Search

To the editor: Germ line–activating mutations of protein tyrosine phosphatase PTPN11 (Shp2), a positive regulator of the Ras signaling pathway,[1][1] account for more than 50% of patients with Noonan syndrome.[2][2] These patients have an increased risk of developing leukemias,[3][3] especially

Germline and somatic gain-of-function mutations in tyrosine phosphatase PTPN11 (SHP-2) are associated with juvenile myelomonocytic leukemia (JMML), a myeloproliferative disease (MPD) of early childhood. The mechanism by which PTPN11 mutations induce this disease is not fully understood. Signaling partners that mediate the pathogenic effects of PTPN11 mutations have not been explored. Here we report that germ line mutation Ptpn11D61G in mice aberrantly accelerates hematopoietic stem cell (HSC) cycling, increases the stem cell pool, and elevates short-term and long-term repopulating capabilities, leading to the development of MPD. MPD is reproduced in primary and secondary recipient mice transplanted with Ptpn11D61G/+ whole bone marrow cells or purified Lineage−Sca-1+c-Kit+ cells, but not lineage committed progenitors. The deleterious effects of Ptpn11D61G mutation on HSCs are attributable to enhancing cytokine/growth factor signaling. The aberrant HSC activities caused by Ptpn11D61G mutation are largely corrected by deletion of Gab2, a prominent interacting protein and target of Shp-2 in cell signaling. As a result, MPD phenotypes are markedly ameliorated in Ptpn11D61G/+/Gab2−/− double mutant mice. Collectively, our data suggest that oncogenic Ptpn11 induces MPD by aberrant activation of HSCs. This study also identifies Gab2 as an important mediator for the pathogenic effects of Ptpn11 mutations.

Gab2 is an important adapter molecule for cytokine signaling. Despite its major role in signaling by receptors associated with hematopoiesis, the role of Gab2 in hematopoiesis has not been addressed. We report that despite normal numbers of peripheral blood cells, bone marrow cells, and c-Kit+Lin−Sca-1+ (KLS) cells, Gab2-deficient hematopoietic cells are deficient in cytokine responsiveness. Significant reductions in the number of colony-forming units in culture (CFU-C) in the presence of limiting cytokine concentrations were observed, and these defects could be completely corrected by retroviral complementation. In earlier hematopoiesis, Gab2-deficient KLS cells isolated in vitro responded poorly to hematopoietic growth factors, resulting in an up to 11-fold reduction in response to a cocktail of stem cell factor, flt3 ligand, and thrombopoietin. Gab2-deficient c-Kit+Lin− cells also demonstrate impaired activation of extracellular signal-regulated kinase (ERK) and S6 in response to IL-3, which supports defects in activating the phosphatidylinositol-3 kinase (PI-3K) and mitogen-associated protein kinase (MAPK) signaling cascades. Associated with the early defects in cytokine response, competitive transplantation of Gab2−/− bone marrow cells resulted in defective long-term multilineage repopulation. Therefore, we demonstrate that Gab2 adapter function is intrinsically required for hematopoietic cell response to early-acting cytokines, resulting in defective hematopoiesis in Gab2-deficient mice.

SHP-2 phosphatase forms a stable protein complex with and is heavily tyrosine-phosphorylated by the oncogenic tyrosine kinase Bcr-Abl. However, the role of SHP-2 in Bcr-Abl–mediated leukemogenesis is unclear. In the present report, we provide evidence that SHP-2 is required for hematopoietic cell transformation by Bcr-Abl. In vitro biological effects of Bcr-Abl transduction were diminished in SHP-2Δ/Δ hematopoietic cells, and the leukemic potential of Bcr-Abl–transduced SHP-2Δ/Δ cells in recipient animals was compromised. Further analyses showed that Bcr-Abl protein (p210) was degraded, and its oncogenic signaling was greatly decreased in SHP-2Δ/Δ cells. Treatment with proteasome inhibitors or reintroduction of SHP-2 restored p210 level in Bcr-Abl–transduced SHP-2Δ/Δ cells. Subsequent investigation revealed that SHP-2 interacted with heat shock protein 90, an important chaperone protein protecting p210 from proteasome-mediated degradation. The role of SHP-2 in the stability of p210 is independent of its catalytic activity. Blockade of SHP-2 expression in p210-expressing cells by antisense or small-interfering RNA approaches decreased p210 level, causing cell death. Inhibition of SHP-2 enzymatic activity by overexpression of catalytically inactive SHP-2 mutant did not destabilize p210 but enhanced serum starvation-induced apoptosis, suggesting that SHP-2 also plays an important role in downstream signaling of p210 kinase. These studies identified a novel function of SHP-2 and suggest that SHP-2 might be a useful target for controlling Bcr-Abl–positive leukemias.

Hematopoiesis initiates in the extraembryonic yolk sac. To isolate various types of precursor cells from this blood cell–forming tissue, yolk sac cells were immortalized by retroviral-mediated expression of the HOX11 homeobox-containing gene. Among the cell lines derived, some were able to spontaneously generate adherent stromal-like cells capable of taking up acetylated low-density lipoprotein, and they could be induced to form tubelike structures when cultured on Matrigel. Although these cell lines were negative for hematopoietic cell surface markers, they gave rise to hematopoietic colonies—containing cells belonging to the monocytic, megakaryocytic, and definitive erythroid lineages—when plated in methylcellulose medium supplemented with hematopoietic growth factors. Low amounts of Flk-1 mRNA could be detected in these cells, and they showed significant responsiveness to vascular endothelial growth factor, stem cell factor, basic fibroblast growth factor, and interleukin 6. They also expressed the transcription factors SCL, GATA2, GATA1, PU.1, and c-myb. These yolk sac–derived cell lines may represent a transitional stage of early hematopoietic development.

Gab2, a newly identified pleckstrin homology domain-containing docking protein, is a major binding protein of SHP-2 tyrosine phosphatase in interleukin (IL)-3–stimulated hematopoietic cells. Its signaling mechanism remains largely unknown. We report here an important regulatory role for Gab2 in β1 integrin signaling pathway that mediates hematopoietic cell adhesion and migration. Cross-linking of the β1 integrin on Ba/F3 cells induced rapid tyrosine phosphorylation of Gab2 and its association with Syk kinase, SHP-2 phosphatase, and the p85 subunit of phosphatidylinositol (PI)-3 kinase. In addition, Gab2 was also constitutively associated with SHP-1 phosphatase via its C-terminal Src homology 2 domain. Overexpression of the pleckstrin homology domain or a mutant Gab2 molecule lacking SHP-2 binding sites resulted in significant reductions in Ba/F3 cell adhesion and migration. Biochemical analyses revealed that enforced expression of Gab2 mutant molecules dramatically reduced β1-integrin ligation-triggered PI3 kinase activation, whereas Erk kinase activation remained unaltered. Furthermore, transduction of primary hematopoietic progenitor cells from viable motheaten mice with these mutant Gab2 molecules also significantly ameliorated their enhanced migration capacity associated with theSHP1 gene mutation. Taken together, these results suggest an important signaling role for Gab2 in regulating hematopoietic cell adhesion and migration.

Background: Cytogenetic and molecular aberrations have improved risk stratification of acute myeloid leukemia (AML) patients, however survival continues to be low (40-50%) and current therapy primarily includes high dose cytotoxic chemotherapy with or without allogeneic stem cell transplant. In AML, metabolomic analysis has identified key differences in glycolysis, gluconeogenesis and tri-carboxylic acid (TCA) pathways between normal and AML cells. Monotherapy with mitochondrial inhibitors like Metformin have met with little success since cancer cells use glycolytic escape mechanisms to overcome the decreased mitochondrial oxidative phosphorylation. Here we demonstrate a novel therapeutic approach for overcoming this route of Metformin resistance. Methods: Metformin, a well-established anti-diabetic drug, is a strong inhibitor of mitochondrial respiration and is also considered an inhibitor of mTOR signaling acting via AMPK activation. Despite its therapeutic potential, cytotoxicity of AML cells with Metformin alone at achievable plasma levels (10-20 mM) is modest due in part to increased glycolytic ATP production as a bypass mechanism. This has limited the effectiveness of Metformin in AML and led us to investigate mechanisms for reversal of this resistance mechanism to improve the pre-clinical efficacy. 6-Benzylthioinosine (6-BT) is a small molecule that we have shown depletes ATP and can cause differentiation in myeloid leukemia cells (at 10µM concentration) with minimal toxicity to normal cells (LD50 >100µM) (Wald et al. Cancer Res, 2008). To improve on cytotoxicity seen with Metformin, we hypothesized that the combination of 6-BT and Metformin would enhance cytotoxicity in AML. The combination of Metformin (10mM) and 6-BT (10 µM) were tested alone or combined. We compared the effects of the combination treatment on mTOR signaling and caspase-3 activation. We also measured changes in reactive oxygen species (ROS), intracellular ATP, extracellular glucose levels and GLUT-1 expression. Glycolytic flux was measured by Extracellular Acidification Rate (ECAR) and oxidative phosphorylation was measured by Oxygen Consumption Rate (OCR) using Seahorse XF Analyzer. All experiments were performed in triplicate and Student’s two tailed t-test was used to calculate p-values (less than 0.05 were considered to be statistically significant). Results: Metformin and 6-BT alone caused inhibition of cell growth (< 3-fold) and modest cytotoxicity (10-40%, SD ± 6.3%) in all AML cell lines tested (MV4-11, MOLM-14, OCI-AML3, Nomo-1 and THP-1). However the combination of 6-BT with Metformin resulted in marked growth inhibition (30-50 fold) and cytotoxicity (90-100%, SD ± 8.3%) in monocytic AML cell lines (MV4-11 and MOLM-14) within 48 hours of exposure, which was partially dependent on caspase-3 induction. Apoptosis induction was also detected by Annexin V assay. Cytotoxic effects were independent of mTOR inhibition since AZD8055 showed no synergy with the 6-BT or the combination. 6-BT has structural similarity with 6-Mercaptopurine (6-MP), however the combination of Metformin and 6-MP also showed no synergy. 6-BT combined with Metformin caused significant (50-70%) reduction in intracellular ATP levels in the monocytic AML lines without significant extracellular glucose depletion suggestive of glycolytic inhibition. As expected, Metformin significantly suppressed oxidative phosphorylation as evident by near complete inhibition of OCR ( Conclusions: The anti-tumor and metabolic effects of Metformin have been limited by the metabolic reprogramming within cells that result in low cytotoxicity. Here we show that the novel combination of 6-BT and Metformin targets this metabolic bypass mechanism resulting in increased cell death. This is the first demonstration that 6-BT, a drug that is selectively taken up and phosphorylated by AML cells, can exert cytotoxicity involving down modulation of glycolysis. We propose that the combination of 6-BT and Metformin represents a new therapeutic strategy that warrants further testing in primary patient samples. Disclosures No relevant conflicts of interest to declare.

Effective therapeutic interventions for juvenile myelomonocytic leukemia (JMML), a fatal childhood malignancy, are lacking. Relapse is the most frequent cause of treatment failure, most likely due to the persistence of leukemic stem cells (LSCs), a small population of self-renewing precursor cells that give rise to the bulk of tumor cells. This reservoir of tumor cells are responsible for long-term maintenance of leukemia growth, and are also a major source of drug resistance. Clearly, a novel approach focused on the unique properties of LSCs is needed. However, it remains a critical challenge how such cells may be eradicated. JMML is known to be caused by genetic mutations in cell signaling proteins involved in the Ras pathway, among which the phosphatase Ptpn11 (Shp2) is most frequently mutated. Ptpn11 mutations (heterozygous) cause greatly increased catalytic activity. We have recently created conditional knock-in mice with the Ptpn11E76K mutation, the most common Ptpn11 mutation found in JMML. Induction of the Ptpn11E76K/+ mutation in these mice (Ptpn11E76K/+/Mx1-Cre+) leads to JMML-like myeloid malignancy with full penetrance by aberrant activation of stem cells and myeloid progenitors. In an effort to understand the biological properties of LSCs in JMML, we studied leukemic hematopoietic stem cells (Lin-Sca-1+c-Kit+Flk2-CD150+CD48- cells)(referred to as LSCs since they reproduce the same disease in sub-lethally-irradiated transplants) in this mouse model. We found that Stat5 was hyper-activated in LSCs in Ptpn11E76K/+/Mx1-Cre+ mice in the chronic phase. Stat5 hyperactivation is likely to be mediated through Jak2 kinase as Jak2 is highly activated in Shp2 E76K-expressing cells. More importantly, these LSCs appear to rely on hyperactivation of Stat5 for maintenance and self-renewal because deletion of Stat5ab in Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl double mutant mice resulted in massive cell death in LSCs while neither Stat5ab knockout nor Ptpn11E76K/+ knock-in alone mice had these stem cell phenotypes. Apoptotic cells in the stem cell population were 6.38±1.42, 4.06±0.51, 10.52±6.88, and 24.50±10.27% in Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, Stat5abfl/fl/Mx1-Cre+, and Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice, respectively. Consequently, the stem cell pool in Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl double mutants was drastically decreased. Numbers of stem cells per femur were 3.36±1.51, 0.94±0.63, 1.15±0.39, and 0.17±0.20 (x103) in Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, Stat5abfl/fl/Mx1-Cre+, and Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice, respectively. Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice died of pan cytopenia within 4-8 weeks of Stat5 deletion while none of other groups of mice did. Repopulation capabilities of the double mutant stem cells were essentially lost in sub-lethally-irradiated recipient animals. Further mechanistic investigations revealed that tyrosine phosphorylation levels (indicative of activity) of Stat3, one of the substrates of the Shp2 phosphatase, were decreased by ~7.50 fold in Ptpn11E76K/+ LSCs as compared to Ptpn11+/+ control cells due to the enhanced dephosphorylation by the hyperactive Shp2 E76K mutant. Thus, diminished Stat3 activity sensitized Ptpn11E76K/+ LSCs to Stat5 depletion-induced cell death. Taken together, this study suggests a crucial role of Stat5 in the maintenance of LSCs in Ptpn11-associated JMML. The synthetic lethality induced by loss of Stat5 in Ptpn11-mutated JMML raises the possibility that clinically-used inhibitors of upstream Jak2 kinase may be effective in eradicating LSCs in this particular subtype of JMML. Disclosures No relevant conflicts of interest to declare.

Shp-1 and Shp-2 are cytoplasmic phosphotyrosine phosphatases with similar structures. Mice deficient in Shp-2 die at midgestation with defects in mesodermal patterning, and a hypomorphic mutation at the Shp-1 locus results in the moth-eaten viable (mev) phenotype. Previously, a critical role of Shp-2 in mediating erythroid/myeloid cell development was demonstrated. By using the RAG-2–deficient blastocyst complementation, the role of Shp-2 in lymphopoiesis has been determined. Chimeric mice generated by injecting Shp-2−/− embryonic stem cells into Rag-2–deficient blastocysts had no detectable mature T and B cells, serum immunoglobulin M, or even Thy-1+ and B220+ precursor lymphocytes. Collectively, these results suggest a positive role of Shp-2 in the development of all blood cell lineages, in contrast to the negative effect of Shp-1 in this process. To determine whether Shp-1 and Shp-2 interact in hematopoiesis, Shp-2−/−:mev/mev double-mutant embryos were generated and the hematopoietic cell development in the yolk sacs was examined. More hematopoietic stem/progenitor cells were detected in Shp-2−/−:mev/mevembryos than in Shp-2−/− littermates. The partial rescue by Shp-1 deficiency of the defective hematopoiesis caused by the Shp-2 mutation suggests that Shp-1 and Shp-2 have antagonistic effects in hematopoiesis, possibly through a bidirectional modulation of the same signaling pathway(s).

Abstract 616 Background: Older investigations suggest that Factor XII (FXII) influences the inflammatory response. FXII deficient patients have reduced leukocyte migration into skin windows. In addition to bradykinin generation, FXII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. In vitro, purified FXIIa corrects neutrophil aggregation and degranulation defects in FXII-deficient plasma. Our laboratory demonstrated that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation, growth and angiogenesis. Since FXII modulates wound angiogenesis, we examined the role for FXII in the inflammatory response and wound repair. Investigations: Exon 3 to 8–deleted FXII knockout mice (FXII−/−) were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited to the injury site per 20X high power field (HPF) was determined and analyzed by Image J (NIH). We observed that on Day 1, FXII−/− mice exhibit significantly decreased recruitment of CD11 b–labeled inflammatory cells to injury sites compared with wild type mice (WT) [p=0.0136]. Similar results are observed when uPAR KO mice were compared to WT [p=0.0001], suggesting that leukocyte migration in part is mediated through this receptor. Next we determined the nature of cells in the wound sites. FXII−/− mice have reduced wound neutrophils (Gr-1 staining) and macrophages (F4-80 staining). On the thioglycolate (TG)-induced peritoneal inflammation assay, the number of peritoneal exudate cells (PECs) was measured in WT and FXII−/− mice 1 and 7 days following instillation. FXII−/− mice exhibit significantly decreased number of PEC's on days 1 and 7. Giemsa-Wright stain of peritoneal lavage fluid with manual differential counts or flow cytometry shows that there is a disproportionate decrease in neutrophil recruitment in peritoneal fluid of FXII−/− mice. Also, FXII−/− macrophages have reduced adherence to plastic. Identical assays performed on uPAR and bradykinin B2 receptor (B2R) KO mice did not reveal the same inflammatory defects; both uPAR and B2R KO mice have mild decreases in PEC cell numbers following TG instillation, but overall their inflammatory response remained intact. Investigations next determined if the leukocyte migration defect seen in FXII−/− mice is related to plasma FXII or an intrinsic leukocyte defect. On adoptive bone marrow (BM) transplantation experiments, WT bone marrow transplanted to FXII−/− mice corrects the TG-induced PEC migration defect after both 1 and 7 days from instillation. Alternatively, transplantation of FXII−/− bone marrow into WT mice produced a leukocyte migration defect. In order to further discriminate the contribution of FXII in leukocyte migration, we produced plasma FXII deficiency in WT mice using FXII siRNA. WT mice treated with FXII siRNA reduced plasma FXII activity to Conclusions: These data indicate that there is a unique pool of FXII in leukocytes distinct from plasma (hepatic) FXII. Leukocyte FXII, not plasma FXII, is responsible for leukocyte function in wounds and inflammation sites. FXII−/− mice have attenuated wound injury and, paradoxically, improved wound healing rates. Modulation of leukocyte FXII is a target for promotion of wound healing. Disclosures: No relevant conflicts of interest to declare.

Abstract 392 SHP2, a protein tyrosine phosphatase implicated in multiple cell signaling processes, plays an essential role in hematopoietic cell development. Our previous studies have demonstrated that this phosphatase is required for erythroid, myeloid, and lymphoid development and that it functions in cytokine signaling in both catalytically-dependent and –independent manners. Notably, germline and somatic mutations (heterozygous) in PTPN11 (encoding SHP2) have been identified in 35% of the patients with juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disorder (MPD). Furthermore, PTPN11 mutations are also found in pediatric myelodysplastic syndromes (10%), B cell lymphoblastic leukemia (B-ALL) (7%), acute myeloid leukemia (AML) (4%), and sporadic solid tumors. These mutations result in hyperactivation of SHP2 catalytic activity. In addition, PTPN11 disease mutations, especially leukemia mutations, enhance the binding of mutant SHP2 to signaling partners. Although previous studies have shown that Ptpn11 mutations induce cytokine hypersensitivity in myeloid progenitors and MPD in mice, it is unclear whether Ptpn11 mutations also play a causal role in the pathogenesis of acute leukemias. If so, the underlying mechanisms and the cell origin of leukemia initiating/stem cells (LSCs) remain to be determined. PTPN11E76K mutation is the most common and most active PTPN11 mutation found in JMML and acute leukemias. However, the pathogenic effects of this mutation have not been well characterized. We created Ptpn11E76K conditional knock-in mice. Global Ptpn11E76K/+ mutation resulted in early embryonic lethality associated with enhanced ERK signaling. Induced knock-in of this mutation in pan hematopoietic cells led to MPD as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors. These animals subsequently progressed to acute leukemias. Intriguingly, in addition to AML, T-ALL and B-ALL were evolved. PTPN11E76K/+ mutation induced LSC development not only in stem cells but also in lineage committed progenitors as tissue-specific knock-in of Ptpn11E76K/+ mutation in myeloid, T lymphoid, and B lymphoid progenitors also resulted in AML, T-ALL, and B-ALL, respectively. Further analyses revealed that Shp2 was distributed to centrosomes and that Ptpn11E76K/+ mutation promoted LSC development partly by causing centrosome amplification and genomic instability. Thus, Ptpn11E76K mutation has non-lineage specific effects on malignant transformation of hematopoietic cells and initiates acute leukemias at various stages of hematopoiesis. This mutation may play an initiating role in the pathogenesis of pediatric acute leukemias. Disclosures: No relevant conflicts of interest to declare.

Abstract 368 Background: Factor XII (FXII) participates in inflammation. FXII deficient patients have reduced leukocyte migration into skin windows. Recently, we have determined that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation and post-natal angiogenesis. Present studies examined if this pathway also influences leukocyte response to injury. Methods: Exon 3- to 8-deleted FXII knockout mice (FXII KO), generously provided by Dr. Frank Castellino, have impaired inflammatory response to injury. FXII KO were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited at the injury site/20 × high power field (HPF) was determined and analyzed by Image J (NIH). We observed that FXII KO on Day 1 exhibit significantly decreased recruitment of CD11b-labeled inflammatory cells to injury sites compared with wild type mice (WT) [mean ± SEM, FXII KO: 40220 ± 3732 vs. WT: 59740 ± 6318 total surface area of CD11b positive cells (pixels)/HPF, p=0.0136]. Similar results are observed when uPAR KO were compared to WT mice [39380 ± 5234 vs. 73310 ± 4688, p=0.0001], suggesting that leukocyte migration is mediated through uPAR. On Day 1 of thioglycolate (TG)-induced peritonitis, FXII KO have a mean of 12.62 × 105/mL peritoneal exudate cells (PEC) vs. 25.94 × 105/mL (p=0.0072) observed in WT. Additionally on Day 7, FXII KO have 10.63 × 105/mL PEC vs. WT 25.38 × 105/mL (p< 0.0001). Likewise on Day 7, uPAR KO have 16.34 × 105/mL peritoneal leukocytes recruited vs. WT 25.38 × 105/mL (p=0.0124). These combined studies indicate that FXII influences the degree of leukocyte inflammation and the inflammatory response is partially mediated through uPAR. Studies next determined if leukocyte recruitment promotes wound healing. On Days 3–5 the rate of wound closure of back punch biopsies is faster in FXII KO than WT (p < 0.0327 by one way ANOVA). Again, we observed that FXII KO on Days 2 and 5 exhibit significantly less CD11b-labeled inflammatory cells to the injury site compared to WT (Day 2: p=0.0004, Day 5: p= 0.0075). Leukocyte subpopulation analysis reveals decreased neutrophil migration (Gr-1 positive cells) into the wound area of FXII KO on Day 2 [FXII KO 5669 ± 1844 vs. WT 40490 ± 8564 total surface area of Gr-1 positive cells (pixels)/ HPF, p=0.0018] and Day 5 [FXII KO 6216 ± 3829 vs. WT 34890 ± 7629 total surface area of Gr-1 positive cells (pixels)/HPF, p=0.0176]. Macrophage recruitment into the wound area, as determined by F4-80 antigenicity, increases over time, but remains significantly reduced in FXII KO mice on Day 2 [FXII KO 3111 ± 1115 vs. WT 19140 ± 5767 total surface area of F4-80 positive cells (pixels)/HPF, p=0.0348] and Day 5 [FXII KO 9377 ± 4772 vs. WT 28340 ± 4411 total surface area of F4-80 positive cells (pixels)/HPF, p=0.0171]. These studies indicate that, in FXII deficiency, there is less neutrophil and macrophage-mediated inflammation and this observation correlates with faster wound healing. Finally studies determined if the reduced inflammation seen in FXII KO is the result of host factors or bone marrow-derived cells. Adoptive bone marrow transplant (BMT) experiments were performed where WT or FXII KO bone marrow was transplanted into FXII KO hosts. Eight weeks following the BMT, mice were subjected to TG-induced peritonitis. KO/KO recipients, have significantly decreased number of PEC on day 1 (17.3 × 105/mL ± 3.894 × 105/mL cells) and day 7 (18.6 × 107/mL ± 4.0 × 107/mL cells) when compared to WT/KO recipients [Day 1: 68.25 × 105/mL ± 13.83 × 105/mL cells (p=0.041), Day 7: 190.5 × 107/mL ± 51.80 × 107/mL cells]. These data indicate that FXII interacts with neutrophils and macrophages to promote the inflammatory response; its absence causes decreased inflammatory cell recruitment. Conclusions: Our data indicate that FXII deficiency disrupts the leukocyte response to injury by reducing inflammatory cell recruitment in two murine models. Paradoxically, reduced leukocyte infiltration into skin wounds promotes healing. These investigations indicate a novel role for FXII in inflammation and wound healing and indicate a unique potential target for inflammation therapeutics. Disclosures: No relevant conflicts of interest to declare.

Abstract 4994 The concept of the leukemic stem cell (LSC) has gained wide acceptance since it was first definitively established using a NOD-SCID xenotransplantation model nearly 15 years ago. LSCs, which are functionally defined as SCID leukemia-initiating cells (SL-ICs), are believed to possess biological properties that render them resistant to conventional chemotherapy. Although there is still much debate over how to phenotypically define LSCs, there is general agreement that LSCs are rare in acute myeloid leukemia (AML), which has hindered efforts to develop LSC-targeted therapies. In order to provide researchers and pharmaceutical companies with an ample supply of LSCs for testing, methods are needed to generate large numbers of LSCs from patient samples. Ex vivo expansion of LSCs in culture is one approach that offers tremendous promise for increasing cell numbers for research and drug development. Conditions that enable efficient expansion of normal hematopoietic stem cells (HSCs) can be used as a starting point for developing an optimal culture system for LSCs. The natural bone marrow microenvironment maintains HSCs in close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D NANEX nanofiber based cell culture substrate. The NANEX substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSC proliferation. Here, we present our recent work with the NANEX platform towards achieving a high yield ex vivo expansion of LSCs. Using common LSC markers, including CD34, CD38, CD117, and CD123, we quantify and characterize NANEX-expanded leukemic cells using flow cytometry. We compare NANEX to standard tissue culture polystyrene and demonstrate that NANEXÔ significantly improves LSC expansion and reduces clonogenic phenotype loss during ex vivo culture. Additionally, we show that NANEXÔ-expanded cells engraft in NOD-SCID mice and, through limiting dilution analysis, quantify the increase in SL-ICs as a result of culture on NANEXÔ. Our data indicates that NANEX technology provides a robust ex vivo expansion of SL-ICs and, with further development, offers great potential for use in LSC-targeted drug development. Disclosures: No relevant conflicts of interest to declare.

Mutations in SHP-2 phosphatase that cause its hyper-activation have been identified in human leukemias, in particular, juvenile myelomonocytic leukemia (JMML) that is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce leukemia are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in catalytic-dependent and -independent manners and that overexpression (5-to-6 fold) of wild-type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through dephosphorylation of STAT5. This raised the possibility that SHP-2-associated JMML was not solely attributed to the increased catalytic activities of SHP-2 GOF mutants. The SHP-2 mutants must have gained other additional functions. To test this possibility, we investigated effects of a GOF mutation (E76K, the most frequent SHP-2 mutation seen in JMML) of SHP-2 on IL-3 signal transduction in great detail by comparing signaling activities of SHP-2 E76K to WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. E76K mutation in the N-terminal Src homology 2 domain significantly increased binding of the mutant SHP-2 to Grb2 and Gab2, two adaptor proteins coupling downstream Erk and PI3 kinase pathways to the proximity of the IL-3 receptor. As a result, IL-3-induced Erk and PI3 kinase pathways were highly activated by SHP-2 E76K mutation. In addition, Jak2 kinase activation was also markedly enhanced and due to the E76K mutation the substrate specificity of SHP-2 toward STAT5 was changed. Dephosphorylation of STAT5 by SHP-2 E76K was alleviated. These studies suggest that in addition to elevated catalytic activity, profound changes in protein-protein interaction capacities of GOF mutant SHP-2 play an important role in the pathogenesis of SHP-2-related leukemias.