Competitive Repopulation Assay Research Articles

Deletion of PTPN1 Promotes the Development of Myelofibrosis

Deletion of chromosome 20q [del(20q)] is a common chromosomal abnormality associated with myeloid neoplasms including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN overlap disorders and acute myeloid leukemia (AML). The del(20q) lesion is often associated with myeloproliferative features; it is present in patients with myelofibrosis (MF) at a high frequency (24%) and thus considered to be one of the most frequent cytogenetic abnormalities in MF (Wassie et al., Br J Haematol. 2015). The del(20q) lesion can also coexist with JAK2V617F mutation in MPN/MF. However, the target tumor-suppressor gene(s) within chromosome 20q involved in the pathogenesis of MF remains unknown.The PTPN1 locus maps to human chromosome 20q13.1-q13.2. PTPN1 (also known as PTP1B) is a ubiquitously expressed non-receptor tyrosine phosphatase that has been linked to metabolism and cancer. Mice deficient in Ptpn1 exhibit resistance to diet-induced obesity and diabetes. Both oncogenic and tumor suppressor functions for PTPN1 have been suggested. PTPN1 can negatively regulate the JAK/STAT signaling, which is frequently found activated in MPN.Here, we report the identification and functional consequences of PTPN1 deletion in the pathogenesis of MF. Deletion of PTPN1 was identified in 14% cases of MF. Conditional deletion of Ptpn1 in the mouse hematopoietic compartment resulted in significant increases in white blood cell and neutrophil counts in the peripheral blood and enlargement of spleen size. Flow cytometric analyses showed significant expansion of myeloid (Gr-1+/Mac-1+) precursors in the bone marrow (BM) and spleens of Ptpn1-deleted mice compared with control animals. Megakaryocytic (CD41+/CD61+) precursors were also significantly increased in the spleens of Ptpn1-deleted mice. Flow cytometric analyses also revealed significant increases in absolute numbers of LSK cells (Lin-Sca1+c-kit+) and its subsets including long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC) and multi-potent progenitors (MPP) in the spleens of Ptpn1-deleted mice. Hematopoietic progenitor colony assays showed significant increases in myeloid (CFU-GM) and megakaryocytic (CFU-Mk) colonies in the BM of Ptpn1-deleted mice compared with control mice BM. Histopathologic analysis demonstrated fibrosis (grade 2) in the BM and spleens of Ptpn1-deleted mice at 52 weeks after induction, whereas control animals did not exhibit fibrosis at that age. Together, these results suggest that deletion of Ptpn1 induces an MPN-like phenotype, which progresses to MF over time. Moreover, transplantation of Ptpn1-deficient BM into lethally irradiated wild-type animals resulted in fibrosis at 18 weeks after transplantation, demonstrating that the effect of Ptpn1 loss in the development of myelofibrosis is cell-intrinsic. Competitive repopulation assays using BM from control or Ptpn1-deficient (CD45.2+) mice with wild type congenic (CD45.1+) mice showed that deletion of Ptpn1 enhances the repopulation capacity of hematopoietic stem cells. Biochemical analyses revealed that depletion of Ptpn1 enhanced JAK2/STAT5, AKT and ERK signaling in the BM of Ptpn1-deleted mice. Furthermore, we observed that deletion of Ptpn1 in Jak2V617F knock-in mice accelerates the development of myelofibrosis. In conclusion, our results establish a tumor-suppressor function for PTPN1 in MF. DisclosuresNo relevant conflicts of interest to declare.

EPCR Guides Hematopoietic Stem Cells Homing to the Bone Marrow Independently of Niche Clearance

Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) are active and essential first steps during embryonic development and in clinical stem cell transplantation. Rare, BM LT-HSCs endowed with the highest self-renewal and durable repopulation potential, functionally express the anticoagulant endothelial protein C receptor (EPCR) and PAR1. In addition to coagulation and inflammation, EPCR-PAR1 signaling independently controls a BM LT-HSC retention-release switch via regulation of nitric oxide (NO) production within LT-HSCs. EPCR+ LT-HSCs are maintained in thrombomodulin+ (TM) periarterial BM microenvironments via production of activated protein C (aPC), the major ligand for EPCR. Restriction of NO production by aPC-EPCR-PAR1 signaling, activates VLA4-mediated adhesion, anchoring EPCR+ LT-HSCs to the BM and protecting them from chemotherapy insult, sparing hematological failure and premature death (Gur-Cohen S. et al, Nat Med 2015).We report that transplanted EPCR+ LT-HSCs preferentially homed ‎to and were retained in the BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing treatment that block aPC binding and attenuate EPCR+ LT-HSC BM homing. Furthermore, short term aPC in vitro pretreatment dramatically augmented EPCR+ LT-HSC BM homing, lodgment and long-term repopulation. PAR1 deficient stem cells were irresponsive to treatment with aPC and displayed reduced BM homing efficiency, all pointing to the aPC-EPCR-PAR1 axis as a crucial mediator of BM LT-HSC homing. Additionally, aPC pretreated EPCR+ LT-HSCs had a striking advantage to competitively home to the BM. Consistently, BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with wild type stem cells in competitive repopulation assays. Importantly, the competitive homing results strongly imply that the BM available niches for newly arrived EPCR+ LT-HSCs are limited. Indeed, aPC pretreated EPCR+ LT-HSCs BM homing reached a plateau, as increasing the transplanted cell dose above 5x106 BM mononuclear cells, did not yield higher donor EPCR+ LT-HSC homing. These results reveal that there is a limited BM space for newly arrived transplanted EPCR+ stem cells to non-irradiated hosts. Importantly, we found that EPCR+ LT-HSCs can engraft the BM of non-conditioned mice with high efficiency, while remaining in a dormant, non-cycling state. Furthermore, the dormant homed EPCR+ LT-HSCs were later awakened and activated solely by treating the engrafted hosts with a low dose 5-FU chemotherapy, or with NO donor SNAP, revealing that preconditioning and clearance of occupied BM HSC niches are not required.To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by unique BM arterioles, and not in the spleen. BM homed EPCR+ LT-HSCs were found adjacent to TM+ arterioles, imposing their retention. Homed BM EPCR+ LT-HSCs highly express full-length TM with intact lectin-like domain, and the BM TM+ endothelium was found to be enriched with a Glycocalyx layer, in particular with Heparan Sulfate Proteoglycan-2 (HSPG-2). HSGP-2 might specifically interact with the lectin-like domain of TM-expressingLT-HSCs, providing BM specific recognition and accelerated homing. Intriguingly, stabilizing TM function by in vitro pretreatment with platelet factor-4 (PF4) bypassed BM-derived cues and increased EPCR+/TM+ LT-HSC homing also to the spleen, suggesting a supportive role for PF4, highly secreted by BM megakaryocytes, in guiding EPCR+/TM+ LT-HSCs to the BM.Herein we define EPCR as a guidance molecule, navigating LT-HSC specifically to BM TM+ aPC-secreting blood vessels, allowing stem cell retention and protection from DNA damaging agents. The BM harbors a limited number of available stem cell niches for newly arrived transplanted EPCR+/TM+ LT-HSCs, and in vitro aPC pretreatment dramatically augments EPCR+/TM+ LT-HSC BM homing. Our findings provide new mechanistic insights and identify key players concerning LT-HSC homing specifically to the BM, leading to better repopulation following transplantation. This up-to-date approach and new knowledge may potentially lead to improved BM transplantation protocols and to prevent chemotherapy resistance of EPCR-expressing cancer stem cell mediated relapse. DisclosuresRuf:Iconic Therapeutics: Consultancy.

JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive.To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs.We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. DisclosuresNo relevant conflicts of interest to declare.

Development of a New Generation, Forward-Oriented Therapeutic Vector for Hemoglobin Disorders

Ameliorating hemoglobin disorders such as sickle cell disease (SCD) using hematopoietic stem cell (HSC) gene therapy is under development. Unlike in other diseases, therapeutic globin vectors have demanding requirements including high-level β-globin expression, tissue specificity among erythroid cells, long-term persistence, and high-level modification at the HSC level. These demanding requirements necessitate the inclusion of complex genetic elements including the locus control region (LCR), β-globin promoter, β-globin gene, and the 3' untranslated region (3'UTR), all now feasible using lentiviral vectors. The additional requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent loss by RNA splicing during viral preparation. This reverse-orientation is in contrast to all other therapeutic vectors under clinical development. Current reverse-oriented globin vectors can drive phenotypic correction in mouse models for both b-thalassemia and SCD, while they are limited by lower viral titers and lower transduction efficiency in primary human HSCs, limiting their prospects, especially in SCD. We hypothesized that the reverse-orientation impedes both viral preparation and vector transduction, as despite deletion of cryptic polyadenylation (polyA) signals to optimize a conventional reverse-oriented globin vector, titers were still 10-fold lower than a standard GFP-vector.We thus designed a forward-oriented globin-expressing vector, which was further optimized by minimizing the size of the LCR, inclusion of a large segment of the β-globin promoter and an enhancer region of the 3’UTR lacking the polyA signal. Viral titers of the forward-oriented vectors (1.0±0.2x10e9 IU/mL) were 6-fold higher than the optimized vector in the reverse orientation (1.6±0.2x10e8 IU/mL, p<0.01), and comparable to a standard GFP-marking vector (1.9±0.2x10e9 IU/mL, p<0.01). The forward-oriented vector demonstrated 3-4 fold higher transduction efficiency among human erythroid cells derived from transduced CD34+ cells in in vitro culture (34±0% vs 13±1%, p<0.01) and in xenografted mice (31±9% vs 7±5%, p<0.05).To evaluate transduction efficiency for long-term HSCs, we transduced rhesus CD34+ cells with our optimized reverse-oriented vector and our forward-oriented vector including GFP or YFP genes (instead of β-globin gene) in a competitive repopulation assay following 10 Gy total body irradiation. In two animals, GFP and YFP signals from both vectors were detected exclusively in red blood cells, documenting tissue specificity. Gene marking levels were 10-fold higher out to 4 years with the forward-oriented vector, compared to the reverse-oriented vector, and were comparable to standard GFP or YFP-marking vectors in 2 other animals.We then replaced the GFP gene with the β-globin gene containing intron 2 in the forward-oriented vector construct. To positively select intron-2-containing β-globin vectors, essential viral components (packaging signal, rev response element (RRE), or central polypurine tract (cPPT)) were deleted in the backbone of the forward-oriented vector, and the deleted viral components were inserted into intron 2 of the β-globin gene. We observed that half of the forward-oriented vectors lost intron 2 during vector preparation when no elements were included into intron 2. Insertion of the RRE resulted in positive selection of intron-2-containing β-globin vectors. We confirmed β-globin expression from the forward-oriented vector by hemoglobin A production in human erythroid cells derived from transduced peripheral blood mononuclear cells from SCD patients. Finally, human β-globin expression was detected in rhesus erythroid cells following transplantation of transduced CD34+ cells in 2 animals.In summary, we have developed a clinically relevant forward-oriented globin-expressing vector, which has 6 fold higher viral titers and 4-10 fold higher transduction efficiency for hematopoietic repopulating cells, as compared to the optimized reverse-oriented vector. RRE insertion allowed positive selection of intron-2-containing β-globin vectors, and human β-globin production was observed in transplanted rhesus macaques with the forward-oriented β-globin vector transduction. These findings bring us closer to a curative gene therapy for hemoglobin disorders. DisclosuresNo relevant conflicts of interest to declare.

Fbxw11 Variants Control the Quiescence of HSCs

Ubiquitination is a posttranslational mechanism that controls diverse cellular processes. Fbxw11, a constituent of the SCF (Skp1-Cul1-F-box) ubiquitin ligase complex, targets for degradation of several important transcription factors, including NF-κB, β-cantenin and ATF4. Fbxw11 may play pivotal roles in many aspects of hematopoiesis through regulating various signal transduction pathways. However, the role of Fbxw11 on HSCs quiescence remains largely unknown. In this study, we cloned three transcript variants (Fbxw11a, Fbxw11c and Fbxw11d) to study the biological function of Fbxw11 in hematopoiesis.To elucidate Fbxw11 distribution in hematopoietic system, hematopoietic cell populations at different stages of differentiation were isolated from bone marrow of 8 week-old mice and Fbxw11 expression was studied by real-time PCR. Expression of Fbxw11 were lower in purified long term hematopoietic stem cells (LT-HSC, LSK CD34- Flt3-), but higher in short term hematopoietic stem cells (ST-HSC, LSK CD34+ Flt3-), hematopoietic stem and progenitor cells (LSK), and various hematopoietic progenitor cells. The results reveal that Fbxw11 is preferentially expressed in more mature progenitor cells. The expression of Fbxw11 in mature blood cells was also studied showing that Fbxw11 was expressed at lower level in neutrophils, higher level in B and T lymphocytes, and moderate level in monocytes.To assess the impact of Fbxw11 on reconstitution capacity of LT-HSCs, we cloned Fbxw11a, Fbxw11c and Fbxw11d into retrovirus system, respectively. LSK cells were infected with MSCV-Fbxw11a/Fbxw11c/Fbxw11d-IRES-GFP or the blank control vector MSCV-GFP. Competitive repopulation assays we performed 48h later after infection, and reconstitution in peripheral blood (PB) was analyzed every 4 weeks. Repopulation of donor cells expressing high level of Fbxw11 variants was significantly lower than those infected with control vector at 1 and 4 months in PB and at 4 months in BM after transplantation. These data indicate that Fbxw11 is negative for the long-term repopulating capacity of HSCs.To further confirm the effects of Fbxw11 variants in hematopoiesis, the effect of Fbxw11 variants on the growth and enumeration of hematopoietic progenitor cells was detected by colony-forming cell assay (CFC). The number of CFU-G, CFU-GM, CFU-GEMM and the total number of CFU were lower in LSK over-expressing Fbxw11 variants when compared with LSK control. To determine the cell-cycle distribution of HSC cells, Hoechst 33342 and Ki67 staining were performed showing that G0 phase LSK cells were decreased when they over-expressing Fbxw11 variants.In conclusion, our data reveal unrecognized roles for Fbxw11 in the regulation of HSPCs. Our findings suggest that Fbxw11 variants have negative effect on reconstitution capacity of LT-HSCs. Fbxw11 variants decrease the reconstitution capacity through promoting cell proliferation, which results in loss of hematopoietic stem cell quiescence. We anticipate that our experiments will facilitate the understanding of hematopoiesis through which Fbxw11-mediated signals control HSC quiescence and functions.The work was supported by the Grants 81300376, 81370634, 81570153 from the National Natural Science Foundation of China (NSFC); 14JCQNJC10600 from the Tianjin Science and Technology Programs; DisclosuresNo relevant conflicts of interest to declare.

Smc3 Haploinsufficiency and Smc3 Deletion Alter Hematopoiesis In Vivo

Recurrent mutations in SMC3, encoding a cohesin subunit, have been identified in acute myeloid leukemia (AML) and other myeloid malignancies by our group and others. SMC3 mutations are heterozygous in AML patients. Missense, nonsense, and splice site mutations have been observed across all domains of SMC3. Given the breath of mutations, it is important to determine whether these represent recurrent loss-of-function mechanisms, or if some might have dominant negative effects. To determine the impact of Smc3 deletion on hematopoiesis, we studied both Smc3 haploinsufficient and Smc3 deficient mice as models of loss-of function and dominant negative phenotypes respectively.The Smc3 haploinsufficient mouse model has a lacZneo gene trap inserted in intron 3-4 of Smc3, which leads to a premature transcription stop and therefore produces a truncated and dysfunctional protein. The homozygous Smc3trap allele is embryonic lethal. The Smc3trap/+ mice have an early growth defect, although their body weight catches up to wild type mice after 6 weeks of age. We found no difference in spleen weights, peripheral blood counts, and bone marrow (BM) compositions between Smc3trap/+ and wild type mice. The Smc3trap/+ BM cells formed similar number of colonies as wild type cells when plated in methylcellulose in vitro and lost self-renewal capabilities after replating for two weeks. Competitive repopulation assay in vivo showed neither advantage nor disadvantage for the Smc3trap/+ BM cells (n=10). Thus, Smc3trap/+ BM cells have normal colony forming capacity in vitro and normal homeostatic feedback in vivo.Further, we generated Smc3 conditionally deficient mice by removing the gene-trap cassette, which retains the loxP sites flanking exon 4 (Smc3fl), and crossing these mice with either Vav1-Cre+/- or ERT2-Cre+/- to delete the allele (Smc3fl/+/Vav1-Cre+/- is constitutively haploinsufficient in hematopoietic cells, whereas Smc3fl/+/ERT2-Cre+/- is only haploinsufficient when induced with tamoxifen). We characterized both models by serial replating assays, flow cytometry assays for hematopoietic stem/progenitor cells (HSPCs), and BM lineage in vitro and found no difference in these mice compared to the Smc3fl/+ control. In contrast to the Smc3fl/+/Mx1-Cre+/- mice (Viny et al. JEM 212 (11): 1819-1832), we observed a significant competitive disadvantage for the Smc3fl/+/ERT2-Cre+/- BM cells (p<0.0001, n=10), most pronounced in Gr1+ myeloid cells in vivo (p<0.0001), implying Smc3 haploinsufficiency alters hematopoiesis in those mice in vivo.We characterized the effects of homozygous Smc3 loss on hematopoiesis in the inducible Smc3fl/fl/ERT2-Cre+/- mice by treating mice with tamoxifen at 6 weeks of age (Smc3fl/fl/Vav1-Cre+/- is embryonic lethal). Deletion of Smc3 led to rapid bone marrow failure and 100% lethality with a median survival of 8 days (n=4, 2 independent experiments). At the time of death, we observed severe reduction in the sizes of spleen (Sp) and thymus (Thy), in total number of BM, Sp, and Thy cells, and in white blood counts, lymphocytes, monocytes, and platelets. The Smc3 deficient BM cells had decreased levels of Smc3 by Western blot. The impact of Smc3 deletion on HSPC functions in vivo was assessed by a competitive repopulation assay of Smc3fl/fl/ERT2-Cre+/- BM cells (p<0.0001, n=10). Recipient mice were treated with tamoxifen after 6-week engraftment. After tamoxifen-mediated deletion, Smc3 deficient cells were rapidly outcompeted in vivo, indicating complete loss of HSPC functions.Collectively, these results suggest that Smc3 is necessary for normal hematopoiesis and for HSPC functions. The AML-associated SMC3 mutations are therefore unlikely to be dominant negative because the complete loss of Smc3 is incompatible with hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

A Small Molecule Inhibitor of Protein Tyrosine Phosphatase-Sigma (PTPσ) Promotes Hematopoietic Stem Cell (HSC) Regeneration

Receptor tyrosine kinases (RTKs), such as c-kit, Flt-3 and Tie2, regulate hematopoietic stem cell (HSC) proliferation, differentiation and maintenance. Substantially less is known regarding the function of receptor protein tyrosine phosphatases (PTPs) in regulating HSC fate. We recently discovered that receptor protein tyrosine phosphatase sigma (PTPσ) is highly expressed by murine and human HSCs. Interestingly, constitutive deletion of PTPσ caused a marked increase in HSC repopulating capacity as measured in primary and secondary competitive repopulation assays (J Clin Invest 2015;125:177-182). Further, negative selection of human cord blood (CB) HSCs for PTPσ surface expression (PTPσ - negative CB HSCs) conferred more than 10-fold increased human CB hematopoietic engraftment through 20 weeks in transplanted NSG mice. Additionally, PTPσ-deficient mice displayed significantly augmented recovery of phenotypic bone marrow (BM) HSCs and colony forming cells at day +10 following 550 cGy total body irradiation. Based on these observations, we hypothesized that PTPσ may function as a negative regulator of HSC self-renewal and regeneration. We sought to develop pharmacologic strategies to inhibit PTPσ function as a means to augment HSC functional capacity. Based on structure-activity-relationship analysis of PTPσ, we screened candidate small molecules for ability to modulate PTPσ function on BM HSCs. We identified a small molecule, 5483071 (Chembridge), as a candidate PTPσ inhibitor. In silico simulation indicated that 5483071 rigidly docked into the binding site of PTPσ intracellular domain through hydrogen bonding and electrostatic interactions. In a model of HSC injury and regeneration, we irradiated C57Bl6 mice with 700 cGy TBI and treated mice systemically with either 10 μcg of 5483071 or water subcutaneously every other day from day +1 to day +14. Irradiated mice treated with 5483071 displayed significantly increased 60 day survival compared to controls (P=0.0007). Irradiated mice treated with 5483071 showed accelerated recovery of BM SLAM+kit+sca-1+lin- HSCs (P=0.02), BM KSL cells (P=0.01), and colony forming cells (CFCs) (P=0.0003). In vitro culture of BM KSL cells with 5483071 significantly increased the levels of activated Rac1 (P=0.0004), which recapitulated the effects of PTPσ deletion on Rac1 activation in HSCs. Importantly, treatment of BM KSL cells from PTPσ -/- mice with 5483071 caused no change in Rac1 activation, suggesting that 5483071 acted specifically on PTPσ and was not mediating effects via inhibition of other phosphatases. Systemic administration of 5483071 to irradiated mice caused an increase in BM KSL cell cycling at 72 hours compared to irradiated control mice (p=0.02), while also decreasing BM KSL cell apoptosis at 24 hours after TBI (p=0.02). Subsequent to these findings, we have generated several new small molecule inhibitors of PTPσ with novel composition of matter and have taken a lead compound into pre-clinical studies for investigational new drug (IND) development. PTPσ represents a novel receptor tyrosine phosphatase that regulates HSC self-renewal and regeneration. Targeted inhibition of PTPσ has high therapeutic potential to promote hematopoietic regeneration in patients receiving myelosuppressive chemotherapy and/or radiotherapy or undergoing myeloablative hematopoietic cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem Cells Fail to Regenerate In Vivo Following Inflammatory Stress

An often-cited defining property of hematopoietic stem cells (HSCs) is their extensive or unlimited in vivo self-renewal capacity. We have recently described a novel mouse disease model forFanconi anemia, in which serial challenge with pro-inflammatory agonists that mimic infection, such aspolyinosinic:polycytidylic acid (pI:C), results in HSC attrition followed by a highly penetrant severe aplastic anemia, closely recapitulating the disease in patients (Walter et al., 2015, Nature).In order to explore the broader implications of these findings in the context of HSC self-renewal, we conducted apI:Cdose escalation regimen using standard C57BL6 mice. A single injection withpI:Cprovoked transient peripheral blood (PB)cytopenias, with the recovery of mature blood cell numbers correlating with HSCs being forced into active cell cycle. Injection with 1-3 rounds ofpI:C(1-3 x 8 injections) led to no discernable sustained impact on blood production as, at 5 weeks post-treatment, PB frequencies were in the normal range, as were the absolute numbers of HSCs and all progenitor compartments in the bone marrow (BM), as determined by flowcytometry.However, in vitro analysis of the proliferation and differentiation potential of 411 individual sorted long-term (LT)-HSCs 5 weeks after 3 rounds of pI:C challenge, revealed a decrease in the frequency of LT-HSCs able to generate progeny in vitro (1.6-fold reduction, p<0.05), and a 2-fold reduction in the total number of progeny produced per HSC, which was even more pronounced inmultilineage potential clones (2.6-fold decrease, p<0.0001) compared touni- or bi-lineage clones. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers with increasing rounds ofpI:C treatment, with a 1.8, 3.4 and 15.3-fold decrease in donorchimerism across all lineages at 6 months post-transplantation (p<0.01) following 1, 2 or 3 rounds ofpI:C treatment, respectively. Notably, robust engraftment (up to 65% donorchimerism, 6 months post-transplantation, p<0.01) was achieved when mice exposed to 3 rounds ofpI:C treatment were used as a recipient for non-treated BM cells in the absence of any irradiation conditioning, while engraftment was always <1% when non-treated controls were used as recipients. This excludes the possibility that the observed progressive depletion of functional HSCs was the result of artifacts associated with a compromised niche or the non-physiologic stress imposed on donor cells during transplantation.In order to test the kinetics of HSC recovery following HSC challenge, BM was harvested from mice at either 5, 10 or 20 weeks after treatment with 3 rounds of pI:C, and both competitive and limiting dilution transplantation assays (Table 1) were used to quantify HSC frequencies. Surprisingly, both assays demonstrated that HSCs failed to regenerate at all following pI:Cchallenge, directly contradicting the canonical view that HSCs possess extensive self-renewal capacity in vivo. The physiologic relevance of this observation was illustrated when we measured the hematologic parameters of aged mice that had been exposed to chronicpI:C treatment in early to mid-life. Although these mice had normal PB counts at 4 weeks post-treatment, at 2 years of age, peripheral bloodcytopenias and bone marrow aplasia became evident (Table 2), recapitulating clinically relevant features of non-malignant aged human hematopoiesis that are never seen in standard laboratory mice. Together, these data suggest that functional HSCs can be progressively and irreversibly depleted in response to environmental agonists, such as infection and inflammation, which force HSCs to reconstitute mature blood cells consumed by such stimuli.This model has clear implications relating to the role of adult stem cells in tissue maintenance and regeneration during ageing, and how stress agonists that are absent in most laboratory animal models, but would be ubiquitous in the wild, are likely key mediators of age-associated disease pathologies. [Display omitted] [Display omitted] DisclosuresFrenette:PHD Biosciences: Research Funding; Pfizer: Consultancy; GSK: Research Funding.

The Role of H2AFY in U2AF1 Mutant Cells and Normal Hematopoiesis

Mutations in genes that are involved in pre-mRNA splicing (i.e., spliceosome genes; U2AF1, SF3B1, SRSF2 and ZRSR2) are particularly frequent in MDS, affecting ~50% of MDS patients. Our group showed that expression of U2AF1(S34F), the most common U2AF1 mutant, causes altered splicing in human and mouse cells and altered hematopoiesis in mice. How U2AF1(S34F) expression contributes to altered hematopoiesis is unclear. We hypothesized that U2AF1(S34F) alters splicing of target genes that affect hematopoiesis. Using three independent sets of RNA-seq data from human acute myeloid leukemia (AML) patients, human CD34+ hematopoietic cells, and mouse common myeloid progenitor cells that express either wild-type or mutant U2AF1, we identified consistent alternative splicing of H2AFY. H2AFY, also known as macroH2A1, is a histone H2A variant implicated in transcription, development, and tumorigenesis. Alternative splicing of H2AFY generates two isoforms, H2AFY1.1 and H2AFY1.2. In many solid cancers, alternative splicing of H2AFY is observed, resulting in reductions in H2AFY1.1 isoform expression. Similarly, in all three RNA-seq datasets and RT-PCR of primary MDS samples, mutant U2AF1 samples express H2AFY1.1 at 30% of the levels observed in samples without a spliceosome mutation (p<0.01). In order to determine whether expression of H2AFY1.1 could rescue a cellular phenotype in U2AF1 mutant cells, we exogenously expressed H2AFY1.1 in doxycycline-inducible U2AF1(S34F) mouse bone marrow cells ex vivo using a lentivirus co-expressing GFP. We observed an attenuation of cell death induced by U2AF1(S34F) expression (%GFP+ live cells relative to uninduced cells: U2AF1(S34F) cells-45% [control virus] vs. 89% [H2AFY1.1 virus], p<0.001; wild-type cells-94% [control virus] vs. 97% [H2AFY1.1 virus], ns). Next, we asked whether loss of H2afy1.1 expression phenocopies any of the hematopoietic alterations observed in U2AF1(S34F) mice (reductions in white blood cell counts [WBC], B-cells and monocytes, or expansion of progenitor cells). To address this question, we first investigated the role of H2afy in normal mouse hematopoiesis. H2afy-/- mice (lacking both H2afy1.1 and H2afy1.2 isoforms) are viable and born at the expected Mendelian frequency. H2afy-/- and H2afy+/-mice exhibited reduced WBC compared to H2afy+/+ littermate mice (7.7, 8.1, and 9.9 K cells/µL, respectively, p<0.01 compared to wild-type mice, N=15 each genotype, 8-12 weeks old), whereas the red blood cell and platelet counts were no different. Flow cytometric analysis of the peripheral blood cell lineages revealed reduced B-cell numbers in H2afy-/- and H2afy+/-mice compared to H2afy+/+ mice (mean 4.0, 4.5, and 5.6 K cells/µL, respectively, p<=0.003). Stem (KLS and KLS-SLAM) and progenitor cells (CMP, GMP, MEP) were not different between genotypes. There was a 2-fold reduction in CFU-pre-B colonies in the H2afy-/- bone marrow cells compared to wild-type controls (p= 0.01, N=10), but CFU-C colonies were no different. To determine whether the reduction in B-cells is hematopoietic cell-intrinsic, we transplanted mouse bone marrow cells collected from H2afy+/+ or H2afy-/- mice into lethally irradiated congenic recipients. At 6 weeks post-transplant, mice who received the H2afy-/- bone marrow cells compared to mice who received H2afy+/+ cells exhibited a marked reduction in WBC (5.0, 9.1 K cells/µL, respectively, p<0.001, N=10) and peripheral blood B-cells (1.9, 5.3 K cells/µL, respectively, p<0.001), but not in myeloid cells. This suggests that hematopoietic expression of H2afy contributes to B-cell development in mice. To assess stem/progenitor cell function in vivo, we performed a competitive repopulation assay. As early as 1 month post-transplant (and persistent to 4 months), there was a reduction in H2afy-/- donor-derived B-cells in the peripheral blood compared to mice receiving H2afy+/+ cells (14%, 58%, respectively, p<0.001, N=14-15). The reduction in peripheral white blood cell counts and B-cells in H2afy-/- mice mirrors the phenotypic changes observed in U2AF1(S34F) mice, suggesting that alterations in H2afy may contribute to altered hematopoiesis in U2AF1 mutant mice. Ongoing studies are designed to address whether the B-cell phenotype in H2afy-/- and U2AF1 mutant mice is due to a specific loss of H2afy1.1 or 1.2 isoform expression in hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

Mitochondria Transfer from Hematopoietic Stem and Progenitor Cells to Pdgfrα+/Sca-1-/CD48dim BM Stromal Cells Via CX43 Gap Junctions and AMPK Signaling Inversely Regulate ROS Generation in Both Cell Populations

Modulation of reactive oxygen species (ROS) levels in hematopoietic stem cells (HSC) is crucial to control HSC quiescence and blood formation. High ROS levels are required for leukocyte formation while low ROS levels are essential to maintain HSC quiescence. However, regulation of ROS content in HSC is poorly understood. Adhesion interactions between HSC and their bone marrow (BM) stromal cells (BMSC) via CXCL12/CXCR4 maintain HSC in a quiescence non-motile state, protecting them from 5-FU chemotherapy insult (Sugiyama, Immunity, 2006). Surface CXCL12 expression by BMSC is dependent on connexin-43 (Cx43) gap junctions mediated cell contact (Schajnovitz, Nat. Immunol., 2011) and BM hematopoietic stem and progenitor cells (HSPC) survive and eliminate excess ROS levels post 5-FU chemotherapy treatment, by transferring ROS to BMSC in a Cx43 dependent manner (Taniguchi, PNAS 2012).Here, we report that ROS content of BM HSPC inversely correlates with ROS levels in adjacent BMSC. Administration of the pro inflammatory cytokine G-CSF results in decreased HSPC Cx43 expression, elevated ROS levels and increased glucose uptake. Conversely, in the BM stromal microenvironment, G-CSF administration generated lower ROS level and reduced glucose uptake. Up-regulation of BM Sphingosine 1-Phosphate (S1P), a downstream target of G-CSF required for ROS production in HSPC, reduced stromal ROS content and proliferation. Accordingly, mice with reduced BM S1P levels (S1Plow) have lower BM content of HSPC, accompanied by reduced ROS, glucose uptake and lactate production in these cells. More importantly, BM from S1Plow mice has a 3 fold increased frequency of primitive ROSlow/ EPCR+ long-term repopulating cells, as evident by immunophenotypic analysis and long-term competitive repopulation assays. Concomitantly, S1Plow mice have increased content of BMSC with higher ROS levels and glucose uptake, leading to higher BM content of colony-forming unit fibroblasts. Our results reveal a dynamic and inverse metabolic relationship between BM HSC and the stroma microenvironment. We hypothesized that the opposite metabolic state of HSPC and BMSC is due to mitochondrial transfer between the two populations. Therefore, we created chimeric mice by transplanting mitochondria labeled GFP (mito-GFP) HSPC to wild type (WT) mice and detected 88% of the host BMSC to contain donor-derived mitochondria, indicating the existence of mitochondria transfer from hematopoietic cells to BMSC in vivo. This transfer is bidirectional, albeit at a lesser degree, as determined in reverse chimeric mice where up to 26% of the donor-derived HSPCs acquired recipient mitochondria. Mitochondrial transfer can be recapitulated also in vitro in an overnight co-culture system of mito-GFP HSPC and primary BMSC, resulting in mitochondrial transfer and increased ROS content in a subpopulation of osteogenic BM PDGFRα+/ Sca-1-/CD48dim stromal cells. Mitochondrial transfer is cell contact dependent and mediated by Cx43 gap junctions. In vitro co-culture of mito-GFPHSPC from Cx43 deficient (KO) mice with WT or Cx43 KO BMSC reduced 50% mitochondrial transfer to PDGFRα+/Sca-1-/CD48dim stromal cells. Contrarily, the mitochondrial transfer from WT HSPC to Cx43 KO stromal cells was not affected, revealing that Cx43 expression on HSPC, but not on BM stromal cells, is specifically required for mitochondrial transfer. Interestingly, in vitro inhibition of AMP-activated protein kinase (AMPK), a crucial metabolic regulator, dramatically increased mitochondrial transfer from HSPC to BMSC. Administration of the AMPK inhibitor BML in vivo increased ROS content of PDGFRα+/Sca-1- BMSC while decreasing it in HSPC, further suggesting that AMPK inhibition regulates mitochondrial transfer and ROS production. Our results imply that mitochondria are scavenged by the BM osteogenic microenvironment to prevent excessive ROS levels in the HSC pool and in parallel to activate bone formation. Altogether, we have discovered a dynamic, inverse metabolic state between BM HSPC and their supporting stromal microenvironment during quiescence, proliferationand differentiation of these two populations. Thus, blood cell production and bone generation take place at the expense of the other. This metabolic seesaw is mediated by mitochondrial transfer from HSPC to osteogenic BM stroma in a HSPC Cx43 gap-junction dependent manner and regulated through AMPK signaling. DisclosuresNo relevant conflicts of interest to declare.

Runx1 Deficiency and MDS-Associated U2af1 Mutation Cooperate for Leukemia Development in a New Mouse Model

The pathogenesis of hematopoietic malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), is very complicated with mutiple genetic alterations required for full-blown disease. Mutations of splicing factor genes, including U2AF1, are found in overal half of MDS patients and 5~10% de novo AML patients. U2AF1 mutations, occuring in roughly 10% of patients with MDS and 3% in de novo AML, are heterozygous and localized almost exclusively in two codons, S34 and Q157, suggesting they are gain-of-function mutations. In addition, our recent studies suggest retention of the wild type U2AF1 allele is required for cell viability (Fei et al., BioRxiv, 2016). Interestingly, RUNX1 is the most commonly co-mutated gene in MDS patients with U2AF1 mutations, and U2AF1 and RUNX1 are also co-mutated in some AML patients. These findings suggest RUNX1 deficiency and U2AF1 mutation cooperate in the pathogenesis of MDS and AML.To test this hypothesis, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f) with mice carrying a newly developed Cre-based conditional U2af1-S34F mutation (U2af1+/S34F) to generate Runx1f/f, Mx1-Cre, U2af1+/S34F mice, which express U2af1-S34F and deficient-Runx1 after inducing Cre from the Mx1 promoter with poly (I:C). We found that U2af1-S34F attenuated the Runx1-/--induced increase of myeloid cells in spleen and bone marrow, as well as the increase of progenitor (lineage-/Sca1-/C-kit+) and stem cells (lineage-/Sca1-/C-kit+). Competitive repopulation assays showed that combining the U2af1 mutation and Runx1 deficiency impaired the transplantation capacity of bone marrow progenitor cells, especially for the myeloid lineage. We further aged these mice to examine the cooperative effects of U2af1-S34F and Runx1 deficiency in disease progression, upon the treatment of a one time, low-dose N-ethyl-N-nitrosourea. Mx1-Cre, U2af1+/S34F mice didn't show survival defect when compared to control mice, while the Runx1f/f, Mx1-Cre mice developed MDS, as previously reported. Interestingly, of fourteen Runx1f/f, Mx1-Cre, U2af1+/S34F mice, two developed AML, and the rest developed MDS. Moreover, transplanted spleen and bone marrow cells from one of four MDS Runx1f/f, Mx1-Cre, U2af1+/S34F mice also developed AML in the recipients. Although Runx1f/f, Mx1-Cre mice also had MDS, their MDS cells did not develop into leukemia after transplantation. Our data therefore suggest that Runx1 deficiency and MDS-associated U2af1 mutation can cooperate in the genesis of AML from MDS. DisclosuresNo relevant conflicts of interest to declare.

Radiation fibrosis resistant SMAD3 -/- mice demonstrate superior donor bone marrow stem cell transplantation capacity by competitive repopulation assay

radiation fibrosis resistant SMAD3 -/- mice demonstrate superior donor bone marrow stem cell transplantation capacity by competitive repopulation assay

Essential role of Ufm1 conjugation in the hematopoietic system.

Protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins plays a pivotal role in a wide range of cellular functions and signaling pathways. The Ufm1 conjugation system is a novel ubiquitin-like system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. Despite its discovery more than a decade ago, its biological functions and working mechanism remains poorly understood. Recent genetic studies using knockout mouse models provide unambiguous evidence for the indispensable role of the Ufm1 system in animal development and hematopoiesis, especially erythroid development. In this short review, we summarize the recent progress on this important protein modification system and highlight potential challenges ahead. Further elucidation of the function and working mechanism of the ufmylation pathway would provide insight into disease pathogenesis and novel therapeutic targets for blood-related diseases such as anemia.

ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.

The ATP-binding cassette transporter ABCG2 is expressed in the interfollicular epidermis and mediates the side-population phenotype in skin cells. However, the role of ABCG2 in skin is unclear. Increased expression levels of ABCG2 were found at the basal layer of transitional epidermis adjacent to cutaneous wounds in human patients, indicating that ABCG2 may be involved in regulating the wound healing process. To investigate the role of ABCG2 in cutaneous wound healing, full-thickness skin wounds were created in ABCG2 knockout (ABCG2-KO) and wild-type mice. The healing process was analysed and revealed that ABCG2 deficiency in skin results in delays in wound closure and impairments in re-epithelialization, as evidenced by reductions in both suprabasal differentiation and in p63-expressing keratinocytes migrating from transitional epidermis to epithelial tongues. The reduction in p63-expressing cells may be due to elevated levels of reactive oxygen species in ABCG2-KO epidermis, which can cause DNA damage and lead to proliferation arrest. To determine whether ABCG2 deficiency affects the potency of epidermal stem/progenitor cells (EPCs), transplantation studies were carried out, which demonstrated that ABCG2-KO EPCs display higher levels of γH2AX and lose the capacity to differentiate into suprabasal keratinocytes. A competitive repopulation assay confirmed that ABCG2 expression is critical for the proper expansion and differentiation of EPCs in cutaneous wounds. As EPCs are known to contribute to the healing of larger wounds, the current findings imply a functional role for ABCG2 in the expansion and differentiation of p63-expressing EPCs. Thus, ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.

EPCR/PAR1 Signaling Navigates Long-Term Repopulating Hematopoietic Stem Cell Bone Marrow Homing to Thrombomodulin-Enriched Blood Vessels

Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) is an active and essential first step in clinical stem cell transplantation. EPCR is expressed by murine BM LT-HSCs endowed with the highest repopulation potential and its ligand, activated protein C (aPC), has anticoagulant and anti-sepsis effects in EPCR+/PAR1+ endothelial cells. We recently found that signaling cascades, traditionally viewed as coagulation and inflammation related, also independently control EPCR+ LT-HSC BM retention and recruitment to the blood via distinct PAR1 mediated pathways. EPCR/PAR1 signaling retains LT-HSCs in the BM by restricting nitric oxide (NO) production and Cdc42 activity, promoting VLA4 affinity and adhesion. Conversely, thrombin/PAR1 signaling overcome EPCR+ LT-HSC BM retention by initiating NO production, leading to TACE-‎mediated EPCR shedding, CXCR4 and PAR1 upregulation and parallel CXCL12 secretion by PAR1+ BM stromal cells, enhancing stem cell migration and mobilization.Since EPCR shedding is essential for BM LT-HSC recruitment, we tested EPCR role in LT-HSC BM homing. EPCR+ LT-HSC exhibited reduced in vitro migration towards CXCL12 and enhanced CXCL12-dependent adhesion to fibronectin. Unexpectedly, transplanted EPCR+ LT-HSCs preferentially homed ‎to the host BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing antibody treatment, which blocks binding to aPC, leading to attenuated EPCR+ LT-HSC homing to the BM but not to the spleen. Importantly, short term aPC pretreatment inhibited NO production and dramatically increased EPCR+ LT-HSC BM homing. Since EPCR navigates LT-HSC to the BM, we studied the role of EPCR signaling in LT-HSC BM repopulation. Mimicking EPCR signaling by in vivo NO inhibition induced preferential expansion of blood and bone-forming stem cells and gave rise to higher donor type EPCR+ LT-HSCs in competitive repopulation assays. Similarly, repeated treatment with aPC expanded BM EPCR+ stem cells and increased competitive LT-repopulation. Importantly, loss of EPCR function reduced HSC long-term repopulation ability while maintaining their short-term repopulation activity. BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with normal stem cells in competitive long-term repopulation assays. Consistent with inferior HSC BM repopulation, Procrlow mice exhibited reduced numbers of BM LT-HSC with reduced adhesion capacity. Additionally, these mice displayed increased HSC frequencies in the blood circulation and the spleen, which were pharmacologically corrected by inhibiting NO generation with L-NAME treatment. BM retention is essential for quiescent HSC protection from chemotherapy. Mice treated with NO donor SNAP, or with blocking EPCR antibody as well as Fr2-/-mice lacking PAR1 expression, were more susceptible to hematological failure and mortality induced by 5-FU treatment compared to control mice. Together, these results indicate a functional aPC/EPCR/PAR1 signaling pathway, regulating EPCR+ LT-HSC BM homing, adhesion and long-term repopulation potential. The thrombin-thrombomodulin (TM) complex converts protein C to its activated form aPC, facilitating high affinity binding to its receptor EPCR. To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by a unique BM endothelial cell (BMEC) subpopulation, but not in the spleen. Moreover, EPCR+ LT-HSCs were found adjacent to TM+/aPC+ BMECs, imposing their adhesion and retention. Interestingly, similar to BMECs, BM EPCR+ LT-HSC also express surface TM, implying the possibility of autocrine aPC generation.Herein we define EPCR as a guidance molecule, navigating slow migrating LT-HSC in the blood flow specifically to TM+ BMEC supporting niches, maintaining NOlow stem cell retention, long-term blood production and protection from myelotoxic insult. Conversely, thrombin/PAR1 signaling oppositely increase NO generation and EPCR shedding allowing increased CXCR4-dependent LT-HSC migration and mobilization. Harnessing EPCR signaling may improve clinical stem cell transplantation, increasing LT-HSC specific BM homing and repopulation by aPC pretreatment, as well as potentially to overcome malignant stem cell chemotherapy resistance. DisclosuresNo relevant conflicts of interest to declare.

Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell (HSC) Self-Renewal Via Control of mTOR Signaling

Elucidation of the mechanisms governing HSC regeneration has been impeded by difficulty in isolating HSCs early following genotoxic injury, such as total body irradiation (TBI). Using multiparametric flow cytometric cell sorting of BM ckit+sca-1+lin- cells coupled with gene expression analysis, we identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed by BM KSL cells at the earliest detectable point of regeneration (day +10) following TBI (3.3-fold, p<0.0001).Grb10 is a member of the imprinted gene family which is predominately expressed in the stem cells of a variety of tissues, including embryonic stem cells, bone marrow, skin and muscle. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) detected in 7-day culture (p=0.03 and p=0.002, respectively). Furthermore, mice competitively transplanted with Grb10-deficient HSCs displayed 10-fold lower donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed a greater than 10-fold deficit in long-term repopulating capacity in Grb10-deficient KSL cells compared to Grb10-expressing KSL cells (p=0.006 for %CD45.1+ donor cells).In order to determine if Grb10 was necessary for HSC maintenance and normal hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8 week old Grb10m/+ (1 mutant allele, 1 wild type allele) had 10-fold decreased Grb10 expression in BM lin-cells. BM CFCs and SLAM+ KSL cells were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006 and p=0.04, respectively). Competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (p=0.003 for %CD45.1+ donor cells). Mice transplanted with BM cells from homozygous Grb10-/- mice showed a similar decrease in donor-derived hematopoietic repopulation compared to mice transplanted with BM cells from Grb10+/+ mice (p=0.02 at 20 weeks post-transplantation). These results confirmed that Grb10 regulates HSC self-renewal capacity in vivo.To determine whether Grb10 regulates HSC regeneration after myelotoxic injury, we irradiated Grb10m/+ mice with 550cGy TBI, and monitored hematopoietic recovery over time in comparison to Grb10+/+ controls. Interestingly, Grb10m/+ mice displayed accelerated hematopoietic regeneration early following TBI. At day+10 after 550cGy, Grb10m/+ mice contained significantly increased numbers of BM SLAM+ KSL cells (p=0.04) and CFCs (p=0.009), compared to Grb10+/+ littermates. Similarly, mice transplanted with BM cells from irradiated, Grb10m/+ mice displayed 5-fold increased donor hematopoietic repopulation at 20 weeks post-transplantation compared to mice transplanted with BM cells from irradiated, Grb10+/+ mice (p=0.006). These data suggest that Grb10 deficiency accelerates hematopoietic recovery in the early period following myelosuppressive radiation injury.Mechanistically, Grb10-deficiency caused an increase in the percentage of BM KSL cells in G1 and G2/S/M phase of cell cycle compared to Grb10+/+ KSL cells (p=0.003). We also observed significantly increased levels of mTOR activation in Grb10m/+ BM KSL cells compared to Grb10+/+ BM KSL cells (p=0.001 for pS6, p=0.001 for pS6k and p=0.02 for p4EBP1). Furthermore, mTOR inhibition via siRNA-mTOR targeting rescued the defect in BM hematopoietic progenitor content (colony forming cells) in Grb10-deficient BM cells (p<0.0001). Taken together, our results suggest that Grb10 is necessary for HSC maintenance in steady state, while, paradoxically, Grb10 inhibition accelerates HSC regeneration early following injury. Furthermore, our data suggest that Grb10 mediates these effects via regulation of mTOR signaling. Selective modulation of Grb10 signaling has the potential to augment HSC self-renewal in steady state and to accelerate HSC regeneration following myelotoxic injury. DisclosuresHimburg:Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

The Class IA PI3 Kinase Isoforms Have Distinct but Overlapping Roles in Adult Hematopoiesis

The PI3 kinase (PI3K) signaling pathway is activated by most hematopoietic growth factors and chemokines that orchestrate normal adult hematopoiesis. Hematopoietic cells express four different catalytic isoforms of PI3K (p110α, β, δ, and γ), each encoded by a different gene (pik3ca, pik3cb, pik3cd and pik3cg, respectively). The Class IA (p110 α, β, δ) and Class IB (p110γ) enzymes use different regulatory subunits and are activated by different sets of upstream signals. While pik3ca and pik3cb are expressed ubiquitously, pik3cd is enriched in leukocytes. The roles of PI3K in adult hematopoiesis have been difficult to investigate using gene targeting in mice, since two of the isoforms (p110α and p110β) are required during embryogenesis. However, mice with germline deletion of pik3cd are viable and fertile with normal blood counts (Jou et al, MCB, 2002. 22(24):8580-91). Using the Mx1-Cre system to conditionally delete pik3ca in adult hematopoietic stem cells (HSCs), we previously showed that p110α is dispensable for adult HSC function (Gritsman et al, J Clin Invest 2014;124(4):1794-1809). We now report that conditional deletion of pik3cb using Mx1-Credoes not affect blood counts, bone marrow cellularity, survival, bone marrow HSC or progenitor numbers, or hematopoietic reconstitution in the transplantation setting. Therefore, none of the individual Class IA PI3K isoforms have critical roles in HSCs. However, it is still unclear whether these isoforms can substitute for each other in adult HSCs, or whether they are truly dispensable for HSC function.To distinguish between these two possibilities and to uncover potentially redundant roles for PI3K isoforms in hematopoiesis, we have generated compound conditional knockout mice with the genotypes: pik3ca-lox/lox;pik3cd-/-;Mx1-Cre ("p110α-/-;p110δ-/-") and pik3ca-lox/lox;pikcb-lox/lox;Mx1-Cre ("p110α-/-;p110β-/-"). Interestingly, compound deletion of p110α and p110δ in HSCs results in significantly reduced white cell counts and anemia (Gritsman et al, Blood (ASH Annual Meeting Abstracts), Nov 2012; 120: 2322). In addition, p110α-/-;p110δ-/- mice have reduced numbers of bone marrow multipotential progenitors (MPPs), common myeloid progenitors (CMPs), and common lymphoid progenitors (CLPs), while HSC numbers are preserved. Furthermore, deletion of both p110α and p110δ leads to reduced B cell repopulation in competitive repopulation assays. This suggests that p110α and δ have redundant roles in HSC differentiation and B cell specification.In contrast, we found that compound deletion of p110α and p110β has no effect on long-term competitive repopulation of any lineages for up to 20 weeks. We also observed normal donor chimerism in the HSC, MPP, and progenitor populations in transplant recipients of p110α-/-;p110β-/- bone marrow. After non-competitive transplantation, recipients of p110α-/-;p110β-/- bone marrow maintained normal blood counts over time. This indicates that, while p110δ is sufficient to support hematopoietic reconstitution of all lineages in the transplantation setting, p110β cannot support normal HSC differentiation by itself, particularly into the B cell lineage. However, p110α-/-;p110δ-/- mice do not develop pancytopenia, and repopulation of the myeloid and T cell lineages is still largely preserved, suggesting that long-term HSC function is intact. To determine whether HSC self-renewal and myeloid reconstitution after transplantation are possible in the absence of all three Class IA PI3Ks, we have generated triple knockout mice with the genotype pik3ca-lox/lox;pikcb-lox/lox;pik3cd-/-;Mx1-Cre ("TKO"). Analysis of the hematopoietic phenotype of TKO mice will be presented. These results will be valuable in predicting future hematologic toxicity in ongoing clinical trials with isoform-selective PI3K inhibitors or pan-PI3K inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Rejuvenation of Aged Vascular Niches to Enhance Hematopoietic Function

The molecular mechanisms regulating the aging of hematopoietic stem cells (HSCs) are poorly understood. To date, most studies describing age-related alterations have focused on HSC-intrinsic alterations, showing that the absolute number of immunophenotypically defined HSCs increases with age but that aged HSCs exhibit decreased long-term reconstitution potential, self-renewing capacity, altered transciptomes, cell-cycling and responses to cellular stress and DNA damage. Furthermore, old HSCs exhibit a significant myeloid bias at the expense of lymphopoiesis, which is thought to predispose the aging hematopoietic to the development of myeloid neoplasms. While these studies show that cell-intrinsic changes contribute to the aging of the hematopoietic system, most have not adequately addressed the effects of the aging microenvironment. A large body of evidence has demonstrated functional interactions between the HSC and its niche, suggesting that local and systemic factors may regulate HSC function; however, the role of the bone marrow (BM) microenvironment in regulating HSC aging has not been fully elucidated. Understanding the relationship between the BM microenvironment and the HSC during aging may aid in efforts to prevent or reverse the age-related functional decline observed in the hematopoietic system.We have shown that Akt-activated endothelial cells (ECs) within the hematopoietic microenvironment, are indispensable for supporting HSC self-renewal during both steady-state and regenerative hematopoiesis and that EC-specific Mapk signaling drives the differentiation of HSCs into lineage-committed progeny. Here, we demonstrate that young BMECs maintain high levels of Akt signaling, whereas aged ECs exhibit preferential signaling through Mapk. Utilizing a novel HSC/EC co-culture system, we demonstrate that aged BMECs co-cultured with hematopoietic stem and progenitor cells (HSPCs) isolated from young mice inhibit the expansion of repopulating HSCs and are unable to expand aged HSPCs that give rise to long-term, multilineage engraftment. Of note, when we co-cultured aged HSPCs with young BMECs we found that we were able to maintain their functional capacity when assessed by competitive repopulation assays. These data suggest that BMECs play an important role in regulating HSC function.Based on these observations, we set out to test if endothelial Mapk inhibits the vascular niche from supporting functional hematopoiesis. We generated a mouse model in which Mapk was selectively overexpressed in ECs (Mapk VCC) and demonstrated that these mice exhibit a defect in phenotypic and functional HSCs, resembling phenotypes similar to aged HSCs. In particular, transplanted HSCs from Mapk VCC mice lead to diminished engraftment ability with an in increase in myeloid contribution at the expense of B and T cells. To directly test if the functional defects in the HSCs were due to the Mapk-activated vascular niche, we isolated BMECs from these mice and found that Mapk-activated ECs have a decreased ability to support the ex vivo expansion of functional HSCs, with less HSCs in quiescence and more differentiation into granulocytic myeloid cells. Transcriptome and proteomic analyses revealed that aged and Mapk-activated BMECs have similar defects in their pro-HSC angiocrine repertoire, suggesting a possible mechanism for their diminished capacity to instruct and maintain a balanced and healthy hematopoietic system. Furthermore, we utilized an endothelial-based cellular therapy approach to rejuvenate the BM microenvironment and demonstrated that transplantation of young BMECs can enhance hematopoietic recovery and restore HSC function following myeloablative injury in aged mice.Taken together, our in vivo animal model and EC/HSC co-culture system will allow us to screen for angiocrine factors that support the functional attributes of the HSC. Additionally, we have unlocked a potential therapeutic application for the transplantation of ECs following myeloablative treatment. Transplantation of BMECs may create a more permissive microenvironment that promotes an increase in the number of engrafted HSPCs following BM transplantation and accelerates the rate of hematopoietic recovery following radiation or chemotherapeutic regimens decreasing the morbidity/mortality associated with life threatening pancytopenias in the elderly. DisclosuresNo relevant conflicts of interest to declare.

Distinct Contributions By Perivascular Niche Cells in Hematopoietic Stem Cell Maintenance

Hematopoietic stem cells (HSCs) self-renew and differentiate into all blood types in response to various demands through life. HSC functions are tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow (BM). Using Nestin-GFP transgenic mice, we have identified Nestin-GFP+ perivascular stromal cells exhibiting a mesenchymal stem/progenitor cell activity as niche cells. Furthermore, we found two types of Nestin-GFP+ cells expressing different surface markers, Nerve/glial antigen 2 (NG2) and Leptin receptor (Lepr) that are associated with arterioles and sinusoid, respectively, in the BM (Kunisaki et al. Nature, 2013). Both arteriolar and sinusoidal niche cells have been reported to show high gene expression of cytokines essential for HSC maintenance such as CXCL12 and stem cell factor (SCF), however, it remains unknown how the distinct niche cells differentially regulate HSC functions. To investigate the mechanisms, we utilized genetic mouse models, in which CXCL12 or SCF can be deleted in specific cell types. CXCL12 deletion in sinusoidal niche cells by using Lepr-cre/Cxcl12fl/− mice mobilized HSCs and lineage− Sca-1+ c-kit+ (LSK) progenitors into spleen (HSC, CT: Control/DL: Deleted: 760±165 / 2193±557 / spleen, n=6, p<0.05) and blood (LSK, CT/DL: 177±36 / 668±156 / mL blood, n=5, p<0.05), but had no effect on HSC numbers in the BM (CT/DL: 1435±101 / 1194±75 / femur, n=6, p=0.085), which is consistent with a previous report (Ding and Morrison, Nature, 2013). Furthermore, assessments of endogenous HSC localization using whole-mount 3D imaging technology revealed that the deletion of CXCL12 in Lepr+ niche cells had no impact on HSC location (KS-test: Two-sample Kolmogorov-Smirnov test, p=0.9981). By contrast, deletion of CXCL12 in NG2-cre derived cells, which recombines efficiently in the entire Nestin-GFP+ non-endothelial stromal fraction including both peri-arteriolar and peri-sinusoidal cells (96.9±1.3%), and overlapping with Lepr+ cells (88.5±1.6%) and CXCL12-abundant reticular cells (90.7±1.4%), led to a robust reduction of HSC numbers in the BM (CT/DL: 1487±87 / 179±40 / femur, n=10, p<0.0001) with HSC and progenitor mobilization into spleen (HSC, CT/DL: 705±262 / 3550±540 / spleen, n=6-8, p<0.01) and blood (LSK, CT/DL: 494±178 / 5357±896 / mL blood, n=5-7, p<0.01). In addition, deletion of CXCL12 in NG2-cre targeted cells led to HSC displacement away from arterioles (KS-test: Two-sample Kolmogorov-Smirnov test; p=0.001). To examine further a role of CXCL12 produced by NG2+ arteriolar niches on HSC maintenance, we generated tamoxifen-inducible NG2-creERTM/Cxcl12fl/− mice. Deletion of CXCL12 postnatally in NG2+ arteriolar niche cells significantly reduced the number of HSCs in the BM (CT/DL: 1617±160 / 960±95 / femur, n=10-13, p=0.0013), which was confirmed functionally by a competitive repopulation assay. Moreover, 3D imaging revealed that HSCs were located further away from arterioles in NG2-creERTM/Cxcl12fl/− marrow (KS-test: p<0.0001), suggesting a role for arteriolar niches in CXCL12-mediated HSC maintenance. As niche cells synthesize several factors, we evaluated the contribution of arteriolar niches in SCF synthesis, a cytokine shown to be critical for HSC maintenance. As expected, deletion of SCF in NG2-cre targeted cells led to a significant reduction of HSC numbers in the BM (CT/DL: 606±85 / 96±23 / femur, n=5-7, p<0.0001). To further evaluate functions of SCF produced by distinct vascular niches, we also compared these mice with deletions using Lepr-cre or tamoxifen-inducible NG2-creERTM mice. We found that deletion of SCF in Lepr-cre targeted cells showed a significant reduction of HSC numbers in the BM (CT/DL: 690±84 / 220±83 / femur, n=3-4, p<0.0118), consistent with previous studies (Ding et al., Nature, 2012), whereas there was no significant change observed in NG2-creERTM/SCFfl/− mice, suggesting that Lepr+ vascular niches rather than NG2+ arteriolar niches are the most important source of SCF in the BM. These results highlight distinct contributions of perivascular cells primarily located in separate vascular niches, arteriolar and sinusoidal, in HSC maintenance and mobilization. DisclosuresNo relevant conflicts of interest to declare.

S6K1 Regulates Self-Renewal of Leukemia Initiating Cells and Normal Hematopoietic Stem Cells

The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult hematopoietic stem cells (HSCs) and regulation of progenitor population. Translocations in MLL have been detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. Various genes, including AF4, AF5, AF9, ELL and ENL have been identified as partners for translocation in MLL-rearranged leukemia. In hematopoietic cells, expression of MLL fusion proteins result in a block of differentiation. AML patients who successfully undergo treatment but relapse suggest the importance of targeting leukemia initiating cells (LICs) within the MLL leukemia. LICs remain in a quiescent state and are capable of survival despite treatment with chemotherapeutic agents or targeted molecular inhibitors. In mouse models, LICs are defined by the capability of successfully propagating disease upon serial transplantation. In order to prevent disease relapse, identification of molecules, which regulate the self-renewal of LICs, is essential.The phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway is a key regulator of self-renewal of both HSCs and LICs. Deletion of Raptor, a subunit of mTORC1, does not affect initiation and progression of acute myeloid leukemia (AML), but Raptor deficiency results in delayed propagation of AML. Upon its activation, mTORC1 phosphorylates and activates p70 ribosomal protein S6 kinase (S6K1) and inhibits the activity of eukaryote translation initiation factor 4E binding protein 1 (4E-BP1). S6K1 has been shown to be hyperactivated in hematopoietic cells expressing oncogenic MLL-AF9 fusion protein.In our study, we have assessed the role of S6K1 in the initiation, progression and propagation of AML using a genetic model of S6K1 knockout mice (S6K1-/-). We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and transplanted them into lethally irradiated recipients. Recipients of both WT and S6K1-/- HSC/Ps bearing MLL-AF9 displayed high white blood cell (WBC) count, splenomegaly and developed AML. There was no difference in survival between the WT and S6K1-/- recipients. In order to determine whether S6K1 regulates the self-renewal of LICs, we transplanted lethally irradiated mice with cells from WT and S6K1-/- primary recipients who developed AML. Recipients of S6K1 deficient AML cells survived significantly longer compared to controls (n=17/group, p<0.001). S6K1 deficient HSC/Ps expressing MLL-AF9 showed reduced activation of Akt as well as decreased mTORC1 activity, suggesting that deletion of S6K1 results in reduced activation of PI-3K-Akt-mTORC1 pathway both upstream and downstream of mTORC1 which indicates that S6K1 might be involve in a feedback loop within this pathway.To determine the role of S6K1 in normal HSC development and maintenance, we analyzed bone marrow derived HSCs in WT and S6K1-/- mice. S6K1 deficiency did not alter the frequency of long term HSCs (LT-HSCs) as defined by CD150+ CD48- Lin- Sca1+ c-Kit+ surface markers, but the absolute number of LT-HSCs were significantly reduced in S6K1 deficient mice (p<0.02). The absolute number of multipotent progenitors (MPPs) (p<0.001), common myeloid progenitors (CMPs) (p<0.01) and megakaryocyte-erythroid progenitors (MEPs) (p<0.01) were also significantly reduced in S6K1 deficient mice. Deficiency of S6K1 resulted in reduced quiescence of LT-HSCs (p<0.05). Expression level of p21, an inhibitor of cell cycle progression, was significantly decreased in LT-HSCs derived from S6K1-/- mice compared to LT-HSCs from control group. To study the role of S6K1 in HSCs' function, we performed competitive repopulation assay. Sorted LT-HSCs from bone marrow cells derived from either WT or S6K1-/- mice were transplanted with competitor cells into lethally irradiated recipients. S6K1 deficient LT-HSCs displayed reduced repopulating ability in secondary recipients (n=9-10/group, p<0.001). Expression level of p21 was downregulated in donor-derived HSCs isolated from secondary recipients of S6K1 deficient HSCs compared to control. Overall, our study establishes S6K1 as a critical regulator of self-renewal of both LICs and HSCs. Deficiency of S6K1 in AML cells results in delayed propagation of disease and deficiency of S6K1 in HSCs results in decreased self-renewal potential. DisclosuresNo relevant conflicts of interest to declare.