Hematopoietic Stem And Progenitor Cells Survival Research Articles

Establishment of human hematopoietic organoids for evaluation of hematopoietic injury and regeneration effect

BackgroundHuman hematopoietic organoids have a wide application value for modeling human bone marrow diseases, such as acute hematopoietic radiation injury. However, the manufacturing of human hematopoietic organoids is an unaddressed challenge because of the complexity of hematopoietic tissues.MethodsTo manufacture hematopoietic organoids, we obtained CD34+ hematopoietic stem and progenitor cells (HSPCs) from human embryonic stem cells (hESCs) using stepwise induction and immunomagnetic bead-sorting. We then mixed these CD34+ HSPCs with niche-related cells in Gelatin-methacryloyl (GelMA) to form a three-dimensional (3D) hematopoietic organoid. Additionally, we investigated the effects of radiation damage and response to granulocyte colony-stimulating factor (G-CSF) in hematopoietic organoids.ResultsThe GelMA hydrogel maintained the undifferentiated state of hESCs-derived HSPCs by reducing intracellular reactive oxygen species (ROS) levels. The established hematopoietic organoids in GelMA with niche-related cells were composed of HSPCs and multilineage blood cells and demonstrated the adherence of hematopoietic cells to niche cells. Notably, these hematopoietic organoids exhibited radiation-induced hematopoietic cell injury effect, including increased intracellular ROS levels, γ-H2AX positive cell percentages, and hematopoietic cell apoptosis percentages. Moreover, G-CSF supplementation in the culture medium significantly improved the survival of HSPCs and enhanced myeloid cell regeneration in these hematopoietic organoids after radiation.ConclusionsThese findings substantiate the successful manufacture of a preliminary 3D hematopoietic organoid from hESCs-derived HSPCs, which was utilized for modeling hematopoietic radiation injury and assessing the radiation-mitigating effects of G-CSF in vitro. Our study provides opportunities to further aid in the standard and scalable production of hematopoietic organoids for disease modeling and drug testing.

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

Heme is required not only in red blood cells for oxygen transport but also in non-erythroid cells for the proper function of hemoproteins. Cytochrome c is one of the hemoproteins and inevitable for mitochondrial oxidative phosphorylation, suggesting that heme possesses important roles for ATP synthesis and mitochondria maintenance. Heme also can skew the differentiation of hematopoietic stem and progenitor cells (HSPCs) by altering the functions of transcription factors. Recent CRISPR Cas9 dropout screens repeatedly showed that some of the heme synthases, such as CPOX, might be inevitable for the survival and/or proliferation of HSPCs. However, still little is known about the roles for heme synthesis in the maintenance (survival, proliferation and differentiation) of HSPCs. To reveal the roles for heme synthesis in HSPCs in vivo, we developed polyI:C inducible hematopoietic cell specific Cpox knock-out mice ( Cpox f/f; Mx1-Cre). PolyI:C treated Cpox f/f; Mx1-Cre mice (KO mice) showed significant increase in the numbers of hematopoietic stem cells (HSCs) and multipotent stem and progenitor cells (MPP2s and MPP3s) but decrease in the numbers of common myeloid progenitors (CMPs) and some of their progenies (i.e., Gr1 positive cells and platelets). In contrast, its effects on erythroid and lymphoid lineages were limited. This result suggests that heme synthesis inhibition in HSPCs (HSCs, MPP2 and MPP3s) can cause their differentiation arrest. On the other hand, competitive transplantation analysis of LSKs (lineage -sca1 +c-kit +) or HSCs revealed that myelopoiesis were markedly decreased compared to control, suggesting that heme synthesis in HSPCs is important for the proper reconstitution of hematopoiesis. These results indicate that cell intrinsic heme synthesis is inevitable for the maintenance of HSPCs and their lineage specific output in vivo. In depth, we revealed that mitochondrial respiratory activity of Cpox KO HSCs were increased compared to control HSCs, suggesting that alterations of mitochondrial status and/or functions by the inhibition of heme synthesis might contribute to the aberrance in Cpox KO HSPCs. Comprehensive gene expression analysis is now ongoing to support this notion. To further reveal the roles for heme synthesis in human HSPCs, we treated human leukemia cell lines (MV4-11, KG1a and U937) with heme synthesis inhibitors (SA or NMPP). Concentration-dependent cell growth inhibitory effect and cell death were observed with increased apoptosis factors (cleaved caspase 3 and cleaved PARP), suggesting that heme synthesis inhibition triggered apoptosis. We also revealed that CPOX inhibition under doxycycline inducible CRISPR Cas9 system caused cell growth inhibitory effect and cell death in these cells. Second mitochondria-derived activator of caspases (Smac) and cytochrome c were released to cytoplasm, and the inhibitor of apoptosis protein (XIAP) was decreased by heme synthesis inhibitions. Therefore, caspase-3 activation by accumulation of cytoplasmic cytochrome c and Smac might be the inducer of this apoptosis pathway. Comprehensive transcriptomic analysis (RNA-seq) revealed that heme synthesis inhibition induced the release of cytochrome c and Smac from mitochondria into cytoplasm by the transcriptomic upregulation of Oxidative Stress Induced Growth Inhibitor 1 (Osgin1) that is the known target of NRF2. Collectively, this study demonstrated the significance of heme synthesis in the maintenance of HSPCs especially in vivo (mouse) and in vitro (human). Dysfunctions of heme synthesis in HSPCs can cause their aberrance by the alterations in their mitochondrial status which can cause apoptosis and/or differentiation arrest. Considering the metabolic differences (i.e., demands for mitochondrial oxidative phosphorylation and subsequent ATP synthesis) between normal HSPCs and leukemia or unperturbed hematopoiesis and perturbed hematopoiesis (i.e., infections and hematopoietic stem cell transplantations), further analysis to reveal the roles for heme synthesis in the maintenance of HSPCs will pave the way to establish optical approaches in these clinical settings.

DNMT3A-Mutated Stem and Progenitor Cells Contribute to Altered T Cell Activation in Clonal Hematopoiesis

Clonal hematopoiesis is characterized by an expansion of blood cells derived from a single mutated hematopoietic stem cell (HSC). When this expansion is driven by a leukemia-associated mutation that exceeds a variant allele frequency (VAF) of 2%, it is termed clonal hematopoiesis of indeterminate potential (CHIP). Approximately half of CHIP cases are driven by mutations in the DNA methyltransferase gene DNMT3A. In patients with CHIP, greater clonal expansion is associated with an increased risk of cardiovascular diseases and hematologic malignancies. While DNMT3A impairment has been shown to alter inflammatory signaling in HSCs and myeloid cells, less is known about the impact of DNMT3A-mutated CHIP on T cell biology. Here, we leverage a cohort of human CHIP samples to assess how the expansion of DNMT3A-mutated cells affects gene expression across blood cell types and generate a new mouse model to assess antigen-specific immune cell interactions. We find evidence that the accumulation of DNMT3A-mutated stem and progenitor cells can cause T cell dysfunction. We first identified CHIP in a cohort of 191 sternum bone marrow samples collected during heart surgery. We detected CHIP in 45 samples (23.6%), including 22 samples with mutated DNMT3A (48.9% of CHIP samples). To mitigate confounding factors that might be introduced by comparing CHIP to healthy individuals, we used ten DNMT3A-mutated CHIP samples for this study, splitting them into a low-VAF (<10%, three samples) and high-VAF (>10%, seven samples) cohorts. We performed single-cell RNA sequencing on these 10 samples, generating high-quality data for 95,245 cells. We clustered and annotated 33 cell types according to the expression of canonical marker genes, representing the full spectrum of hematopoietic progenitors to myeloid, erythroid and lymphoid lineages. To evaluate how greater DNMT3A clonal expansion affects immune cell states, we performed differential gene expression analysis in the high-VAF compared to the low-VAF cohort with DESeq2 using a pseudobulk approach followed by gene set enrichment analysis using Hallmark pathways. We found that TGF-beta signaling, known for its immunosuppressive role, is upregulated in hematopoietic stem and progenitor cells (HSPCs), myeloid and T cell populations in the high-VAF compared to the low-VAF cohort. Both interferon alpha and gamma signaling pathways are consistently downregulated in HSPCs and multiple myeloid cell types as well as T cells. TNF-alpha signaling is upregulated in HSPCs and myeloid cells, consistent with recent observations linking TNF-alpha to the survival and proliferation of HSPCs and myeloid cells. Overall, greater expansion of DNMT3A-mutated cells is associated with variable gene expression changes in progenitor and myeloid cell types, whereas gene expression changes in T cells are consistent with diminished activation (Figure 1). To evaluate mechanisms contributing to altered T cell activation in DNMT3A-mutated CHIP, we combined our Dnmt3a-fl-R878H/+ inducible mouse model with the ovalbumin (OVA) trackable antigen system. Endogenously expressed OVA is processed into a peptide that is loaded onto MHCI and recognized by transgenic CD8+ T cells from OT-I mice. We crossed the Dnmt3a-fl-R878H/+ mouse with a mouse expressing OVA under the actin promoter, resulting in Dnmt3a-mutated mice with constitutive OVA expression. To interrogate antigen-specific T cell responses, we co-cultured Dnmt3a-mutated OVA+ lineage negative (Lin-) stem and progenitor cells with CD8+ T cells from OT-I mice. By measuring T cell proliferation using CFSE dilution, we observed an increase in T cell proliferation when CD8+ T cells were co-cultured with Dnmt3a-mutated OVA+ Lin- cells compared to wildtype OVA+ Lin- cells (Figure 2). These results suggest that Lin- Dnmt3a-mutated cells have an increased capacity for antigen-specific T cell activation. In conclusion, our studies demonstrate that greater expansion of DNMT3A-mutated CHIP is associated with reduced expression of activation signatures in T cells. In apparent contrast, Dnmt3a-mutated mouse cells have an increased capacity for antigen-specific T cell stimulation. Since chronic antigenic stimulation causes T cell exhaustion, our findings are consistent with a model in which the expansion of DNMT3A-mutated progenitor and myeloid cells causes chronic stimulation and dysfunction of T cells.

Heme-deficient primitive red blood cells induce HSPC ferroptosis by altering iron homeostasis during zebrafish embryogenesis.

The crosstalk between hematopoietic lineages is important for developmental hematopoiesis. However, the role of primitive red blood cells (RBCs) in the formation of definitive hematopoietic stem and progenitor cells (HSPCs) is largely unknown. Primitive RBC deficiencies in mammals always lead to early embryonic lethality, but zebrafish lines with RBC deficiencies can survive to larval stage. By taking advantage of a zebrafish model, we find that the survival of nascent HSPCs is impaired in alas2- or alad-deficient embryos with aberrant heme biosynthesis in RBCs. Heme-deficient primitive RBCs induce ferroptosis of HSPCs through the disruption of iron homeostasis. Mechanistically, heme-deficient primitive RBCs cause blood iron-overload via Slc40a1, and an HSPC iron sensor, Tfr1b, mediates excessive iron absorption. Thus, iron-induced oxidative stress stimulates the lipid peroxidation, which directly leads to HSPC ferroptosis. Anti-ferroptotic treatments efficiently reverse HSPC defects in alas2 or alad mutants. HSPC transplantation assay reveals that the attenuated erythroid reconstitution efficiency may result from the ferroptosis of erythrocyte-biased HSPCs. Together, these results illustrate that heme-deficient primitive RBCs are detrimental to HSPC production and may provide potential implications for iron dysregulation-induced hematological malignancies.

Evaluation of Unrestricted Somatic Stem Cells Therapy in an Ex-Vivo and in-Vivo Acquired Severe Aplastic Anemia Model

Background: In acquired severe aplastic anemia (aSAA), oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Liao and Cairo et al (Cell Transplantation, 2014) have demonstrated that cord blood-derived unrestricted somatic stem cells (USSCs) could significantly reverse the abnormal/inverted ratio of CD4:CD8 T cells, increases the number of FOXP3A positive Treg cells and decrease the level of IFN-γ in an animal model of recessive dystrophic epidermolysis bullosa (RDEB). Alternative therapies are in great need for patients with aSAA as immunosuppressive therapy (IST) overall responses are sub-optimal. Objectives:To determine the effect of USSCs on HSPC survival, differentiation and phosphorylation pathways in an ex-vivo culture of aSAA with human CD34+ cells.To determine the effect of USSCs administration on blood cell count recovery, bone marrow (BM) cellularity and survival in an in-vivo inflammatory cytokine mediated mice model of aSAA. Design/Methods: Human HSPC were cultured with SCF, FLT3 and human TPO. HSPCs alone, with and without IFN-γ and TNF-α served as controls. USSCs (transwell and direct) were added to the culture. HSPC were harvested and assayed for their survival at day 7 and 14. Annexin-V+/PI+ were referred to as late apoptotic cells. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO2. Multi-lineage differentiation capacity was assessed with a selective colony forming units (CFU) assay and compared between experimental groups. Signaling pathways were determined using phosphor-flow cytometric analysis of pSTAT1. For the immune mediated mouse model of aSAA, 8-week-old C57BL/6 mice were irradiated with 6.5Gy TBI. Spleen lymphocytes were collected from FVB donors, and injected 4 hours post-radiation into B6 recipient mice via the retro-orbital vein, at 1-1.5 × 106 cells/recipient in 100μL PBS. Each experimental group (PBS control and USSCs) included 20 mice per group. Mice were continuously monitored for their survival. Peripheral blood (PB) was analyzed using a VETSCAN® HM5 Analyzer. Mean ± SEM of the blood counts was compared between experimental groups. Mice femur bones were extracted and cleaned of soft tissue, fixed and sectioned. Paraffin sections were stained with Hematoxylin and Eosin (H&E) to assess morphology. Results: The myelosuppressive effect of IFN-γ and TNF-α was significantly rescued (4-fold increase) by the addition of USSCs (transwell) at day 7. After 28 days direct co-culture with USSCs increased CD34+ survival as well (p<0.001) from 5 to 35 cells/µl. A significantly higher percent of viable cells (73% versus 63%) was observed among the USSCs treated cells (P<0.05). Phosphor-flow cytometric analysis demonstrated an almost 50% decrease in pSTAT1 in the presence of USSCs (p<0.05). Based on CFU analysis, CD34+ cells cultured in the presence of USSCs were able to produce more CFU (52, SD±3.7) at day 7 (p<0.01) compared to IFN-γ and TNF-α treated cells (16, SD±2.8). Also, USSCs were associated with more BFU (p<0.05) at day 14. In our immune mediated aplastic anemia mice model, treatment with USSCs was associated with substantial prolonged mice survival (p=0.02) (Figure 1a). Evaluation of PB counts demonstrated increased hemoglobin levels at day 14 in the USSCs treated mice (p<0.05) 5.6g/dL (SD±0.6), compared to PBS treated mice 2.1g/dL (SD±0.1). BM evaluation of USSCs treated mice versus PBS control displayed higher cellularity in the USSCs treated sections (Figure 1b). Conclusions: Cellular therapy, such as USSCs, may be instrumental in the treatment of aSAA, specifically by increasing BFU-E and eventually hemoglobin levels. Future studies will need to be done to investigate the exact mechanisms of action and the clinical efficacy of this novel cellular therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IFN-γ Inhibitors and Ruxolitinib Therapy for Acquired Severe Aplastic Anemia in an Ex-Vivo Model

Background:Severe aplastic anemia (SAA) is a life-threatening disorder that is associated with multiple etiologies, both inherited and acquired. In acquired SAA, oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Currently, the first line treatment for patients who have a suitable HLA matched donor is a hematopoietic progenitor cell transplant (HPCT). When HPCT is not possible, due to lack of a closely matched HLA donor and/or concomitant co-morbidities, then the treatment of choice is immunosuppression with anti-thymocyte globulin, cyclosporine and eltrombopag (ELT)(Georges et al, Blood, 2018). Alvarado et al (Blood, 2019) recently demonstrated that ELT bypasses the inhibitory effect of IFN-γ by alternatively activating TPO signaling. However, ELT cannot overcome other IFN-γ mediated effect through JAK-STAT1 phosphorylation or apoptosis via Fas/FasL. Alternative therapies are in great need for patients with aSAA as treatment response is sub-optimal.Objective:To determine the effects of IFN-γ neutralizing antibodies or Ruxolitinib on HSPCs survival, proliferation and differentiation in an ex vivo culture of human CD34+ cells in the presence of IFN-γ and TNF-α.Design/Methods:Human CD34+ HSPCs were isolated from cord blood, based on CD34 microbeads magnetic selection (Miltenyl Biotec, Germany). The CD34+ cells were cultured in StemSpan Serum-Free Medium II (STEMCELL Technologies) supplemented with 5 ng/mL human stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and 5 ng/mL recombinant human TPO. A 1x10 5 CD34+ HSPCs were seeded in a 96 well plate. HSPC alone, with and without IFN-γ (100 ng/mL) and TNF-α (10 ng/mL), were the negative and positive control, respectively. Specific IFN-γ neutralizing antibody, B27 (BD Pharmingen), MD1 (BioLegend) and B133.5 (ImmunoTools) or Ruxolitinib (Jakafi, Incyte) were added to the culture and HSPC were harvested and assayed for their survival at day 7 and 14. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO 2. Also, we assessed the multi-lineage differentiation capacity with a selective colony forming units (CFU) assay. Fourteen days after co-culture of each experimental treatment, 500 CD34+ cells were seeded in 6-well plates Smart Dish™ (STEMCELL Technologies). Big burst forming units of erythroid (BFU-E), CFU of granulocyte and megakaryocyte (GM) and granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) were counted and compared between the experimental groups. The signaling pathways were determined using phospho flow-cytometric analysis of pSTAT1, pSTAT3, pSTAT5. All statistically analyzed data is represented as mean ± SD. Differences between groups were analyzed by multiple 2-tailed unpaired Student t tests using Excel. Statistically significant differences were represented as *P <0.05, ** P <0.01 and *** P<0.001.Results:The number of CD34+ cells at day 7 and day 14 of culture in the presence of IFN-γ and TNF-α was significantly lower (21% ± 3 (p<0.0006) and 15% ± 6 (p<0.02), respectively, than that of the control (Fig 1). Importantly, the myelosuppressive effect of IFN-γ and TNF-α was significantly rescued by the addition of Ruxolitinib (at day 14, p<0.05) or IFN-γ neutralizing antibodies (B27 and B133.5, respectively) (day 7 and 14, p<0.01). Improvement in HSPC survival ranged between 1.5-3.3 fold compared to our negative control. Based on the CFU analysis, the CD34+ cells cultured in the presence of B27, B133.5 or Ruxolitinib, were able to produce more CFU at day 7 (p<0.01, p<0.01 and p<0.05 respectively)(Fig 2A). Additionally, Both B27 (p < 0.01) and Ruxolitinb (p ≤ 0.001) were found to produce more CFU-GM on day 14 (Fig 2B). Phospho flow-cytometry demonstrated a significant decrease in STAT1 phosphorylation of CD34+ cells in the presence of B27 and B133.5 (p<0.05, p<0.001, respectively).Conclusions:Our preliminary studies supports the potential benefits of utilizing IFN-γ neutralizing antibodies or Ruxolitinib to improve HSPC survival, proliferation and differentiation in aSAA. Future studies will need to be done to investigate the exact mechanisms of action and the effects of IFN-γ neutralizing antibodies in an animal model of aSAA. [Display omitted] DisclosuresCairo: Amgen: Speakers Bureau; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau. OffLabel Disclosure:Ruxolitinib was used to inhibit JAK-STAT signaling pathway in an Ex-Vivo model of Aplastic Anemia. Drug wasn't supplied by drug company.

Microtubule Polymerization Inhibition Enhances Human Hematopoietic Stem Cell Homing and Engraftment

Hematopoietic stem cells (HSCs) serve as the origin of the hematopoietic system, with the ability to differentiate into all blood cell lineages and self-renewal to sustain the hematopoiesis throughout life. Hematopoietic stem cell transplantation (HSCT) currently represents the most effective therapeutic strategies to treat hematological and non-hematological diseases. However, limited numbers of HSCs or poor homing capabilities into the bone marrow are still major hurdles for successful HSCT. Moreover, graft failure and delayed reconstitution due to inefficient engraftment, remains an important complication because of the high morbidity and mortality. Although ex vivo expansion of HSCs has been well studied for decades, which displays huge potentials for clinical application, exploration of novel targets to improve HSC homing and engraftment will provide new insights to enhance HSCT efficacy.To explore the chemical compounds enhancing the capabilities of homing and engraftment, we used CXCR4 (CD184) as the readout bio-marker, which is considered as the most essential chemokine receptor of HSPCs interacting with CXCL12 (SDF1) secreted in BM niche to support HSPCs homing, migration, proliferation and survival. We first performed chemical screening of 139 small molecules that can increase CD184 expression on cord blood (CB) CD34 + hematopoietic stem and progenitor cells (HSPCs). We concluded that treatment of CB CD34 + HSPCs for 16 hours with Lexibulin (Lex) or Vinblastine Sulfate (VS), both of which were microtubule polymerization (MP) inhibitors, could significantly promote the CD184 expression.Next, we optimized the MP inhibitors treatment conditions including dosage, treatment duration and culture time prior to treatment. The results proved that treatment with Lex or VS for 16 hours at 1μM was the optimal conditions to significantly enhance the CD184 expression of CD34 + HPSCs and LT-HSCs (CD34 +CD90 +CD45RA -), while maintaining robust cell survival, when compared with the DMSO control group. Moreover, we found that only when HSPCs were under culture within two days prior to small molecules treatment, CD184 expression was significantly increased by MP inhibitors while maintaining high viability, compared with DMSO control group.In order to assess the in vivo repopulating potential of the CB CD34 + HSPCs post treatment with MP inhibitors, we transplanted CB-HSPCs 16 hours post-treatment with Lex and VS respectively into irradiated nonobese diabetic (NOD)/Prkdc scid/IL-2Rγ null (NPG) mice. All transplanted mice of MP inhibitors-treated groups presented efficient engraftment, in multiple immune organs at 4-16 weeks post-transplantation, suggesting greater engraftment potential than the mock group, as measured by human CD45 of total CD45. Furthermore, hematopoietic reconstitution analysis indicated that the MP inhibitors -treated cells maintained different lineage distribution in peripheral blood (PB), bone marrow (BM) and spleen. Moreover, the equivalent phenotypes of pre- and post-treatment reveal the better reconstitution by MP inhibitors was independent of HSC-enrichment, Thus, short-term MP inhibitors treatment of CB CD34 + HSPCs enhances their homing and long-term engraftment.In conclusion, we demonstrated that short-term microtubule polymerization inhibition on human CB CD34 + HSPCs could not only enhance CD184 cell surface expression but also the capabilities of in vivo human HSCs homing and reconstitution via screening chemical compounds to increase CD184 expression and the following function evaluation study. Vinblastine Sulfate and Lexbulin were applied or registered as anti-cancer drugs for clinical use. Our study also indicates that MP inhibitors pretreatment of cells possesses significant translational implications, designating MP inhibitors as promising drug candidates to facilitate clinical HSCT. [Display omitted] DisclosuresFang: EdiGene, Inc.: Current Employment.

Lnk/Sh2b3 Deficiency Restores Hematopoietic Stem Cell Function and Genome Integrity in Fanconi Anemia

▪Although initially identified over 85 years ago, Fanconi Anemia (FA) remains a fatal genetic disease. Patients with FA exhibit developmental malformations, bone marrow failure, and increased cancer susceptibility. Twenty-one FA genes cooperate in a genome stability pathway that is essential for repair of DNA interstrand crosslinks (ICLs) and attenuation of replication stress. Cells derived from FA patients are hypersensitive to ICL-inducing agents such as Mitomycin C (MMC), a hallmark of FA. In addition, FA hematopoietic stem and progenitor cells (HSPCs) show growth retardation and increased apoptosis culminating in hematopoietic stem cell (HSC) exhaustion and bone marrow failure. The poor growth of HSPCs also poses a significant obstacle in gene therapy for FA patients. Moreover, unrepaired DNA damage increases genome instability and promotes leukemia and cancer. Of note, interventions to mitigate HSPC defects in FA do not exist, aside from allogeneic bone marrow transplantation.Remarkably, we reveal here that loss of the adaptor protein Lnk (also called Sh2b3) suppresses FA associated phenotypes without further accelerating neoplastic transformation. LNK is a negative regulator of HSC self-renewal in part by attenuating cytokine signaling, in particular thrombopoietin (TPO)-activated JAK2 signaling in HSCs. Fancd2-/-;Lnk-/- mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals. The reconstituting ability of Fancd2-/-;Lnk-/- HSCs is also largely restored to WT levels in serial transplantation assays. In addition, Lnk deficiency restored the growth and survival of FA HSPCs in ex vivo culture. More importantly, Lnk deficiency mitigated chromosomal aberrations associated with FA upon proliferation. To determine if Lnk deficiency mitigates ICL repair, we examined MMC sensitivity in primary HSPCs that were dually null for Lnk and FancD2. Our data suggest that LNK does not play an overt role in ICL repair. Rather, Lnk deficiency in HSPCs mitigated replication stress by stabilizing replication forks and reduces genome instability associated with FA. Hence, our data suggest that Lnk deficiency alleviates replication stress-induced DNA damage and genome instability, thereby restoring cell proliferation and survival of Fancd2-/- HSPCs ex vivo and HSC functions in vivo.Last and importantly, we began to test if LNK inhibition restores human FA HSPCs. We first generated FA-like HSPCs by knocking down FANCD2 in normal umbilical cord blood (UCB) CD34+ cells. Our results demonstrated that shRNA-mediated depletion of LNK in human CD34+ cells rescued progenitor cell numbers and growth in FA-like HSPCs. Take together, our findings reveal a previously unappreciated role of cytokine/JAK signaling in alleviating replication stress and promoting replication-associated genome maintenance. Our studies also illuminate replication stress as a major underlying origin of bone marrow failure in FA patients and have implications for therapeutic strategies to treat FA associated bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Phosphatidylinositol-3 kinase signaling controls survival and stemness of hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild-type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single-cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multipotent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs.

Connexin-43 Is a Negative Regulator of Mitochondrial Fission, Mitophagy and Apoptosis of Dividing Hematopoietic Stem Cells through the Drp1-Pink1 Axis

Lifelong production of blood by hematopoietic stem and progenitor cells (HSPC) depends on stem cells self-renewal and their maintenance by bone marrow (BM) microenvironment. BM chemotherapy or whole body irradiation frequently causes hematological abnormalities in cancer patients. HSC within the BM exhibit quiescent state with low mitochondrial reactive oxygen species (ROS) and hyperpolarizing membrane potential, however upon stress hematopoiesis HSC exit quiescence and either self-renew or differentiate in mature cells. Understanding the mechanism controlling hematopoiesis regeneration upon replicative stress is expected to provide molecular targets for amelioration of chemotherapy induced toxicity. Mitochondria recognize and respond to many stresses by modifying mitochondrial dynamics and macroscopic autophagy (mitophagy), and maintains HSC function both at intrinsic and extrinsic level (Ho et al., Nature 2017). Upon myeloablative stress, hematopoietic Connexin 43 (H-Cx43) preserves HSPC survival and efficient blood formation by transfer of damaging excess ROS to BM stromal cells (BMSC), preventing HSC apoptosis and lethal hematology failure (Taniguchi-Ishikwawa E et al., PNAS 2012), and its intermediate C-loop domain has been shown to act as negative regulator of the beclin complex of microautophagy (Eloy Bejarano et al, Nat Cell Biol. 2014). Since HSC apoptosis depends on mitochondrial activation and mitochondrial homeostasis is maintained by coordinated regulation of mitochondrial fission/fusion and mitophagy, we hypothesized that mitochondrial Cx43 may exert a cell-autonomous activity over the mitochondrial turnover upon cell division in the regeneration phase after myelotoxic chemotherapy. To analyze the role of Cx43 in HSC mitochondrial dynamics and fate, we created HSC mitochondrial reporter mice by crossing Cox8 mitochondrial signal localization peptide-Dendra2 fusion protein transgenic (mito-Dendra2) mice with Vav-Cre; Cx43f/f (H-Cx43Δ/Δ)mice. While quiescent Cx43Δ/Δ HSC do not demonstrate any major phenotype, snapshot high resolution microscopy demonstrated that Cx43 deficiency enhances mitochondrial superoxide levels, depolarizes membrane potential and results in larger mitochondria accumulation (~2 fold) after forced division of HSC. In addition, time lapsed cinematography of red-fluorescent, photo-converted mito-Dendra2 demonstrated that Cx43-deficient mitochondria in cycling HSC split into multiple smaller fragments. Phosphorylation of Drp1 (Ser616) and its accumulation with mitochondria is higher in dividing Cx43Δ/ΔHSC, suggesting that Cx43 is a negative regulator of mitochondrial fission and its deficiency facilitates mitochondrial fragmentation. Inhibition of mitochondrial fission with overexpression of the dominant-negative Drp1 mutant (K38A) or the Drp1 inhibitor Mdivi1 prevents the effect of Cx43 deficiency on mitochondrial hyper-fragmentation. The fate of the smaller fragments is mitophagy as demonstrated by increased co-localization of Cx43 deficient mitochondria with the ubiquitin kinase Pink1, the autophagy reporter's p62 and LC3 punctae, and the lysosomal membrane proteins Lamp1/2 in HSC. Genetic silencing of Atg7 or addition of the autophagolysosome inhibitor bafilomycin A1 to dividing HSC prevents the induction of mitophagy in dividing Cx43Δ/Δ HSC. Overexpression of structure-function mutants in Cx43Δ/Δ HSC (Cys-less, with 6 point mutations in Cysteine residues replaced by Alanine, resulting in impaired hemichannel docking or the truncated C-terminus mutant D257 resulting in impaired signaling and intramolecular interactions needed for channel gating) demonstrated that both the formation of whole intercellular channel and the presence of C-terminus once Cx43 channel formed are indispensable for the negative regulatory role of Cx43 on mitophagy. Our results thus identify gap junction protein Cx43 as an important regulator of mitochondrial fission and mitophagy in dividing HSC which may be a target for preservation of hematopoietic stem cell regenerative capacity. DisclosuresNo relevant conflicts of interest to declare.

Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells.

Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver-derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

Reduced Erg Dosage Impairs Survival of Hematopoietic Stem and Progenitor Cells.

ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Ergkd ) in which Erg expression can be conditionally restored by Cre recombinase. Ergkd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Ergkd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin- Sca-1+ c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Ergkd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785.

Glucose-Dependent Insulinotropic Polypeptide Receptor Deficiency Leads to Impaired Bone Marrow Hematopoiesis.

The bone marrow (BM) contains controlled specialized microenvironments, or niches, that regulate the quiescence, proliferation, and differentiation of hematopoietic stem and progenitor cells (HSPC). The glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone that mediates postprandial insulin secretion and has anabolic effects on adipose tissue. Previous studies demonstrated altered bone microarchitecture in mice deficient for GIP receptor (Gipr-/- ), as well as the expression of high-affinity GIP receptor by distinct cells constructing the BM HSPC niche. Nevertheless, the involvement of GIP in the process of BM hematopoiesis remains elusive. In this article, we show significantly reduced representation and proliferation of HSPC and myeloid progenitors in the BM of Gipr-/- mice. This was further manifested by reduced levels of BM and circulating differentiated immune cells in young and old adult mice. Moreover, GIP signaling was required for the establishment of supportive BM HSPC niches during HSPC repopulation in radioablated BM chimera mice. Finally, molecular profiling of various factors involved in retention, survival, and expansion of HSPC revealed significantly lower expression of the Notch-receptor ligands Jagged 1 and Jagged 2 in osteoblast-enriched bone extracts from Gipr-/- mice, which are important for HSPC expansion. In addition, there was increased expression of CXCL12, a factor important for HSPC retention and quiescence, in whole-BM extracts from Gipr-/- mice. Collectively, our data suggest that the metabolic hormone GIP plays an important role in BM hematopoiesis.

5-hydroxytryptamine synthesized in the aorta-gonad-mesonephros regulates hematopoietic stem and progenitor cell survival.

The in vitro or ex vivo production of transplantable hematopoietic stem cells (HSCs) holds great promise for the treatment of hematological diseases in the clinic. However, HSCs have not been produced from either embryonic or induced pluripotent stem cells. In this study, we report that 5-hydroxytryptamine (5-HT; also called serotonin) can enhance the generation of hematopoietic stem and progenitor cells (HSPCs) in vitro and is essential for the survival of HSPCs in vivo during embryogenesis. In tryptophan hydroxylase 2-deficient embryos, a decrease in 5-HT synthesized in the aorta-gonad-mesonephros leads to apoptosis of nascent HSPCs. Mechanistically, 5-HT inhibits the AKT-Foxo1 signaling cascade to protect the earliest HSPCs in intraaortic hematopoietic clusters from excessive apoptosis. Collectively, our results reveal an unexpected role of 5-HT in HSPC development and suggest that 5-HT signaling may be a potential therapeutic target for promoting HSPC survival.

Prostaglandin E2 Promotes the Sequential Recovery of Bone Marrow Vasculature and the Megakaryocyte Lineage Following Radiation Injury

The sinusoidal vascular niche is the site of terminal megakaryopoiesis and platelet production in the bone marrow. The association of megakaryocytes (MKs) with the sinusoidal endothelium and microenvironmental cues from the vascular niche are critical for efficient thrombopoiesis. Thrombocytopenia is an important clinical problem that can cause significant morbidity for patients receiving genotoxic treatments such as chemotherapy and radiation therapy. Following myeloablative bone marrow injury, the recovery of both hematopoietic stem and progenitor cells (HSPCs) and the MK lineage is dependent on marrow vascular recovery (Hooper et al. Cell Stem Cell 2009, Avecilla et al. Nature 2004). Accordingly, we reasoned that improving recovery of the vasculature might serve as a rational therapeutic strategy for thrombocytopenia stemming from cytotoxic treatments. We focused our efforts on the arachidonic acid derivative prostaglandin E2 (PGE2), which regulates numerous physiologic processes including microvascular endothelial cell proliferation and angiogenesis. Within the context of the bone marrow, PGE2 also has diverse roles in HSPC expansion, survival, and trafficking. Importantly, systemic administration of the long-acting analogue 16,16-dimethyl PGE2 (dmPGE2) improves hematopoietic recovery and enhances overall survival following total body irradiation (TBI) in mice (Hoggatt et al. Blood Cells 2013). Intriguingly, following sublethal TBI, dmPGE2-induced recovery of peripheral platelets precedes restoration of red blood cells and neutrophils (Porter et al. Stem Cells 2013). We hypothesized that the preferential recovery of platelets following dmPGE2 administration is mediated by accelerated vascular recovery in the marrow. We tested this in a murine model of sublethal radiation injury in which we had previously characterized MK lineage kinetics following 4Gy TBI (Niswander et al. Blood 2014). First, the kinetics of MK progenitors (MKPs), maturing MKs, and platelets were determined at days 5, 7, and 10 in mice receiving 6mg/kg dmPGE2 or vehicle at 48 and 72 hours post-4Gy TBI. Mice receiving dmPGE2 had an early decrease in both MKPs and platelets, with no change in MKs at day 5 post-TBI. However, by day 10, dmPGE2-treated mice demonstrated significantly enhanced recovery of both MKs and circulating platelets compared to controls, but no difference in the number of immunophenotypic HSPCs. Having confirmed enhanced recovery of the MK lineage with dmPGE2 treatment, we next sought to characterize vascular recovery. Marked injury and dilation of the marrow sinusoidal vasculature occurs rapidly following TBI. Immunohistochemistry with morphometric analysis revealed no change in vascular dilation at day 5 post-TBI with dmPGE2 treatment. However, mice receiving dmPGE2 exhibited a more than 25% improvement in vascular dilation that was first evident at day 7 and was sustained at day 10 post-TBI. While vascular dilation post-TBI has been posited to correlate with loss of surrounding hematopoietic cells, improvement in vascular dilation with dmPGE2 treatment is contrastingly evident in the setting of slightly fewer (day 7) or equivalent (day 10) numbers of nucleated marrow cells compared to irradiated control mice. These data suggest that dmPGE2-mediated recovery of the vasculature coincides temporally with the accelerated onset of MK lineage recovery. In addition, immunohistochemical morphometric analyses indicate that dmPGE2-treated mice had an 80% increase in maturing MKs in physical contact with the vascular endothelium, the site of platelet production, at day 10 post-TBI. Taken together, these data support the concept that dmPGE2-mediated marrow vascular recovery following radiation injury enhances the recovery of the MK lineage and accelerates the production of circulating platelets. Modulation of vascular recovery with dmPGE2 may be a promising therapeutic strategy for thrombocytopenia secondary to myeloablative bone marrow injury. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Standard sub-thermoneutral caging temperature influences radiosensitivity of hematopoietic stem and progenitor cells.

The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs). To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI). Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C) resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to β-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the β-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon β-adrenergic signaling. Since β-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI.

Related-to-receptor tyrosine kinase receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice

Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a noncanonical Wnt ligand receptor termed 'related-to-receptor tyrosine kinase' (Ryk). Expression of Ryk on HSPCs invivo is associated with a lower rate of proliferation, and, following treatment with fluorouracil (5-FU), the percentage of Ryk(+/high) HSPCs increased and the percentage of Ryk(-/low) HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling invivo resulted in increased hematopoietic-stem-cell proliferation and decreased hematopoietic-stem-cell function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures:No relevant conflicts of interest to declare.

Maintenance Of Bone Marrow Residency and Self-Renewal Of Leukemic Stem Cells By Cdc42 Gtpase

The leukemic stem cell (LSC) depends on specific interactions with extracellular matrix, soluble factors, and cellular components of the microenvironment, or niche. These interactions promote LSC self-renewal and survival, thus contributing to chemoresistance and treatment failure. Understanding the signaling pathways that promote LSC maintenance in response to niche interactions may reveal novel targets for therapy. Recent studies indicate a critical role for the small Rho GTPase, Cdc42, in the maintenance of normal hematopoietic stem and progenitor cells (HSPCs). Cdc42 coordinates actin cytoskeleton organization, adhesion, migration, self-renewal, cell polarity, proliferation, and survival of normal HSPCs in response to niche signaling through multiple cell surface receptors, including CXCL12/CXCR4, SCF/KIT, and fibronectin/integrin. Cdc42 activity is increased in both murine and human models of MLL-AF9 (MA9) acute myeloid leukemia (AML). Cdc42 expression is also increased in human patient AML samples across cytogenetic subtypes, compared to normal hematopoietic cell subsets, in analysis of curated datasets in the HemaExplorer database. In earlier work, we have shown that Cdc42 inhibition leads to peripheral mobilization of leukemia cells out of the marrow niche (Blood 114, 13). In the present study, we investigate whether Cdc42 inhibition also disrupts intrinsic LSC self-renewal. To interrogate Cdc42 in LSC self-renewal, MA9 cell lines were established following transduction of bone marrow HSPCs harvested from tamoxifen-inducible Cdc42 knockout mice, with Cre null donors as controls. Upon tamoxifen (TAM) treatment, Cdc42KO-MA9 cells had decreased CFU and small, diffuse colony morphology. Mice transplanted with untreated MA9 cells were divided to receive injections of TAM vs control. The Cdc42KO-MA9 cohort remains alive at over 180 days post-transplant, whereas vehicle control mice died of AML with latency similar to Cre null MA9 cell recipients (p<0.005). In vivo deletion of Cdc42 from MA9 leukemia in secondary recipients prolonged disease latency (p<0.005). AML cells recovered from vehicle control mice showed decreased growth in culture, reduced CFU content, and increased apoptosis following treatment with TAM to delete Cdc42. Cdc42KO-MA9 leukemia cells also had higher side scatter and Gr-1 expression, and decreased c-Kit, suggesting differentiation. These data indicate that Cdc42 is required for murine LSC maintenance. We used Tet-inducible shRNA targeting of Cdc42 in human cell lines expressing MA9 and mutant NRas (MA9/NRas). Cdc42 knockdown reduced MA9/NRas colony-forming ability, blocked actin polymerization and migration in response to CXCL12, and induced apoptosis. MA9/NRas cells co-expressing inducible Cdc42 shRNA and luciferase were transplanted into NSGS mice on doxycycline chow to induce knockdown. Bioluminescence imaging showed delayed AML progression in the knockdown group compared to non-targeting shRNA and regular chow controls. Thus, Cdc42 deficiency in human MA9 LSC reproduces the phenotype seen in the mouse genetic model. We used a novel small-molecule Cdc42-activity specific inhibitor, CASIN, to test pharmacologic inhibition of Cdc42 in AML. Consistent with knockdown data, in vitro CASIN treatment blocked MA9 cell colony-forming ability, actin polymerization, and migration. CASIN treatment led to specific induction of apoptosis in MA9 cells, while normal human umbilical cord blood CD34+ cells showed no significant toxicity in the dose range tested. Together, these studies show that Cdc42 signaling is critical to intrinsic LSC self-renewal and engagement of the niche, and Cdc42 inhibition represents a rational therapeutic principle to target LSC maintenance. Disclosures:No relevant conflicts of interest to declare.

Nfix is a novel regulator of murine hematopoietic stem and progenitor cell survival

AbstractHematopoietic stem cells are both necessary and sufficient to sustain the complete blood system of vertebrates. Here we show that Nfix, a member of the nuclear factor I (Nfi) family of transcription factors, is highly expressed by hematopoietic stem and progenitor cells (HSPCs) of murine adult bone marrow. Although short hairpin RNA–mediated knockdown of Nfix expression in Lineage−Sca-1+c-Kit+ HSPCs had no effect on in vitro cell growth or viability, Nfix-depleted HSPCs displayed a significant loss of colony-forming potential, as well as short- and long-term in vivo hematopoietic repopulating activity. Analysis of recipient mice at 4 to 20 days posttransplant revealed that Nfix-depleted HSPCs are established in the bone marrow, but fail to persist due to increased apoptotic cell death. Gene expression profiling of Nfix-depleted HSPCs reveals that loss of Nfix expression in HSPCs is concomitant with a decrease in the expression of multiple genes known to be important for HSPCs survival, such as Erg, Mecom, and Mpl. These data reveal that Nfix is a novel regulator of HSPCs survival posttransplantation and establish a role for Nfi genes in the regulation of this cellular compartment.