represses multiple pathways whose elevated activity causes HSC exhaustion. Thus, CITED2 promotes pathways necessary for HSC maintenance and suppresses those detrimental to HSC integrity.

Dual Inhibition of FLT3 and AXL by Gilteritinib Overcomes Hematopoietic Niche-Driven Resistance Mechanisms in FLT3-ITD Acute Myeloid Leukemia.

PURPOSE: AXL has been shown to play a pivotal role in the selective response of FLT3-ITD acute myeloid leukemia (AML) cells to FLT3 tyrosine kinase inhibitors (TKI), particularly within the bone marrow microenvironment. EXPERIMENTAL DESIGN: Herein, we compared the effect of dual FLT3/AXL-TKI gilteritinib with quizartinib through in vitro models mimicking hematopoietic niche conditions, ex vivo in primary AML blasts, and in vivo with dosing regimens allowing plasma concentration close to those used in clinical trials. RESULTS: We observed that gilteritinib maintained a stronger proapoptotic effect in hypoxia and coculture with bone marrow stromal cells compared with quizartinib, linked to a dose-dependent inhibition of AXL phosphorylation. In vivo, use of the MV4-11 cell line with hematopoietic engraftment demonstrated that gilteritinib was more effective than quizartinib at targeting leukemic cells in bone marrow. Finally, FLT3-ITD AML patient-derived xenografts revealed that this effect was particularly reproducible in FLT3-ITD AML with high allelic ratio in primary and secondary xenograft. Moreover, gilteritinib and quizartinib displayed close toxicity profile on normal murine hematopoiesis, particularly at steady state. CONCLUSIONS: Overall, these findings suggest that gilteritinib as a single agent, compared with quizartinib, is more likely to reach leukemic cells in their protective microenvironment, particularly AML clones highly dependent on FLT3-ITD signaling.

α-Tocopherol Acetate Attenuates Mitochondrial Oxygen Consumption and Maintains Primitive Cells within Mesenchymal Stromal Cell Population.

We present here the data showing, in standard cultures exposed to atmospheric O2 concentration, that alpha-tocopherol acetate (α-TOA) has a positive impact on primitive cells inside mesenchymal stromal cell (MstroC) population, by maintaining their proliferative capacity. α-TOA decreases the O2 consumption rate of MStroC probably by impacting respiratory chain complex II activity. This action, however, is not associated with a compensatory increase in glycolysis activity, in spite of the fact that the degradation of HIF-1α was decreased in presence of α-TOA. This is in line with a moderate enhancement of mtROS upon α-TOA treatment. However, the absence of glycolysis stimulation implies the inactivity of HIF-1α which might - if it were active - be related to the maintenance of stemness. It should be stressed that α-TOA might act directly on the gene expression as well as the mtROS themselves, which remains to be elucidated. Alpha-tocopherol acetate (α-TOA), a synthetic vitamin E ester, attenuates electron flow through electron transport chain (ETC) which is probably associated with a moderate increase in mtROS in Mesenchymal Stromal Cells. α-TOA action results in enhancement of the proliferative capacity and maintenance of the differentiation potential of the mesenchymal stem and progenitor cells.

Single-cell profiling reveals the trajectories of natural killer cell differentiation in bone marrow and a stress signature induced by acute myeloid leukemia.

Natural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

Targeting Tyrosine Kinases in Acute Myeloid Leukemia: Why, Who and How?

Acute myeloid leukemia (AML) is a myeloid malignancy carrying a heterogeneous molecular panel of mutations participating in the blockade of differentiation and the increased proliferation of myeloid hematopoietic stem and progenitor cells. The historical "3 + 7" treatment (cytarabine and daunorubicin) is currently challenged by new therapeutic strategies, including drugs depending on the molecular landscape of AML. This panel of mutations makes it possible to combine some of these new treatments with conventional chemotherapy. For example, the FLT3 receptor is overexpressed or mutated in 80% or 30% of AML, respectively. Such anomalies have led to the development of targeted therapies using tyrosine kinase inhibitors (TKIs). In this review, we document the history of TKI targeting, FLT3 and several other tyrosine kinases involved in dysregulated signaling pathways.

Metabolic and Innate Immune Cues Merge into a Specific Inflammatory Response via the UPR.

Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondrial reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR.

Targeting the RNA m6A Reader YTHDF2 Selectively Compromises Cancer Stem Cells in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.

Hematopoietic niche drives FLT3-ITD acute myeloid leukemia resistance to quizartinib via STAT5-and hypoxia-dependent upregulation of AXL.

Internal tandem duplication in Fms-like tyrosine kinase 3 (FLT3-ITD) is the most frequent mutation observed in acute myeloid leukemia (AML) and correlates with poor prognosis. FLT3 tyrosine kinase inhibitors are promising for targeted therapy. Here, we investigated mechanisms dampening the response to the FLT3 inhibitor quizartinib, which is specific to the hematopoietic niche. Using AML primary samples and cell lines, we demonstrate that convergent signals from the hematopoietic microenvironment drive FLT3-ITD cell resistance to quizartinib through the expression and activation of the tyrosine kinase receptor AXL. Indeed, cytokines sustained phosphorylation of the transcription factor STAT5 in quizartinib-treated cells, which enhanced AXL expression by direct binding of a conserved motif in its genomic sequence. Likewise, hypoxia, another well-known hematopoietic niche hallmark, also enhanced AXL expression. Finally, in a xenograft mouse model, inhibition of AXL significantly increased the response of FLT3-ITD cells to quizartinib exclusively within a bone marrow environment. These data highlight a new bypass mechanism specific to the hematopoietic niche that hampers the response to quizartinib through combined upregulation of AXL activity. Targeting this signaling offers the prospect of a new therapy to eradicate resistant FLT3-ITD leukemic cells hidden within their specific microenvironment, thereby preventing relapses from FLT3-ITD clones.

MiR-10a and HOXB4 are overexpressed in atypical myeloproliferative neoplasms.

BACKGROUND: Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients. METHODS: MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2'deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34+ cells with lentiviral vectors encoding the pri-miR-10a precursor. RESULTS: MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors. CONCLUSIONS: MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion.

Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells.

Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non-SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non-SP cells. From a cell regulation point of view, we showed that SP activity depended on O2 concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia-induced factors HIF-1α and HIF-2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non-SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy.

Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence.

Albeit tyrosine kinase inhibitors anti-Abl used in Chronic Myeloid Leukemia (CML) block the deregulated activity of the Bcr-Abl tyrosine kinase and induce remission in 90% of patients, they do not eradicate immature hematopoietic compartments of leukemic stem cells. To elucidate if autophagy is important for stem cell survival and/or proliferation, we used culture in low oxygen concentration (0.1% O2 for 7 days) followed back by non-restricted O2 supply (normoxic culture) to mimic stem cell proliferation and commitment. Knockdown of Atg7 expression, a key player in autophagy, in K562 cell line inhibited autophagy compared to control cells. Upon 7 days at 0.1% O2 both K562 and K562 shATG7 cells stopped to proliferate and a similar amount of viable cells remained. Back to non-restricted O2 supply K562 cells proliferate whereas K562 shATG7 cells exhibited strong apoptosis. Using immunomagnetic sorted normal and CML CD34+ cells, we inhibited the autophagic process by lentiviral infection expressing shATG7 or using a Vps34 inhibitor. Both, normal and CML CD34+ cells either competent or deficient for autophagy stopped to proliferate in hypoxia. Surprisingly, while normal CD34+ cells proliferate back to non restricted O2 supply, the CML CD34+ cells deficient for autophagy failed to proliferate. All together, these results suggest that autophagy is required for CML CD34+ commitment while it is dispensable for normal CD34 cells.

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1/Hoxa9-driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation.

Adult hematopoietic stem cells lacking Hif-1α self-renew normally.

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed ß subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance.

Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance.

Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance.

CBL controls a tyrosine kinase network involving AXL, SYK and LYN in nilotinib-resistant chronic myeloid leukaemia.

A tyrosine kinase network composed of the TAM receptor AXL and the cytoplasmic kinases LYN and SYK is involved in nilotinib-resistance of chronic myeloid leukaemia (CML) cells. Here, we show that the E3-ubiquitin ligase CBL down-regulation occurring during prolonged drug treatment plays a critical role in this process. Depletion of CBL in K562 cells increases AXL and LYN protein levels, promoting cell resistance to nilotinib. Conversely, forced expression of CBL in nilotinib-resistant K562 cells (K562-rn) dramatically reduces AXL and LYN expression and resensitizes K562-rn cells to nilotinib. A similar mechanism was found to operate in primary CML CD34(+) cells. Mechanistically, the E3-ligase CBL counteracts AXL/SYK signalling, promoting LYN transcription by controlling AXL protein stability. Surprisingly, the role of AXL in resistance was independent of its ligand GAS6 binding and its TK activity, in accordance with a scaffold activity for this receptor being involved in this cellular process. Collectively, our results demonstrate a pivotal role for CBL in the control of a tyrosine kinase network mediating resistance to nilotinib treatment in CML cells.

γδ T cells confer protection against murine cytomegalovirus (MCMV).

Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αß and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αß T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε-/- mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27- γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag-/-γc-/- mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αß T cell compromised patients.