Low-density Mononuclear Cells Research Articles

Pharmacologic Inhibition of PI3Kδ Prolongs Survival of Mutant Shp2E76K-Expressing Mice

Juvenile myelomonocytic leukemia (JMML) is an aggressive overproduction of cells in the myeloid lineage, characterized by hypersensitivity to granulocyte macrophage colony-stimulating factor (GM-CSF). There is a clear need for improved chemotherapies, as the current treatments are ineffective. The most commonly mutated gene in JMML patients is PTPN11, which encodes the protein tyrosine phosphatase Shp2. One of the downstream targets of Shp2 is phosphoinositide 3-kinase (PI3K), and we have previously focused on the hematopoietic-specific catalytic subunit p110δ (PI3Kδ). The PI3Kδ inhibitor idelalisib is FDA-approved for patients with B cell malignancies. However, the effectiveness of PI3Kδ inhibition in JMML and other myeloid malignancies has not been studied. Therefore, following our previous work in vitro which demonstrated reduced proliferation of GOF Shp2-expressing murine cells and primary JMML cells (Goodwin et. al, Blood 2014), we assessed the effect of PI3Kδ inhibition on GOF Shp2-expressing mice in vivo as the next step in exploring PI3Kδ inhibition as a potential treatment in JMML.We treated Shp2E76K/+;LysMcre+ mice between 12 and 20 weeks of age with 30mg/kg of the PI3Kδ inhibitor GS-9820 (hereafter PI3Kδ inhibitor) or vehicle BID via oral gavage for 21 days. First, we evaluated mice directly after completing 21 days of treatment (16 hours following final vehicle or PI3Kδ inhibitor dose). The PI3Kδ inhibitor-treated mice had significantly reduced spleen-to-body weight ratio in comparison to the vehicle-treated mice. We measured the colony-forming ability of bone marrow low-density mononuclear cells (LDMNCs) in response to GM-CSF and found only a slight reduction in drug-treated mice, indicating that pharmacologic inhibition of PI3Kδ does not permanently correct GM-CSF hypersensitivity. To investigate how PI3Kδ inhibitor treatment induced the functional effect of reduced spleen size, we phenotypically analyzed the bone marrow and peripheral blood for hematopoietic progenitor cells (lineage-Sca1+cKit+, LSK) and for mature myeloid cells (Gr1+Mac1+). The frequency of LSK cells was significantly reduced in the bone marrow of PI3Kδ inhibitor-treated mice. There was no difference in the frequency of Gr1+Mac1+ in the bone marrow compartment, but there was a significant increase in the peripheral blood of PI3Kδ inhibitor-treated mice. Furthermore, the Mac1+ mean fluorescence intensity in the peripheral blood was significantly higher in PI3Kδ inhibitor-treated mice. Collectively, these findings suggest that PI3Kδ inhibition induced stem/progenitor terminal differentiation and reduced the self-renewal and hyperproliferation of immature myeloid cells.A second cohort of treated mice was followed for overall survival. Survival of the PI3Kδ inhibitor-treated mice was significantly prolonged compared to their vehicle-treated counterparts. Serial peripheral blood WBC counts were similar between the two groups until approximately 20 weeks and 34 weeks after the start of treatment, when WBC counts trended upward in the vehicle-treated and drug-treated animals, respectively. These time points coincided with a sharp decrease in the number of surviving mice in the corresponding group. When moribund, mice were euthanized to assess the cause of death. The PI3Kδ inhibitor-treated animals had no difference in splenomegaly and leukocytosis compared to the vehicle-treated animals at the time of euthanasia, and composition of the bone marrow and spleen in moribund animals was not significantly different, suggesting that mice of the two groups ultimately succumbed to the same disease. Taken together, these findings suggest that time-limited PI3Kδ inhibition delays onset of mutant Shp2-induced leukemia, but does not alter the ultimate course of disease. These findings imply that continued PI3Kδ inhibition may be needed for optimal treatment.Our results demonstrate that in vivo administration of a hematopoietic-specific PI3Kδ inhibitor increases the survival of GOF Shp2-expressing mice. The more limited side effect profile of PI3Kδ inhibition compared to pan-PI3K inhibition may provide a therapeutic window to simultaneously target both the Ras-Erk and PI3K-Akt pathways in the treatment of JMML. This work supports the rationale of using PI3Kδ inhibitors in the treatment of patients with JMML and possibly other myeloid cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

PTPN11D61Y/+; Vavcre+ Animals Commonly Succumb to Leukemia Relapse Despite Robust Engraftment of Transplanted Wild-Type Hematopoietic Stem and Progenitor Cells

Juvenile Myelomonocytic Leukemia (JMML) is a rare childhood myelodysplastic / myeloproliferative overlap disorder. JMML exhibits myeloid populations with mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. While allogeneic hematopoietic stem cell transplant (HSCT) is the treatment of choice for children with JMML, 50% of children succumb to leukemia relapse; however, the mechanism leading to this high relapse rate is unknown. We hypothesized that the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, leading to poor engraftment of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and thus promoting leukemia relapse. Using Vav1 promoter-directed Cre, we generated a mouse model of JMML that conditionally expresses gain-of-function PTPN11D61Yin utero during development. While PTPN11D61Y/+; VavCre+embryos did not demonstrate in utero lethality, we observed a modest reduction of PTPN11D61Y/+; VavCre+ mice at the time of weaning compared to predicted Mendelian frequencies. Further, surviving PTPN11D61Y/+; VavCre+ mice developed elevated peripheral blood leukocytosis and monocytosis as early as 4 weeks of age compared to PTPN11+/+; VavCre+ controls. To address the hypothesis that an aberrant bone marrow microenvironment in the PTPN11D61Y/+ mice leads to poor engraftment of wild-type donor cells following transplant, we examined engraftment of wild-type hematopoietic stem and progenitor cells (HSPCs) in the PTPN11D61Y/+; VavCre+ mice and monitored animals for disease relapse. 16-24 week-old diseased PTPN11D61Y/+; VavCre+ and control PTPN11+/+; VavCre+ mice were lethally irradiated (11 Gy split dose) and transplanted with 5 x 105 CD45.1+ wild-type bone marrow low density mononuclear cells (LDMNCs), which simulates a limiting stem cell dose commonly available in a human HSCT setting. 6 weeks post-HSCT, PTPN11D61Y/+; VavCre+recipients demonstrated an unexpected elevated CD45.1+ donor cell contribution in peripheral blood compared to the control PTPN11+/+; VavCre+ recipients. However, despite superior engraftment in the PTPN11D61Y/+; VavCre+ recipients, these mice had a significantly shorter median survival post-HSCT due to a resurgence of recipient CD45.2-derived leukemic cells. We repeated the experiment using a high dose of CD45.1+ LDMNCs (10 x 106 cells) to determine if providing a saturating dose wild-type cells could prevent the relapse of recipient-derived leukemogenesis and normalize the survival of the PTPN11D61Y/+; VavCre+recipients. While this saturating dose of wild-type cells resulted in high peripheral blood chimerism in both the PTPN11D61Y/+; VavCre+ and PTPN11+/+; VavCre+ recipients, the PTPN11D61Y/+; VavCre+ animals nevertheless demonstrated significantly reduced overall survival. When we examined the cause of mortality in the HSCT-treated PTPN11D61Y/+; VavCre+mice, we found enlarged spleens, hypercellular bone marrow, and enlarged thymuses. Flow cytometry revealed that the majority of cells in the peripheral blood, bone marrow, and spleen were recipient-derived CD45.2+ CD4+ CD8+ T cells. To verify that the disease was neoplastic in origin, secondary transplants into CD45.1/.2 recipients were performed from two independent primary PTPN11D61Y/+; VavCre+and two independent primary PTPN11+/+; VavCre+ controls. Secondary recipients of bone marrow from PTPN11D61Y/+; VavCre+ animals rapidly succumbed to a CD45.2-derived T-cell acute lymphoid leukemia (T-ALL). Previous studies demonstrated that wild-type PTPN11 is needed to protect the integrity of the genome by regulating Polo-like kinase 1 (Plk1) during the mitosis of the cell cycle (Liu et al., PNAS, 2016). We now demonstrate that even when PTPN11 mutant animals are provided with saturating doses of wild-type HSCs, dysregulated residual recipient cells are able to produce relapsed disease. Collectively, these studies highlight the propensity of residual mutant PTPN11 cells to transform after being subjected to mutagenic agents that are commonly used for conditioning regimens prior to allogeneic HSCT. These findings suggest that modified pre-HSCT conditioning regimens bearing reduced mutagenicity while maintaining adequate cytoreductive efficacy may yield lower post-HSCT leukemia relapse in children with PTPN11mutations. Disclosures No relevant conflicts of interest to declare.

Effect of Bone Marrow-derived Mesenchymal Stem Cells and Umbilical Cord Blood-CD34+ cells on Experimental Rat liver Fibrosis

Background and Objective: Liver disease is one of the major causes of death in many countries. Hence, the development of effec- tive therapies for liver fibrosis is a major aim of medical research. So this study was designed to investigate the therapeutical role of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) transplantation in the experimental rat liver fibrosis. Design and Method: Bone marrow-derived MSCs were isolated from femoral and tibial bones of male albino rats, then were grown and propagated in culture for 2 weeks and were characterized morphologically and by detection of CD29 by real time-PCR. Hu- man umbilical cord blood cells were obtained after full-term caesarean delivery from healthy donors after written informed consent. Low-density mononuclear cells were separated over Ficoll- Paque (Gibco-Invitrogen, Grand Island, NY), and then CD34+ HSC was isolated using a magnetic cell sorter (MiniMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then infused intraperi- toneally in rats that received CCl4 injection to induce liver fibrosis. Rats were divided into 4 groups: control, CCl4, CCl4 plus MSC, and CCl4 plus CD34+. Liver tissue was examined histopathologically for all groups. The expression of collagen I and metalloproteinase-2 genes as a marker of liver fibrosis was measured by real time RT- PCR. Results: The results of the present study showed that both MSCs and CD34+ have a significant antifibrotic effect as evidenced by the significant decrease in liver collagen gene expression as well as the decrease in MMP-2 (p < 0.05) compared to the CCl4 group.

Ameliorative Potential of Mesenchymal Stem Cells and Hematopoietic Stem Cells against CCl&amp;lt;sub&amp;gt;4&amp;lt;/sub&amp;gt; Induced Chromosomal Abnormalities in Experimental Rat Liver Fibrosis

Background and Objective: The liver is responsible for metabolism and detoxification of the most of components that enter the body. Once the liver became injured, its efficient treatment with famous chemical drugs was limited. Therefore, interest concerned the use of alternative medicines for the treatment of hepatic disease has been arisen. The present study was aimed to investigate the therapeutic effect of the two types of stem cells against chromosomal aberrations in bone marrow (BM) cells of rats treated with carbon tetrachloride (CCl4). Design and Method: BM-derived mesenchymal stem cells (MSCs) were isolated and propagated in culture for 2 weeks and were characterized morphologically. Human umbilical cord blood (UCB) cells were obtained after full-term caesarean delivery from healthy donors. Low-density mononuclear cells were separated over Ficoll-Paque, and CD34+ hematopoietic cells were isolated using a magnetic cell sorter. Rats were divided into 4 groups: control, CCl4, CCl4 plus MSC, and CCl4 plus CD34+. Liver tissue was examined histopathologically for all groups. Results: The results of the present study indicated a significant increase (p ＜ 0.05) in the number of metaphases with different types of chromosomal aberrations in CCl4 group. Treatment of the animals with BM-MSCs and UCB-CD34+ cells improved both genotoxicity and histopathological changes induced by CCl4.

Phosphatidylinositol-3-Kinase p110d Uniquely Promotes Gain-Of-Function PTPN11-Induced GM-CSF Hypersensitivity In a Model Of Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a fatal leukemia affecting children under the age of 4 years and is characterized by myelomonocytic cell overproduction and hypersensitivity to GM-CSF. The only curative therapy is allogeneic stem cell transplantation; however, half of children relapse after this aggressive therapy. Approximately 85% of JMML patients bear loss-of-function (LOF) mutations in NF1 or CBL or gain-of-function (GOF) mutations in KRAS, NRAS, or PTPN11. Typically, these mutations are non-overlapping, with the net effect being Ras hyperactivation. Children bearing somatic GOF mutations within PTPN11, which encodes the protein tyrosine phosphatase, Shp2, exhibit the poorest prognosis. GOF Shp2 (Shp2D61Y and Shp2E76K) induces hyperactivation of both the Ras-MEK- Erk and PI3K-Akt pathways. While the Ras-MEK-Erk pathway clearly contributes to the pathogenesis of JMML, we hypothesize that the PI3K-Akt pathway cooperates with the Ras-MEK-Erk pathway to promote JMML. Recently published work indicates that genetic disruption of the PI3K regulatory subunit, p85a, reduces GOF Shp2-induced hypersensitivity to GM-CSF. However, as PI3K regulatory subunits cannot be easily inhibited pharmacologically, we examined the contribution of class IA PI3K catalytic subunits in GOF Shp2-induced JMML.Shp2D61Y/+;Mx1Cre+ mice were crossed with mice bearing conditional knockout of p110a (Pik3caflox/flox) or bearing a kinase dead mutant of p110d (Pik3cdD910A/D910A). Shp2D61Y/+;Mx1Cre-, Shp2D61Y/+;Mx1Cre+, Shp2D61Y/+;Mx1Cre+; Pik3caflox/flox, and Shp2D61Y/+;Mx1Cre+; Pik3cdD910A/D910A mice were treated with polyI;polyC, and 8 weeks post-treatment, animals were euthanized followed by evaluation of spleen size, hypersensitivity of bone marrow low density mononuclear cells (LDMNCs) to GM-CSF, frequency of bone marrow phenotypically-defined common myeloid, granulocyte-monocyte, and megakaryocyte-erythroid progenitors (CMPs, GMPs, and MEPs), and GM-CSF-stimulated Erk and Akt activation. Genetic disruption of p110a failed to normalize GOF Shp2-induced splenomegaly, GM-CSF hypersensitivity in proliferation assays and methylcellulose-based progenitor assays, or hyperphosphorylation of Erk or Akt. In contrast, genetic ablation of p110d kinase activity significantly reduced spleen size, normalized progenitor hypersensitivity to GM-CSF, and reduced both Akt and Erk hyperactivation. Additionally, genetic inhibition of p110d normalized the skewed hematopoietic progenitor distribution reported in the Shp2D61Y/+;Mx1Cre+ mice, while genetic disruption of p110a failed to do so. This unique function of p110d in the context of GOF Shp2-expressing mice is significant, as p110d expression is restricted to hematopoietic cells and p110d bears transforming properties independent of Ras.While previously published work indicates that the PI3K p110a and p110d inhibitor, GDC-0941, inhibits proliferation of GOF Shp2-expressing cells, we tested if the potent p110d-specific inhibitor, GS-9820, is similarly effective. GOF Shp2-expressing bone marrow LDMNCs treated with GS-9820 demonstrated significantly reduced proliferation in a dose-dependent fashion, while GS-9820 failed to inhibit the proliferation of WT Shp2-expressing cells. GS-9820 treatment decreased Akt phosphorylation (S473 and T308) as well as reduced Erk phosphorylation, indicating that p110d inhibition also reduces signaling within the Ras-MEK-Erk pathway. While PI3K activates the canonical Akt-mTORC1 pathway, it also positively feeds back to the Ras-MEK-Erk pathway via activation of Rac-Pak-MEK; therefore, we evaluated if p110d inhibition adds to or is redundant with MEK inhibition. Treatment of GOF Shp2-expressing hematopoietic cells with the MEK inhibitor, PD0325901, effectively reduced proliferation, and addition of GS-9820 further significantly reduced proliferation, indicating that p110d works cooperatively with MEK to promote GOF Shp2-induced disease.Collectively, our findings suggest that PI3K catalytic subunit p110d functions in a Ras-MEK-Erk pathway-independent manner to promote GOF Shp2-induced hypersensitivity to GM-CSF, and suggest that PI3K p110d inhibition in combination with MEK inhibition may be a novel, optimal approach for the treatment of JMML. Disclosures:No relevant conflicts of interest to declare.

The Protein Tyrosine Phosphatase, Shp2, Positively Contributes to FLT3-ITD-Induced Malignant Disease in Vivo and Co-Localizes with Nuclear Phospho-STAT5 in FLT3-ITD-Expressing Leukemic Cells.

Abstract 2420Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in individuals with acute myeloid leukemia (AML). Based on the finding that the protein tyrosine phosphatase, Shp2, interacts with WT FLT3 tyrosine (Y) 599, which is commonly duplicated in FLT3-ITDs, we hypothesized that increased recruitment of Shp2 to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and aberrant STAT5 activation. Co-immunoprecipitation studies demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Additionally, we found that genetic disruption of Ptpn11, the gene encoding Shp2, significantly reduced N51-FLT3-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation in vitro. To investigate these findings further, Lin- bone marrow cells from Shp2flox/flox;Mx1Cre+ animals were retrovirally transduced with N51-FLT3, sorted to homogeneity, and transplanted into lethally irradiated congenic recipients. Transplanted animals were treated with polyI:polyC to delete Shp2 or with phosphate buffered saline (PBS control) 4 – 6 weeks following transplantation, and animals were followed temporally. The majority of PBS-treated animals (16/18) died of hematologic malignancy. In contrast, animals with Shp2 deletion (polyI:polyC-treated, n=16) succumbed to malignant disease less frequently (10/16), demonstrated a significantly prolonged survival (p=0.024 by log-rank test), and had smaller spleen sizes compared to the PBS-treated animals. Notably, Y599 has been shown to recruit Shp2 to WT FLT3 and mutation of Y599 to phenylalanine (F) within WT FLT3 causes a reduction in FL-stimulated cell proliferation. Thus, we generated point mutants including N51-Y599F1 bearing the Y to F mutation at the first Y599 and N51-Y599F1/2 bearing Y to F mutation at both the first and duplicated Y599. Murine bone marrow low density mononuclear cells were transduced with each construct and subjected to 3H-thymidine incorporation and immunoblot for proliferation and STAT5 activation, respectively. While mutation of the first Y599 alone failed to reduce proliferation or STAT5 phosphorylation, mutation of both the first and duplicated Y599 significantly reduced cellular proliferation and phospho-STAT5 levels. To investigate molecular mechanisms underlying how constitutive association of Shp2 with STAT5 may promote FLT3-ITD-induced leukemogenesis, we utilized the human FLT3-ITD positive AML-derived cell line, MV411. While previous studies have demonstrated nuclear localization of Shp2 in AML samples, the role of nuclear Shp2 in leukemia has never been investigated. We utilized in situ immunofluorescence to examine nuclear distribution of Shp2 and potential co-localization with phospho-STAT5. Strong nuclear expression of Shp2 was observed in MV411 cells, and upon merging of images, nuclear Shp2 co-localized strongly with nuclear phospho-STAT5, suggesting that Shp2 may work with STAT5 within the nucleus to enhance gene expression promoting leukemogenesis. We chose to examine the BCL2L1 promoter, a STAT5-responsive promoter which regulates expression of the prosurvival protein, Bcl-XL. Using chromatin immunoprecipitation assays, we found Shp2 is present at functional interferon-g activation sites (GAS) within the BCL2L1 promoter. Furthermore, knockdown of Shp2 in MV411 cells resulted in reduced phospho-STAT5 levels and reduced BCL2L1 promoter-directed luciferase expression. Moreover, using a novel small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was significantly reduced in a dose-dependent manner. Our findings suggest that constitutive association of Shp2 with N51-FLT3 promotes hyperproliferation and that either genetic disruption of Shp2 expression or mutation of the Shp2 binding sites on N51-FLT3 significantly abrogates N51-FLT3-induced hyperproliferation, STAT5 hyperactivation, and N51-FLT3-induced hematologic malignancy in vivo. Furthermore, Shp2 and STAT5 appear to work functionally in the nucleus to promote STAT5-responsive, pro-leukemogenic gene expression. Collectively, these studies demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML. Disclosures:No relevant conflicts of interest to declare.

The protein tyrosine phosphatase, Shp2, positively contributes to FLT3-ITD-induced hematopoietic progenitor hyperproliferation and malignant disease in vivo

Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia. We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knock-down of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to acute myeloid leukemia.

The effects of WNT activator on chondrocytogenesis of human bone marrow stromal cells

Objective To investigate the effects of a WNT signaling pathway activator, SB-216763,on chondrocytogenesis of human bone marrow stromal cells (hMSCs) in vitro. Methods Fresh bone marrow samples were obtained with institutional review board (IRB) approval as discarded femoral tissue. Low-density mononuclear cells were isolated by density centrifugatian on Ficoll/Histopaque 1077. Adherent hMSCs were expanded 2 passages. Upon near-confluence of hMSCs ,medium was changed to α-MEM with chondrogenic supplements (1% ITS~(+1), 10~(-8) mol/L dexamethasone,50 mg/L ascorbate-2-phosphate, 40 mg/L proline,100 mg/L pyruvate) with or without 5 μmoL/L SB-216763. After 10 days,the cultures were stained with 0.1% alcian blue in 3% acetic acid (pH=1.0) for sulfated glycosaminoglycans and the number of alcian-blue-positive nodules was counted with 4×magnification, hMSCs from five women and one man, aged 57-77 years,wero used to measure content of sulfated glycesaminoglycans by absorbance (A). After 14 days, the effects of SB-216763 on the expression of chondrocyte signature genes were quantified with reverse transeription polymerase chain reaction (RT-PCR). Results During the process of induction differentiation of hMSCs into chondrocytes ,SB-216763 could produce more cartilage nodules (133.17±16.58) than the control group (9.67±1.87 ) ( P<0.01). SB-216763 stimulated the production of sulfated glycosaminogly-cans by 1.6 to 2.6-fold as indicated by alcian blue staining (P<0.01). Consistent with the matrix data, analysis of chondrocyte signature genes showed that the expression of SOX9, collagen Ⅱ, and aggrecan was enhanced by 5 μmol/L SB-216763. Conclusion The WNT signaling pathway activator,SB-216763 stimu-lates in vitro chondrocytogenesis and cartilage matrix production by hMSCs. Key words: MSCs; Chondrocyte; Differentiation; WNT signaling pathway

Targeting the Protein Phosphatase, Shp2, Reduces FLT3-ITD-Induced Hyperproliferation of Murine Hematopoietic Progenitors.

Abstract 827Internal tandem duplications in the FMS-like receptor tyrosine kinase receptor (FLT3-ITDs) are present in approximately 25% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperproliferative cellular growth and promiscuous activation of STAT5; however, the molecular mechanism underlying aberrant activation of these signaling molecules is largely unknown. Upon FLT3 ligand (FL) stimulation, tyrosine 599 (Y599) of WT-FLT3 recruits the protein tyrosine phosphatase, Shp2 and induces cell proliferation and Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73-FLT3, via Y599, contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT-FLT3, N51-FLT3, or N73-FLT3, co-immunoprecipitation assays demonstrated that Shp2 associates with WT-FLT3 in a FL-dependent manner, while Shp2 is constitutively associated with N51-FLT3 and N73-FLT3 independent of FL-stimulation. To investigate the functional role of Shp2 in FLT3-ITD-induced leukemogenesis, Baf3 cells expressing WT-FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III–directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Immunoblot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. To further test the role of Shp2 in FLT3-ITD-induced transformation, Shp2flox/flox mice were crossed with Mx1Cre transgenic mice to produce Shp2flox/flox Mx1Cre+ mice. Shp2flox/floxMx1Cre+ and Shp2flox/floxMx1Cre- pairs were treated with 3 intraperitoneal injections of polyI/polyC to induce recombination of the Shp2 allele and generate a null allele in the Mx1Cre+ animals. PCR and immunoblot analysis revealed ablation of the flox allele and reduction of Shp2 protein, respectively, in Shp2flox/floxMx1Cre+ bone marrow as soon as 2 weeks post injection. Low density mononuclear cells (LDMNCs) isolated from polyI/polyC-treated Shp2flox/floxMx1Cre- and Shp2flox/floxMx1Cre+ mice were transduced with either pMSCV-WT-FLT3 or pMSCV-N51-FLT3. Transduced cells were sorted for EGFP (enhanced green fluorescent protein) expression and subjected to thymidine incorporation assays. Shp2flox/floxMx1Cre+ cells transduced with N51-FLT3 demonstrated significantly reduced proliferation at baseline and in response to FL stimulation compared to Shp2flox/floxMx1Cre- cells expressing N51-FLT3. To pharmacologically examine the role of Shp2 in FLT3-ITD-induced-hyperproliferation and constitutive activation of Stat5, we utilized a Shp2 inhibitor, II-B08, identified from a library of indole salicylic acid derivatives. II-B08 exhibits an IC50 of 5.5 μM for Shp2 and demonstrates excellent cellular activity. Bone marrow LDMNCs retrovirally transduced with WT-FLT3 or N51-FLT3 were incubated in the absence of or in the presence of increasing concentrations of II-B08 and plated into thymidine incorporation assays. A minimal and non-significant reduction in proliferation was observed in WT-FLT3 expressing cells in the presence of II-B08; however N51-FLT3 expressing cells demonstrated a highly significant reduction in cellular proliferation in a concentration-dependent fashion. In addition to these functional analyses, biochemical studies revealed a reduction in Stat5 activation in N51-FLT3 expressing cells treated with II-B08 compared N51-FLT3 expressing cells treated with DMSO in two independent experiments. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3-ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias. Disclosures:No relevant conflicts of interest to declare.

Genetic Disruption of the Scaffolding Protein, Kinase Suppressor of Ras 1 (Ksr1), Differentially Regulates GM-CSF-Stimulated Hyperproliferation in Hematopoietic Progenitors Expressing Activating PTPN11 Mutants D61Y and E76K.

Abstract 1895Poster Board I-918Juvenile myelomoncytic leukemia (JMML) is a lethal childhood disease characterized by the in vitro phenotype of hematopoitic progenitor hypersensitivity to granulocyte-macrophage-colony-stimulating factor (GM-CSF). At the molecular level, Ras hyperactivation is implicated based on the majority of JMML patients bearing either loss-of-function NF1 mutations or gain-of-function RAS or PTPN11 mutations. We demonstrated previously that the Shp2 gain-of-function mutants Shp2E76K and Shp2D61Y induce constitutively elevated and sustained activation of Erk. Signal transduction among Raf1/MEK/Erk kinases is mediated through direct phosphorylation, but scaffolding proteins also play an important role in regulating the location, strength, and duration of Raf1/MEK/Erk signaling. One of the best-defined scaffolding proteins that positively facilitates the Raf1/MEK/Erk cascade is Kinase Suppressor of Ras (Ksr). In its inactivated state, Ksr is phosphorylated and constitutively associated with MEK. In response to growth factor stimulation or Ras activation, Ksr is dephosphorylated (serine 392), translocates to cell membrane, recruits Raf1 and Erk, and, thus, promotes Erk activation. We hypothesized that Ksr contributes to the hyperproliferation and GM-CSF hypersensitivity of mutant Shp2-expressing cells. Upon examination of phosphorylated Ksr levels, we observed lower GM-CSF-stimulated phospho- Ksr levels in the Shp2D61Y- and Shp2E76K-expressing macrophage progenitors compared to cells expressing empty vector or WT Shp2. Consistently, in co-immunoprecipitation assays, we found that upon GM-CSF stimulation, macrophage progenitors expressing Shp2D61Y or Shp2E76K demonstrated an increased physical association between phospho-Erk and Ksr, suggesting that Ksr promotes enhanced Erk activation in mutant Shp2-expressing cells and may contribute functionally to GM-CSF hypersensitivity of mutant Shp2-expressing cells. To examine this hypothesis, we subjected retrovirally transduced WT and Ksr1-/- bone marrow low density mononuclear cells (LDMNCs) to 3H-thymidine incorporation assays and found that GM-CSF-stimulated proliferation of Ksr1-/- cells expressing Shp2E76K was significantly reduced, but not entirely normalized to the level of WT Shp2-expressing cells. In contrast, the proliferation of Ksr1-/- cells expressing Shp2D61Y was unchanged compared to WT cells expressing Shp2D61Y. To examine the effect of genetic disruption of Ksr1 on GM-CSF-stimulated activation of Erk and Akt, western blot analysis was performed using retrovirally transduced WT and Ksr1-/- bone marrow LDMNCs, as described above. Activation of phospho-Erk was similarly reduced in both Shp2E76K- and Shp2D61Y-expressing cells upon genetic disruption of Ksr1 both at baseline and in response to GM-CSF. However, in contrast, Akt activation was increased, rather than decreased, in both Ksr1-/- Shp2E76K- and Shp2D61Y-expressing cells, suggesting that a compensatory mechanism in the absence of Ksr leads to enhanced signaling through the phospho-inositol-3-kinase (PI3K) pathway in mutant Shp2-expressing cells. Taken together, these findings suggest that the E76K mutant is dependent on Ksr-mediated Erk activation for GM-CSF-stimulated hyperactivation and that the compensatory upregulation of Akt activation in the absence of Ksr may contribute to the incomplete correction of GM-CSF hypersensitivity. Regarding the D61Y mutant, although Erk activation is reduced in the absence of Ksr, the lack of GM-CSF hypersensitivity correction suggests that the Shp2D61Y-expressing cells are more sensitive to the compensatory upregulation of Akt activation. Disclosures:No relevant conflicts of interest to declare.

Increased C-Jun and Reduced GATA2 Expression Promotes Aberrant Monocytic Differentiation and Expansion Induced by Activating PTPN11 Mutants

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation and expansion despite being subjected to conditions that normally support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression using quantitative RT-PCR indicates equal expression of PU.1 in mutant Shp2- and WT Shp2-expressing hematopoietic progenitors; however, GATA2 expression is substantially reduced and c-Jun expression is increased in mutant Shp2-expressing progenitors. Based on these findings, we hypothesized that mutant Shp2-induced Ras hyperactivation produces constitutive c-Jun expression permitting, as a co-activator of PU.1, excessive monocytic differentiation and reduced GATA2 expression. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (amino acids 330 – 407, DGATA2), which is needed to compete with c-Jun for binding to PU.1, would normalize mutant Shp2-induced monocytic differentiation and expansion. To examine this hypothesis, we utilized retroviral co-transduction of bone marrow Lin-enriched cells to generate six experimental groups:pMIEG3-Shp-2WT plus pCD4 (empty vector);pMIEG3-Shp2WT plus pCD4-GATA2;pMIEG3-Shp2WT plus pCD4-ΔGATA2;pMIEG3-Shp-2E76K plus pCD4;pMIEG3-Shp-2E76K plus pCD4-GATA2;pMIEG3-Shp-2E76K plus pCD4-ΔGATA2.Cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2 mutant E76K and a secondary empty vector produced significantly more CFU-M than cells expressing WT Shp2. Upon co-transduction with GATA2, the number of CFU-M generated from E76K-expressing cells was significantly reduced. In contrast, cotransduction of ΔGATA2 lacking the C-terminal zinc finger failed to normalize the number of CFU-M produced by E76K-expressing cells. Mechanistically, we reasoned that if the c-Jun-PU.1 interaction contributes to the mutant Shp2-induced aberrant monocytic differentiation, we would observe increased expression directed by the macrophage colony-stimulating factor receptor (MCSFR) promoter, originally characterized to be activated by PU.1 and co-activator c-Jun. Consistently, MCSFR promoter-directed luciferase expression exhibited higher levels in NIH3T3 cells expressing Shp2E76K compared to those expressing WT Shp2. Furthermore, co-immunoprecipitation assays using nuclear extracts demonstrate increased c-Jun-PU.1 complexes in mutant Shp2-expressing hematopoietic progenitors, while chromatin immunoprecipitation assays demonstrate an increased association of the c-Jun-PU.1 complex at the MCSFR promoter. Moreover, CD34+ JMML progenitors express significantly higher levels of c-JUN than CD34+ cord blood progenitors from healthy newborns, substantiating the disease relevance of these mechanistic findings. These data suggest that one fundamental sequela of Ras hyperactivation in mutant Shp2-expressing cells is elevated c-Jun expression and an increased c-Jun-PU.1 interaction promoting monocytic differentiation and, thus, overproduction of myelomonocytic cells, the hallmark of JMML. These findings imply that pharmaceutical interventions that inhibit c-Jun expression or function could theoretically inhibit mutant Shp2-induced monocytic differentiation and, thus, serve as a novel approach for treatment of JMML.

High Frequency of Tumor Suppressor Genes Promoter Hypermethylation in Peripheral Blood of Patients with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias, with very little established about their pathogenesis. DNA methylation is a common modification thought to be relevant to carcinogenesis. The epigenetic silencing, through methylation of CpG islands within the promoter regions, is one of the mechanisms by which numerous genes are inactivated in MDS and acute myeloid leukemias (AML). In particular, the promoter of CDKN2B (encoding p15INK4b), has been shown to be hypermethylated in poor-risk subtypes of MDS and often predicts transformation to AML. In this study, we examined the promoter methylation status of 7 genes in peripheral blood (PB) cells collected from 19 patients affected by untreated MDS. We investigated genes involved in cell cycle regulation (p14ARF, p15INK4b, p16INK4a), apoptosis (SPARC, DAPK1), differentiation (RARb) and response to growth factors (RASSF1A) previously reported as being abnormally hypermethylated in acute leukemia and in a variety of solid malignancies. DNA was extracted from PB low-density mononuclear cells and promoters methylation status was analyzed by means of methylation-specific PCR assay (MSP). MSP distinguishes unmethylated from methylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated (but not methylated) cytosines to uracil, and subsequent PCR using primers designed for either methylated or unmethylated DNA. Sodium bisulfite modification was performed using the Methylamp DNA Modification Kit (Epigentek) following manufacturer's instructions. Our series included 4 RA, 3 RARS, 3 RCMD, 2 RCMD/RS, 5 RAEB-1, 1 RAEB-2 and 1 CMML-1; according to the IPSS, 17 patients (89%) were classified as either low- or intermediate-1 risk. Median age was 72 years (range 49–86). Cytogenetics were normal in 12 patients (63%), while in 3 patients showed the 5q- abnormality. Median CBC counts were: WBC 3.1 × 109/L (0.9–12.5), Hb 10 g/dl (6.4–15), PLT 106 × 109/L (9–406). The cell cycle regulator p15INK4b was found to be the most frequently hypermethylated gene (14/19 pts, 74%) in our MDS series, followed by the p14ARF (13/19 pts, 68%). The latter was hypermethylated in 100% of our 5q- cases. Ninety-five percent (18/19 pts) of the MDS samples were found to be hypermethylated in at least one of the seven genes, with the only exception being the patient with CMML (unmethylated in all the tested genes). Hypermethylation of the cell cycle regulator p16INK4a was detected in 47% of samples. In 3 patients (16%) we found hypermethylation in all genes but RASSF1A, which indeed was found unmethylated in all but one of the samples analyzed (5%). The tumor suppressor gene SPARC was hypermethylated in 58% of patients (including 2 out of the 3 cases of 5q-), whereas 42% and 47% of samples, respectively, showed hypermethylation of the RARb and DAPK1 genes promoter. Although in a limited number of samples, we detected a differential rate of hypermethylation of the tested genes in peripheral blood of MDS patients. Altogether, our results seem to confirm high frequency of hypermethylation genes involved in regulation of cell cycle and apoptosis in MDS. We conclude that mononuclear cells from peripheral blood represents an easily accessible sample for detection and monitoring of genes promoter hypermethylation in myelodysplastic syndromes.

Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

Activating PTPN11 mutants promote hematopoietic progenitor cell-cycle progression and survival

Mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp2, are commonly found in juvenile myelomonocytic leukemia (JMML). We hypothesized that PTPN11 mutations promote cell-cycle progression and confer enhanced survival to hematopoietic progenitors. Murine bone marrow low-density mononuclear cells were transduced with pMIEG3, pMIEG3-WT Shp2, pMIEG3-Shp2D61Y, or pMIEG3-Shp2E76K followed by cell-cycle and survival functional analysis as well as biochemical analysis for key cell-cycle and programmed cell-death regulatory proteins. A higher proportion of hematopoietic progenitors bearing the gain-of-function Shp2 mutants were residing in the S or G2 phase of the cell cycle in response to low doses of granulocyte-macrophage colony-stimulating factor compared to cells transduced with empty vector (MIEG3) or with WT Shp2. Likewise, Shp2D61Y- or Shp2E76K-expressing hematopoietic cells demonstrated reduced apoptosis based on Annexin-V staining and produced increased progenitor colonies after 48 hours in minimal media compared to cells transduced with empty vector or WT Shp2. To differentiate enhanced survival vs hyperproliferation, cells were stained with PKH26 to distinguish undivided cells from divided progeny. Shp2D61Y- or Shp2E76K-expressing PKH26+ cells similarly demonstrated reduced apoptosis. Upon biochemical analysis, expression of Akt- and Erk-responsive cell-cycle and programmed cell-death regulatory proteins were altered, including increased levels of cyclin D1, Bcl2, and BclXL and reduced levels of p27, p21, and Bim. Collectively, these data demonstrate that gain-of-function Shp2 mutants promote hematopoietic progenitor cell-cycle progression and survival and imply that agents targeting the cell cycle or promoting apoptosis may have therapeutic potential in JMML.

Adult Bone Marrow–derived Cells Do Not Acquire Functional Attributes of Cardiomyocytes When Transplanted into Peri-infarct Myocardium

The cardiomyogenic potential of adult bone marrow (BM) cells after being directly transplanted into the ischemically injured heart remains a controversial issue. In this study, we investigated the ability of transplanted BM cells to develop intracellular calcium ([Ca(2+)](i)) transients in response to membrane depolarization in situ. Low-density mononuclear (LDM) BM cells, c-kit-enriched (c-kit(enr)) BM cells, and highly enriched lin(-) c-kit(+) BM cells were obtained from adult transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP), and injected into peri-infarct myocardiums of nontransgenic mice. After 9-10 days the mice were killed, and the hearts were removed, perfused in Langendorff mode, loaded with the calcium-sensitive fluorophore rhod-2, and subjected to two-photon laser scanning fluorescence microscopy (TPLSM) to monitor action potential-induced [Ca(2+)](i) transients in EGFP-expressing donor-derived cells and non-expressing host cardiomyocytes. Whereas spontaneous and electrically evoked [Ca(2+)](i) transients were found to occur synchronously in host cardiomyocytes along the graft-host border and in areas remote from the infarct, they were absent in all of the >3,000 imaged BM-derived cells that were located in clusters throughout the infarct scar or peri-infarct zone. We conclude that engrafted BM-derived cells lack attributes of functioning cardiomyocytes, calling into question the concept that adult BM cells can give rise to substantive cardiomyocyte regeneration within the infarcted heart.

The C-Terminal Zinc Finger of GATA2 Is Necessary for Normalization of Aberrant Myelomonocytic Differentiation Induced by Activating PTPN11 Mutations.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation despite being subjected to conditions that typically support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression revealed that GATA2 expression, needed for mast cell differentiation, is dramatically reduced, while, surprisingly, PU.1 expression is unchanged in cells expressing activating Shp2 mutants. In addition to lineage-specific transcription factors such as PU.1, however, c-jun also promotes monocytic differentiation by functioning as a co-activator of PU.1. Thus, we hypothesized that activating Shp2 mutations (Shp2D61Y or Shp2 E76K) induce increased c-jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression in hematopoietic progenitors. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (GATA2del330–407), which is needed for disruption of the PU.1-c-jun interaction, would normalize aberrant myelomonocytic differentiation induced by activating Shp2 mutants. Consistent with our hypothesis, quantitative RT-PCR studies revealed 5-fold higher c-jun levels in cells expressing Shp2D61Y or Shp2E76K compared to cells expressing WT Shp2. We next utilized retroviral co-transduction of murine bone marrow LDMNCs to generate six groups:1.pMIEG3-Shp2WT plus pCD4 (empty vector);2.pMIEG3-Shp2WT plus pCD4-GATA2;3.pMIEG3-Shp2WT plus pCD4-GATA2del330-407;4.pMIEG3-Shp2E76K plus pCD4;5.pMIEG3-Shp2E76K plus pCD4-GATA2; and6.pMIEG3-Shp-2E76K plus pCD4-GATA2del330-407.Transduced cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2E76K plus pCD4 produced significantly more CFU-M than cells co-transduced with WT Shp2 plus pCD4. Upon co-transduction with GATA2, the number of CFU-M generated from Shp2E76K-expressing cells was significantly reduced and was similar to that observed in cells expressing WT Shp2. In contrast, co-transduction of GATA2del330-407 failed to normalize the number of CFU-M produced by Shp2E76K-expressing cells. Quantitative RT-PCR verified ectopic GATA2 and GATA2del330-407 expression in the co-transduced cells. These findings demonstrate that restoration of GATA2 expression normalizes the propensity toward monocytic differentiation induced by Shp2E76K. The lack of correction conferred by GATA2del330-407 in combination with the observed increased c-jun expression support a model in which GATA2 and c-jun compete for binding to PU.1 to direct cell differentiation decisions in hematopoietic progenitors bearing activating Shp2 mutants. Collectively, these findings imply that normalization of transcription factor expression may provide a novel approach to differentiation-mediated therapy in JMML.

Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.

Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood. 109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Drive Cardiac and Endothelial Fate in Term Placenta Human Mesenchymal Stem Cells and Enhance Cardiac Repair in Infarcted Rat Hearts

We have developed a mixed ester of hyaluronan with butyric and retinoic acid (HBR) that acted as a novel cardiogenic/vasculogenic agent in human mesenchymal stem cells isolated from bone marrow, dental pulp, and fetal membranes of term placenta (FMhMSCs). HBR remarkably enhanced vascular endothelial growth factor (VEGF), KDR, and hepatocyte growth factor (HGF) gene expression and the secretion of the angiogenic, mitogenic, and antiapoptotic factors VEGF and HGF, priming stem cell differentiation into endothelial cells. HBR also increased the transcription of the cardiac lineage-promoting genes GATA-4 and Nkx-2.5 and the yield of cardiac markerexpressing cells. These responses were notably more pronounced in FMhMSCs. FMhMSC transplantation into infarcted rat hearts was associated with increased capillary density, normalization of left ventricular function, and significant decrease in scar tissue. Transplantation of HBR-preconditioned FMhM-SCs further enhanced capillary density and the yield of human vWF-expressing cells, additionally decreasing the infarct size. Some engrafted, HBR-pretreated FMhMSCs were also positive for connexin 43 and cardiac troponin I. Thus, the beneficial effects of HBR-exposed FMhMSCs may be mediated by a large supply of angiogenic and antiapoptotic factors, and FMhMSC differentiation into vascular cells. These findings may contribute to further development in cell therapy of heart failure.

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human–rat chimeras

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human–rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9–11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.

24 Ectopic Expression of GATA2 Normalizes Aberrant Myelomonocytic Differentiation Induced by Activating PTPN11 Mutations.

Purpose of Study: Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp-2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp-2 mutants preferentially undergo myelomonocytic differentiation despite being subjected to conditions that select for mast cell development (Chan et al., Blood 106, Abstract #3519, 2005). Consistently, GATA-2 expression is dramatically reduced in cells expressing activating Shp-2, suggesting that Shp-2 gain-of-function mutants alter hematopoietic lineage-specific transcription factors and thus shift myeloid differentiation toward the myelomonocytic lineage. We hypothesized that ectopic GATA2 expression would normalize the aberrant myelomonocytic differentiation induced by activating Shp-2 mutants.