Migration Of Hematopoietic Stem Cells Research Articles

Retraction: Role of Microvascular endothelial cells on proliferation, migration and adhesion of hematopoietic stem cells.

This Retraction follows an Expression of Concern relating to this article previously published by Portland Press.This article is being retracted from Bioscience Reports at the request of the authors following receipt of a notification from a reader, alerting the authors to the duplications in Figure 4A as well as duplicated figures from another published article by the same authors in Zhongguo Shi Yan Xue Ye Xue Za Zhi (10.19746/j.cnki.issn1009-2137.2019.02.046).The authors wish to retract the article. The Editor-in-Chief and Editorial Board agree with the retraction.

Expression of Concern: Role of Microvascular endothelial cells on proliferation, migration and adhesion of hematopoietic stem cells.

The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates.

Id1 and Id3 Maintain Steady-State Hematopoiesis by Promoting Sinusoidal Endothelial Cell Survival and Regeneration

Investigating mechanisms that regulate endothelial cell (EC) growth and survival is important for understanding EC homeostasis and how ECs maintain stem cell niches. We report here that targeted loss of Id genes in adult ECs results in dilated, leaky sinusoids and a pro-inflammatory state that increases in severity over time. Disruption in sinusoidal integrity leads to increased hematopoietic stem cell (HSC) proliferation, differentiation, migration, and exhaustion. Mechanistically, sinusoidal ECs (SECs) show increased apoptosis because of reduced Bcl2-family gene expression following Id gene ablation. Furthermore, Id1-/-Id3-/- SECs and upstream type H vessels show increased expression of cyclin-dependent kinase inhibitors p21 and p27 and impaired ability to proliferate, which is rescued by reducing E2-2 expression. Id1-/-Id3-/- mice do not survive sublethal irradiation because of impaired vessel regeneration and hematopoietic failure. Thus, Id genes are required for the survival and regeneration of BM SECs during homeostasis and stress to maintain HSC development.

Role of microvascular endothelial cells on proliferation, migration and adhesion of hematopoietic stem cells.

Background: The present study investigated the effects of microvascular endothelial cells (MECs) on the chemotaxis, adhesion and proliferation of bone marrow hematopoietic stem cells (HSCs) ex vivo. Methods and Results: MECs were collected from the lung tissue of C57BL/6 mice, and HSCs were isolated with immunomagnetic beads from bone marrow of GFP mice. MECs and HSCs were co-cultured with or without having direct cell–cell contact in Transwell device for the measurement of chemotaxis and adhesion of MECs to HSCs. Experimental results indicate that the penetration rate of HSCs from the Transwell upper chamber to lower chamber in ‘co-culture’ group was significantly higher than that of ‘HSC single culture’ group. Also, the HSCs in co-culture group were all adherent at 24 h, and the co-culture group with direct cell–cell contact had highest proliferation rate. The HSC number was positively correlated with vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) levels in supernatants of the culture. Conclusions: Our study reports that MECs enhance the chemotaxis, adhesion and proliferation of HSCs, which might be related to cytokines SDF-1 and VEGF secreted by MECs, and thus MECs enhance the HSC proliferation through cell–cell contact. The present study revealed the effect of MECs on HSCs, and provided a basis and direction for effective expansion of HSCs ex vivo.

Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ Cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential

Historical efforts at expansion of umbilical cord blood (UCB) derived CD34+ hematopoietic stem cells (HSCs) ex vivo with cytokines yielded large numbers of progenitors for transplantation but impaired their long-term engraftment. We used nicotinamide (NAM), an allosteric inhibitor of NAD-enzymes, to create omidubicel, an investigational cell therapy designed to improve the expansion of CD34+ HSCs for bone marrow transplant. A Phase 1/2 clinical study in patients with high-risk hematologic malignancies showed rapid neutrophil engraftment in patients compared to historical controls [1]. We hypothesized that NAM treatment maintains the stemness and engraftment potential of omidubicel, which is associated with clinical benefit [2]. We performed transcriptome analysis to discern the mechanism of action of NAM after ex vivo expansion of CD34+ cells in omidubicel compared to CD34+ controls. Analysis revealed that HSCs expanded with cytokines alone led to an increase in genes responsible for cell proliferation, differentiation, and apoptosis, and in the production of reactive oxygen and nitrogen species (ROS, RNS), and matrix metalloproteinases (MMPs), all of which were attenuated by NAM. Transcription factor (TF) enrichment analysis demonstrated that NAM modulated TFs critically involved in activation of pathways for HSC differentiation, apoptosis, and migration. Specifically, NF-kB, C-Jun, LXR/RXR and PPARα/RXRα, and mTor signaling were all reduced in NAM-treated CD34+ cells compared to controls. NAM treatment upregulated genes linked to cellular metabolism including the Sirtuin family genes, TCA cycle genes, and HIF1a. NAM upregulated genes responsible for telomerase expression further validating our hypothesis that NAM preserves cell stemness. In summary, these data provide further scientific rationale for the favorable clinical engraftment and patient outcomes observed in the Phase 1/2 clinical study of omidubicel. An international, randomized, multi-center Phase 3 study of omidubicel in patients with high-risk hematologic malignancies is underway [2]. [1] Horwitz ME. et. al., J Clin Oncol. 2019 Feb 10;37(5):367-374. [2] ClinicalTrials.gov identifier NCT02730299.

Pluronic-F127/Platelet Microvesicles nanocomplex delivers stem cells in high doses to the bone marrow and confers post-irradiation survival

Platelet microvesicles (pMVs) are submicron-sized heterogeneous vesicles released upon activation and contain several membrane receptors and proteins (CD41, CD61, CD62, CXCR4, PAR-1, etc.). We have revealed their ability to adhere to the triblock copolymer pluronic-F127 (PF127) and form a platelet microvesicular nanocloud which has the potential to enhance the transvascular migration of hematopoietic stem cells across the sinusoidal endothelium to the bone marrow. Besides, the pMVs nanoclouds bestow survival benefits when present on the cells used for infusion, particularly with PF127-stabilized with chitosan-alginate (PF127-CA HSCs). The vesicles were found to be firmly associated with PF127 in the nanocloud, which was detected by confocal laser scanning microscopy. The abrogation of CXCR4/SDF-1 axis regulating the transmigration of the cells by antagonist AMD3100 revealed that the enriched CXCR4 receptors on pMVs robustize the transmigration of the infused cells. The homing of the cells led to effective engraftment and faster regeneration of the critical blood lineages, which elicited 100% survival of the mice receiving lethal doses of radiation. The Human Long-Term Culture Initiating Cells (LTC-ICs), Severe Combined Immunodeficient (SCID) - Repopulating Cells (SRCs) and Colony Forming Cells (CFCs) responsible for the regeneration, but present in extremely low numbers in the infused cell dose, have enabled the cells to reach the bone marrow in high numbers. This potential of the PF127 to sequester the pMVs and its application to achieve over 10-fold delivery of HSCs across the trans-endothelial checkpoint has so far not been reported. Thus, this mechanistic innovation is a potential post-exposure life-saving regimen capable of circumventing the irreparable damage to the bone marrow caused by lethal doses of radiation.

Interactions of Hematopoietic Stem Cells with Bone Marrow Niche.

Hematopoietic stem cells (HSCs) are ultimately responsible for the lifelong renewal of all blood cell lineages. In the bone marrow (BM), HSCs reside in specialized microenvironments referred to as the "niche." HSC niche consists of complex components including heterogeneous cell populations, growth factors, and extracellular matrix molecules. The crosstalk between HSCs and their niche is essential to regulate the survival, self-renewal, migration, quiescence, and differentiation of HSCs. The application of mice models with endogenous ablation of specific cell types, advanced imaging technologies, high-throughput single-cell RNA sequencing, and single-cell mass cytometry methods have provided deep insights into communications between HSCs and niche cells. In this chapter, we have focused on three important cell types in the BM niche: mesenchymal stem cells (MSCs), osteoblasts (OBs), and endothelial cells (ECs). In order to address the interaction between HSCs and these three cell populations in BM niche, we have described methodology for (1) collecting total BM from femur and tibia of C57BL/6 mice; (2) analyzing or sorting of MSCs, OBs, and ECs based on the selection of surface markers CD45, Ter119, CD31, Sca1, and CD51 with flow cytometry; and (3) co-culturing the sorted cells with purified HSCs for further functional assays of HSCs.

Short-term ex-vivo exposure to hydrogen sulfide enhances murine hematopoietic stem and progenitor cell migration, homing, and proliferation

ABSTRACT For successful transplantation of Hematopoietic Stem cells (HSCs), it is quite necessary that efficient homing, engraftment and retention of HSC self-renewal capacity takes place, which is often restricted due to inadequate number of adult HSCs. Here, we report that short-term ex-vivo treatment of mouse bone marrow mononuclear cells (BMMNCs) to Sodium Hydrogen Sulfide (NaHS, hydrogen sulfide-H2S donor) can be used as a possible strategy to overcome such hurdle. H2S increases the expression of CXCR4 on HSPCs, enhancing their migration toward SDF-1α in-vitro and thus homing to BM niche. . Additionally, in-vitro studies revealed that H2S has a role in activating mitochondria, thus, pushing quiescent HSCs into division. These results suggest a readily available and cost-effective method to facilitate efficient HSC transplantation.

A 3D Tissue-wide Digital Imaging Pipeline for Quantitation of Secreted Molecules Shows Absence of CXCL12 Gradients in Bone Marrow.

Technological limitations have hampered understanding of how individual molecules, including putative stem cell regulators, are distributed throughout tissues and stem cell niches. Here, we report adaptation of the proximity ligation assay (PLA) for large-volume, in situ imaging of individual proteins with multipleadditional fluorescent channels with integrated 3D quantification strategies and software. Using this platform, we quantified the bone marrow (BM) distribution of individual CXCL12 chemokine proteins, both before and after their depletion by granulocyte-colony stimulating factor (G-CSF) treatment. We found ubiquitous CXCL12 distributions with local enrichments but no long-range gradients, in contrast to current assumptions about how CXCL12 controls migration of hematopoietic stem and progenitor cells (HSPCs) within BM. This pipeline for discrete digital quantitative, large-volume, multicolor imaging, with up to single-molecule sensitivity, may be broadly applied to any antibody epitope and tissue, enabling further insights into molecular organization of tissues and cellular interactions.

Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ Cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential

Historical efforts at expansion of umbilical cord blood (UCB) derived CD34+ hematopoietic stem cells (HSCs) ex vivo with cytokines yielded large numbers of progenitors for transplantation but impaired their long-term engraftment ability. We used nicotinamide (NAM), an allosteric inhibitor of NAD-enzymes, to create omidubicel, an investigational cell therapy designed to improve the expansion of CD34+ HSCs for bone marrow transplant. A Phase 1/2 clinical study of omidubicel in patients with high-risk hematologic malignancies showed rapid neutrophil engraftment and a more favorable immune reconstitution profile in patients compared to historical controls.1 We hypothesized that NAM treatment maintains the stemness and engraftment potential of omidubicel, which is associated with clinical benefit.2 We performed transcriptome, transcription factor (TF), and pathway analysis by next generation sequencing (NGS) to discern the mechanism of action of NAM and to elucidate the pathways leading to the preservation of engraftment after ex vivo expansion of omidubicel compared to CD34+ cells grown in the absence of NAM. Transcriptome analysis revealed that treatment of CD34+ cells with cytokines alone (stem cell factor [SCF], thrombopoietin [TPO], IL-6, and FLT3 ligand) led to an increase in pathways responsible for cell proliferation and differentiation, apoptotic stress, and production of reactive oxygen species (ROS), and matrix metalloproteinases (MMPs), all of which were attenuated by NAM. TF enrichment analysis of NAM-upregulated genes and downregulated genes demonstrated that NAM modulated several TFs critically involved in pathways of HSC cell self-renewal, differentiation, apoptosis and migration. Specifically, NF-kB, C-Jun, LXR/RXR and PPARα/RXRα, and AMPK-mTor signaling were all reduced in NAM-treated CD34+ cells compared to controls. Reduced expression of key genes involved in the production of ROS and reactive nitrogen species (RNS) including NADPH-oxidase-related genes (CYBB, NCF2 and NCF4) and iNOS, suggested that NAM-expanded CD34+ cells were less exposed to oxygen and nitrogen free radical stress than controls. NAM also downregulated the expression of several matrix metalloproteinases (MMP) genes including MMP7, MMP9, MMP12 and MMP19. NAM-induced downregulation of MMPs may explain the increase in engraftment in patients receiving omidubicel. Pathway analysis of differentially expressed (DE) genes was conducted using ingenuity (IPA) software. IPA analysis of DE genes showed significant downregulation of growth factor activating pathways including SCF, TPO, FLT, and GM-CSF and Endothelin-1 and P2Y Purigenic Receptor, which was confirmed by a reduction in cell cycling rates of labeled cells. IPA analysis also pointed to genes in 3 key cellular pathways that were downregulated by NAM: stress induction of apoptosis, production of ROS and RNS, and production of MMPs. NAM treatment also uniquely upregulated genes linked to cellular metabolism including the Sirtuin family genes, TCA cycle genes, and HIF1a. Interestingly, NAM upregulated genes responsible for telomerase expression further validating our hypothesis that NAM preserves cell stemness. In summary, NGS transcriptome analysis revealed that ex vivo expansion of UCB derived CD34+ cells in the presence of NAM attenuated TFs responsible for proliferation and differentiation of stem cells. In addition, NAM treatment downregulated genes regulating the production ROS, RNS, and MMPs and upregulated genes controlling metabolism and senescence, thus allowing for the expansion of CD34+ cells with preserved function and long-term engraftment ability. Our gene expression data leads to a better understanding of the mechanisms by which NAM modulates CD34+ cells in omidubicel to preserve their function. These data provide further scientific rationale for the favorable clinical engraftment and patient outcomes observed in the Phase 1/2 clinical study of omidubicel.1 An international, randomized, multi-center Phase 3 study of omidubicel in patients with high-risk hematologic malignancies is underway.2 [1]Horwitz M.E., et. al., J Clin Oncol. 2019 Feb 10;37(5):367-374. [2] ClinicalTrials.gov identifier NCT02730299. Disclosures Lodie: Gamida Cell: Employment, Equity Ownership. Adams:Gamida Cell: Employment, Equity Ownership. Yackoubov:GAMIDA CELL: Employment, Other: unexecuted shares of the company . Peled:Gamida Cell: Employment, Equity Ownership.

Deconvolution of Hematopoietic Commitment Decisions By Genome-Wide Analysis of Progressive DNA Methylation Changes

Recent advances in single cell transcriptome analyses have resulted in the derivation of new models to describe the hierarchical organization of the mammalian hematopoietic system. While such an approach appears to be effective at discerning the trajectory of differentiation from hematopoietic stem cells (HSCs) to a given mature lineage, it remains a challenge to identify definitive points where specific lineage fates become restricted. The characterization of molecular events that correspond to such commitment decisions is critical to our interrogation of the existence and nature of serial bifurcation steps that are hypothesized to underlie the process of hematopoiesis. 5-Methylcytosine is a stable epigenetic modification, whose remodeling at specific CpG residues appears to be integral to the process of enforcing lineage-restricted gene expression programs. We have previously observed that the remodeling of the DNA methylome appears to be both progressive and irreversible during the process of hematopoietic differentiation, suggesting that this modification could be used to unambiguously identify molecular marks of lineage commitment. In order to pursue this concept further, we used tagmentation-based whole-genome bisulfite sequencing to generate a genome-wide DNA methylation map of murine hematopoiesis. This map encompasses 26 different FACS-purified populations, ranging from LT-HSCs through to terminally differentiated blood cell lineages. Across all cell populations studied, we identified 147,232 differentially methylated regions (DMRs). In line with our previous data, hierarchical clustering of these DMRs revealed coordinately regulated regions that show progressive and unidirectional lineage-specific DNA methylation dynamics during hematopoietic differentiation that would be indicative of a molecular mechanism of cell fate restriction. Single cell DNA methylome analysis indicated that methylation programming may be exclusive for a specific lineage within each cell analyzed, supporting the use of this data to establish the discreet points at which lineage commitment occurs. Along these lines, lineage-specific DMRs could already be identified within the early hematopoietic stem and multipotent progenitor compartments, supporting the concept that lineage restriction occurs early during differentiation and providing a potential molecular basis for so called lineage-priming/bias. Indeed, a phylogenetic tree of the entire hematopoietic system could be constructed purely based on methylation remodeling events that took place in the Lin-, Sca-1+, c-Kit+ compartment. To gain further insight into how the DNA methylation programming relates to regulation of gene expression, we generated a comprehensive single cell transcriptome map encompassing the entire hematopoietic component of the bone marrow. Integration of DNA methylation dynamics with single cell gene expression dynamics provided evidence for anti-correlation between the transcriptional program and DNA methylation. However, loss of DNA methylation was not invariably associated with an increase in gene expression, suggesting that DNA methylation has more a permissive rather than an instructive role in regulating gene expression programs. We next applied our data set to the exploration of the phenomenon of myeloid lineage bias that has been described in aged mice, by investigating the methylomes of young and aged HSCs. Compared to young HSCs, we identified 3,275 DMRs in aged HSCs, which were predominantly associated with loss of DNA methylation and affected genes involved in HSC adhesion and migration, such as Vwf and ITGB3. Remarkably, 46 % of aging DMRs overlapped with the methylome programs identified in hematopoietic differentiation and were enriched for genes involved in integrin signaling, platelet activation and aggregation. This data suggests that HSC aging results in remodeling of the DNA methylome in vivo, in a manner that is associated with a differentiation bias towards the megakaryocytic lineage. Together, our work provides a rich resource to investigate DNA methylation changes in normal or diseased hematopoiesis, across a broad range of conditions such as aging. Disclosures Lipka: InfectoPharm GmbH: Employment.

PTEN in Regulating Hematopoiesis and Leukemogenesis.

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. By counteracting the PI3K/AKT/mTOR pathway, PTEN plays an essential role in regulating hematopoietic stem cells (HSCs) self-renewal, migration, lineage commitment, and differentiation. PTEN also plays important roles in suppressing leukemogenesis, especially T-cell acute lymphoblastic leukemia (T-ALL). Herein, we will review the function of PTEN in regulating hematopoiesis and leukemogenesis and discuss potential therapeutic approaches against leukemia with PTEN mutations.

Hematopoietic stem cell metabolism and stemness.

Hematopoietic stem cells (HSCs) are considered to originate from the aorta-gonad-mesonephros, migrate into fetal liver for a rapid expansion, and eventually reside into a unique hypoxic bone marrow niche, where they maintain their homeostasis throughout their life span. HSCs have been widely used for the treatment of many begin or malignant hematopoietic disorders. However, the unavailability of sufficient amount of HSCs still impedes their applications in the clinic. It is urgent to understand how HSC stemness or cell fates are determined at different developmental stages. Although many intrinsic and extrinsic factors (niche components) have been identified in the regulation of HSC origination, expansion, migration, and localization, the underlying mechanisms remain largely unknown. In this article, we summarize current views on the metabolic profiles of HSCs and related regulatory networks, which shows that intrinsic metabolic regulation may be critical for the cell fate determinations of HSCs: HSCs utilize glycolysis as their major energy sources; mitochondrial respiration is also required for the homeostasis of HSCs; amino acids, lipids, or other nutrient metabolisms also have unique roles in sustaining HSC activities. Mechanistically, many important regulatory pathways, such as MEIS1/HIF1A, MYC, PPM1K/CDC20, and ROS signals, are identified to fine-tune the nutrient metabolisms and cell fate commitments in HSCs. Nevertheless, more effort is required for the optimization or establishment of sensitive and specific metabolic techniques/systems for the metabolism studies in HSCs with limited cell numbers and exploring the metabolic profiles and fundamental regulatory mechanisms of different types of nutrients at each developmental stage of HSCs.

2020 - GPRASP FAMILY MEMBERS REGULATE MURINE HEMATOPOIETIC STEM CELL MIGRATION, HOMING AND NICHE RETENTION POST-TRANSPLANT VIA CONTROL OF CXCR4 STABILITY

Hematopoietic stem cell (HSC) transplantation (HSCT) represents the only curative therapy for most hematologic disease including leukemia. Recent studies suggest that “stress hematopoiesis”, including that which occurs post-HSCT, is subject to distinct regulation compared steady-state hematopoiesis. Better understanding the molecular mechanisms that regulate transplanted HSCs and their engraftment is necessary to improve HSCT. Thus, we seek to identify novel molecular regulators of HSCT. Our data reveal high expression of multiple GPRASPs (G-Protein Coupled Receptor Associated Sorting Proteins) (e.g. Gprasp1 and Gprasp2) in HSCs relative to downstream progenitors. ShRNA-mediated gene knock-down transplantation-based studies revealed that both GPRASP1 and GPRASP2 act as negative regulators of HSCT, showing 4-fold increase in peripheral blood and bone marrow reconstitution and significantly enhance survival and quiescence in HSCs ex vivo and after transplant. GPRASPs regulate the post-endosomal trafficking of GPCRs to the lysosome. CXCR4, a GPCR that regulates HSCs function, contains a putative GPRASP-binding motif in its C-terminus, implicating it as a possible target of GPRASP-mediated degradation. Indeed, Gprasp1 or Gprasp2 loss increased both total CXCR4 and cell membrane-localized CXCR4 in HSPCs. Consequently, Gprasp1 or Gprasp2 loss increased HSPC migration toward SDF-1. Further, Gprasp1 or Gprasp2 loss significantly enhanced acute homing and HSCs niche retention post-transplant. These GPRASP-knockdown effects disappear when Cxcr4 is genetically deleted. In sum, we report for the first time that multiple GPRASPs function as barriers to HSCT by effecting HSC migration, homing and niche retention via regulation of CXCR4 stability and localization.

Toll-like receptor signaling in hematopoietic stem and progenitor cells.

The innate immune system is essential in the protection against microbial infection and facilitating tissue repair mechanisms. During these stresses, the maintenance of innate immune cell numbers through stress-induced or emergency hematopoiesis is key for our survival. One major mechanism to recognize danger signals is through the activation of Toll-like receptors (TLRs) on the surface of hematopoietic cells, including hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC), and nonhematopoietic cells, which recognize pathogen-derived or damaged-induced compounds and can influence the emergency hematopoietic response. This review explores how direct pathogen-sensing by HSC/HPC regulates hematopoiesis, and the positive and negative consequences of these signals. Recent studies have highlighted new roles for TLRs in regulating HSC and HPC differentiation to innate immune cells of both myeloid and lymphoid origin and augmenting HSC and HPC migration capabilities. Most interestingly, new insights as to how acute versus chronic stimulation of TLR signaling regulates HSC and HPC function has been explored. Recent evidence suggests that TLRs may play an important role in many inflammation-associated diseases. This suggests a possible use for TLR agonists or antagonists as potential therapeutics. Understanding the direct effects of TLR signaling by HSC and HPC may help regulate inflammatory/danger signal-driven emergency hematopoiesis.

Editorial introductions

Editorial introductions

Megakaryocyte volume modulates bone marrow niche properties and cell migration dynamics.

All hematopoietic cells that develop in the bone marrow must cross the endothelial barrier to enter the blood circulation. Blood platelets, however, are released by bigger protrusions of huge progenitor cells, named megakaryocytes, and enter the blood stream as so-called proplatelets before fragmenting into mature platelets. Recently, a second function of megakaryocytes has been identified, as they modulate the quiescence of hematopoietic stem cells, mostly via different soluble factors. We know from light sheet fluorescence microscopy images that megakaryocytes are distributed throughout the bone marrow facing a dense vascular network. Here, we used such three-dimensional images to provide a realistic simulation template reflecting the in vivo cell-vessel distributions resulting in reliable whole-bone analysis in silico. Combining this approach with an automated image analysis pipeline, we found that megakaryocytes influence migration of neutrophils and hematopoietic stem cells, and thus act as biomechanical restrainers modulating cell mobility and extravasation. Indeed, as a consequence of increased megakaryocyte volumes in platelet-depleted mice neutrophil mobility was reduced in these animals.

PS979 PI3K INHIBITION COMBINED WITH STANDARD CHEMOTHERAPY EFFECTIVELY INHIBITS KIT MUTANT AML

Background:AML patients currently demonstrate a dismal overall survival rate (5y OS 25%) in response to standard chemotherapy regimens. The c‐KIT‐encoded receptor protein tyrosine kinase for stem cell factor (KIT) is recurrently mutated in AML (5%). KIT mutations frequently cooccur in AML patients with inv(16)/t(16/16) (15%) and t(8;21) (38%) driver aberrations where they confer inferior prognosis. KIT activation, promoted by the stem cell factor, is essential for survival, migration, and proliferation of normal hematopoietic stem and progenitor cells (HSPCs). KIT mutations in AML, like the KIT(D816Y) in exon 17, confer conformational changes leading to constitutive activation of PI3K and its downstream signalling pathways, which ultimately promotes cell growth, survival and chemotherapy resistance.Aims:In the present we applied the inv(16)/KIT(D816Y) mouse AML model to investigate whether therapeutic targeting of PI3K in combination with standard chemotherapy would inhibit growth and survival of AML synergistically.Methods:Gene Expression Profiling of sorted normal BM progenitor cells and inv(16)/KIT(D816Y) AML cells as well as Western blot analyses demonstrated high PI3K pathway activity in AML cells (i.e. PI3K‐beta and PI3K‐delta) implicating that the inv(16)/KIT(D816Y) AML model would allow to explore the therapeutic potential of PI3K inhibition alone and in combination with standard chemotherapy in a preclinical AML trial program.Results:Colony forming‐cell (CFC) assays were applied to assess the inhibitory effect of a range of PI3K inhibitors and standard chemotherapeutics on clonogenic growth of inv(16)/KIT(D816Y) AML cells vs wild‐type BM cells. Among all tested drugs the pan‐PI3K inhibitor BKM120 (IC50 360 nM), Doxorubicin (IC50 5 nM) and Ara‐C (Cytarabine) (IC50 28 nM) were selected and subjected to subsequent drug combination tests, which demonstrated high synergy scores for BKM120+Doxorubicin (Delta Score = 21) and for BKM120+Ara‐C (Delta Score = 6).We subsequently conducted in vivo pre‐clinical AML trials using varying doses of BKM120 alone and in combination with varying doses of a Doxorubicin/Ara‐C 3+5 chemotherapy regimen. Treatment with BKM120 per oral gavage demonstrated in vivo inhibition of the PI3K pathway, decreased AML burden in bone marrow (BM), spleen, and peripheral blood (PB), and reduced splenomegaly in a dose dependent manner (BKM120: 50 mg/kg/d and 30 mg/kg/d). As expected, the Doxorubicin/Ara‐C 3+5 chemotherapy regimen also inhibited in vivo AML growth in a dose dependent manner (DA‐1: 0,75 mg/kg/d1–3 + 25 mg/kg/d1–5, DA‐2: 1,5 mg/kg/d1–3 + 50 mg/kg/d1–5).Combinatorial treatment with BKM120 and Doxorubin/Ara‐C, increased survival of mice with inv(16)/KIT(D816Y) AML significantly (i.e. 30–42d) compared to untreated mice (17–18d, p>0,001) and mice subjected to single treatment with either BKM120 (22–28d, p = 0,004) or chemotherapy (21–22d, p>0,001).Summary/Conclusion:The present study implicates that targeted inhibition of the KIT/PI3K signaling axis enhances the therapeutic efficiency of standard chemotherapy in KIT mutant AML. Significantly, our findings provide a rationale for development of precision medicine strategies that target cancer‐specific vulnerabilities such as oncogenic signaling in individual AML patients in order to enhance their sensitivity toward conventional chemotherapy, leading to improved clinical outcome.

MiR-125a enhances self-renewal, lifespan, and migration of murine hematopoietic stem and progenitor cell clones

Expansion of hematopoietic stem cells (HSCs) is a ‘holy grail’ of regenerative medicine, as successful stem cell transplantations depend on the number and quality of infused HSCs. Although many attempts have been pursued to either chemically or genetically increase HSC numbers, neither clonal analysis of these expanded cells nor their ability to support mature blood lineages has been demonstrated. Here we show that miR-125a, at the single cell level, can expand murine long-term repopulating HSCs. In addition, miR-125a increases clone longevity, clone size and clonal contribution to hematopoiesis. Unexpectedly, we found that miR-125a expanded HSCs clones were highly homogenously distributed across multiple anatomical sites. Interestingly, these miR-125a overexpressing cells had enhanced mobility and were more frequently detected in the spleen. Our study reveals a novel, cell-intrinsically controlled mechanism by which HSC migration is regulated.

Translating HSC Niche Biology for Clinical Applications

Over the final 3 decades of the twentieth century, the advent and evolution of hematopoietic stem cell transplantation (HSCT) to cure refractory malignancies and severe non-malignant diseases created a need to better understand basic scientific mechanisms underlying the maintenance and migration of hematopoietic stem cells (HSC). Over the past 20 years, researchers have discovered complex overlapping bone marrow (BM) HSC niches that utilize a number of signaling pathways to tightly regulate HSC physiology. Here, we review recent efforts to define critical mediators of HSC niche function in health and disease, and how these discoveries are now enabling the development of the next generation of cellular therapies for cancer and severe non-malignant diseases. A number of cellular interactions and molecular pathways critical for HSC mobilization, donor HSC engraftment after HSCT, and HSC/progenitor cell recovery following chemotherapy have recently been identified. Additional studies have defined mechanisms by which cancer and bone marrow failure states disrupt the normal function of these pathways. Translational investigators are now leveraging these discoveries to develop preclinical and clinical strategies to target the niche for regenerative and cancer therapy. Ongoing research to define fundamental aspects of HSC niche biology will lead to further refinements and precision approaches to improve the safety and efficacy of clinical cell therapy.