Description of a knock-in mouse model of JAK2V617F MPN emerging from a minority of mutated hematopoietic stem cells.

The major weakness of most knock-in JAK2V617F mouse models is the presence of the JAK2 mutation in all rather than in a few hematopoietic stem cells (HSC), such as in human "early-stage" myeloproliferative neoplasms (MPN). Understanding the mechanisms of disease initiation is critical as underscored by the incidence of clonal hematopoiesis of indeterminate potential associated with JAK2V617F. Currently, such studies require competitive transplantation. Here, we report a mouse model obtained by crossing JAK2V617F/WT knock-in mice with PF4iCre transgenic mice. As expected, PF4iCre;JAK2V617F/WT mice developed an early thrombocytosis resulting from the expression of JAK2V617F in the megakaryocytes. However, these mice then developed a polycythemia vera-like phenotype at 10 weeks of age. Using mT/mG reporter mice, we demonstrated that Cre recombination was present in all hematopoietic compartments, including in a low number of HSC. The frequency of mutated cells increased along hematopoietic differentiation mimicking the clonal expansion observed in essential thrombocythemia and polycythemia vera patients. This model thus mimics the HSC compartment observed in early-stage MPN, with a small number of JAK2V617F HSC competing with a majority of JAK2WT HSC. PF4iCre;JAK2V617F/WT mice are a promising tool to investigate the mechanisms that regulate clonal dominance and progression to myelofibrosis.

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.

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.

CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

Hif-2α is not essential for cell-autonomous hematopoietic stem cell maintenance.

Local hypoxia in hematopoietic stem cell (HSC) niches is thought to regulate HSC functions. Hypoxia-inducible factor-1 (Hif-1) and Hif-2 are key mediators of cellular responses to hypoxia. Although oxygen-regulated α-subunits of Hifs, namely Hif-1α and Hif-2α, are closely related, they play overlapping and also distinct functions in nonhematopoietic tissues. Although Hif-1α-deficient HSCs lose their activity on serial transplantation, the role for Hif-2α in cell-autonomous HSC maintenance remains unknown. Here, we demonstrate that constitutive or inducible hematopoiesis-specific Hif-2α deletion does not affect HSC numbers and steady-state hematopoiesis. Furthermore, using serial transplantations and 5-fluorouracil treatment, we demonstrate that HSCs do not require Hif-2α to self-renew and recover after hematopoietic injury. Finally, we show that Hif-1α deletion has no major impact on steady-state maintenance of Hif-2α-deficient HSCs and their ability to repopulate primary recipients, indicating that Hif-1α expression does not account for normal behavior of Hif-2α-deficient HSCs.

The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.

α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect-the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

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.

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.

JMJD6 promotes self-renewal and regenerative capacity of hematopoietic stem cells.

Lifelong multilineage hematopoiesis critically depends on rare hematopoietic stem cells (HSCs) that reside in the hypoxic bone marrow microenvironment. Although the role of the canonical oxygen sensor hypoxia-inducible factor prolyl hydroxylase has been investigated extensively in hematopoiesis, the functional significance of other members of the 2-oxoglutarate (2-OG)-dependent protein hydroxylase family of enzymes remains poorly defined in HSC biology and multilineage hematopoiesis. Here, by using hematopoietic-specific conditional gene deletion, we reveal that the 2-OG-dependent protein hydroxylase JMJD6 is essential for short- and long-term maintenance of the HSC pool and multilineage hematopoiesis. Additionally, upon hematopoietic injury, Jmjd6-deficient HSCs display a striking failure to expand and regenerate the hematopoietic system. Moreover, HSCs lacking Jmjd6 lose multilineage reconstitution potential and self-renewal capacity upon serial transplantation. At the molecular level, we found that JMJD6 functions to repress multiple processes whose downregulation is essential for HSC integrity, including mitochondrial oxidative phosphorylation (OXPHOS), protein synthesis, p53 stabilization, cell cycle checkpoint progression, and mTORC1 signaling. Indeed, Jmjd6-deficient primitive hematopoietic cells display elevated basal and maximal mitochondrial respiration rates and increased reactive oxygen species (ROS), prerequisites for HSC failure. Notably, an antioxidant, N-acetyl-l-cysteine, rescued HSC and lymphoid progenitor cell depletion, indicating a causal impact of OXPHOS-mediated ROS generation upon Jmjd6 deletion. Thus, JMJD6 promotes HSC maintenance and multilineage differentiation potential by suppressing fundamental pathways whose activation is detrimental for HSC function.

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.

Ca2+tapulting HSCs into action.

In this issue of JEM, Umemoto et al. (https://doi.org/10.1084/jem.20180421) demonstrate that calcium influx stimulates mitochondrial metabolism and initiates proliferation in hematopoietic stem cells (HSCs). Extracellular adenosine, sourced from surrounding hematopoietic progenitors, inhibits this calcium influx, thereby suppressing mitochondrial metabolism and promoting HSC quiescence. This is the first demonstration that a calcium-mitochondria pathway regulates HSC division.

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.

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.

Lineage tracing of Pf4-Cre marks hematopoietic stem cells and their progeny.

The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15-60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage.

Slow-cycling/quiescence balance of hematopoietic stem cells is related to physiological gradient of oxygen.

OBJECTIVE: Regulation of hematopoiesis depends on cytokines, cellular interactions, transcription, and metabolic factors. Among the latter, O(2) has been neglected for a long time. Recently, an increasing number of publications evidenced the regulatory role of physiological low O(2) concentrations (0.1-5%; similar to those in bone marrow) on the in vitro behavior of hematopoietic stem cells. This brief review utilizes the article of Eliasson and colleagues in this Journal to summarize the major results and questions about the relationships between O(2) and hematopoiesis. MATERIALS AND METHODS: In order to be concise and interesting for readers unfamiliar with this field, we selected only the most significant data that either reinforce or contradict the conclusions of Eliasson et al., but we also provide references of reviews with a more detailed bibliography. RESULTS: A critical analysis of some key publications provides partial answers to three important questions: is the term hypoxia appropriate to describe physiological low O(2) concentrations? Is a very low O(2) level sufficient to control the quiescence/slow cycling balance of hematopoietic stem cells? Is the O(2) concentration able to modify the effect of cytokines on hematopoietic stem cells? CONCLUSIONS: We propose to use in situ normoxia instead of the confusing term hypoxia when working with normal cells at physiological low O(2) concentrations. We suggest that a very low O(2) concentration is necessary but not sufficient to induce hematopoietic stem cell quiescence. We review some articles showing that O(2) variations modify the effect of cytokines.