Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Claudins are integral tight junction (TJ) proteins that contribute to cell polarity and regulate paracellular permeability to ions and solutes. Claudin 18 (CLDN18) is one of the most highly expressed claudin family members in lung alveolar epithelium. To investigate the role of CLDN18 in alveolar homeostasis, we recently generated Cldn18−/− mice that demonstrate increased lung solute permeability and alveolar fluid clearance (AFC) compared to wild type (WT) controls. Lungs of Cldn18−/− mice are markedly enlarged due to increased abundance and proliferation of alveolar epithelial type II (AT2) cells, known progenitors of distal lung epithelium, with resultant parenchymal expansion. Cldn18−/− AT2 cells grown with MLg fibroblasts in 3‐dimensional (3D) culture show increased colony forming efficiency (CFE), suggesting increased progenitor capacity. Given its known role in regulating stem/progenitor cell proliferation and organ size, we investigated a potential role for Yes‐associated protein (YAP) signaling in mediating the proliferative phenotype of Cldn18−/− lung progenitors. Progenitor cell activation was accompanied by activation of YAP, as evidenced by increased nuclear YAP, increased expression of YAP target genes in lungs of Cldn18−/− mice, and increased YAP and decreased phospho‐YAP (p‐YAP) by western analysis in isolated Cldn18−/− AT2 cells and by immunofluorescence in Cldn18−/− AT2 cells in 3D culture. Treatment with the YAP inhibitor verteporfin (VP, 100 mg/kg) reduced AT2 cell proliferation in vivo (EdU+NKX2.1+/total cells: 1.36 ± 0.10% vehicle vs 0.93 ± 0.06% VP, p<0.05) and decreased lung size (lung dry weight/body weight ratios (mg/g): 3.56 ± 0.08 vehicle vs 2.93 ± 0.13 VP, p<0.05) in Cldn18−/− mice. Inhibition of YAP with VP (0.75 μM) or shRNA decreased colony size (~50% vs control) and number (30–50% vs control) and reduced proliferation (Ki67+ cells at 10.7 ± 0.4% VP vs 29.2 ± 3.7% vehicle) of Cldn18−/− AT2 cells in 3D culture, while overexpression of CLDN18 decreased YAP nuclear localization, cell proliferation, CFE and YAP activity. These results reveal a novel role for YAP signaling in regulation of distal lung epithelial progenitor cell homeostasis and identify a role for TJ proteins, in particular CLDN18, in regulating YAP activity and organ size. Overall, they suggest a mechanism whereby growth‐promoting signals are transduced from TJ to the nucleus that has important implications for modulating stem/progenitor cell function and regeneration following injury.Support or Funding InformationNational Institutes of Health, Hastings and Whittier Foundations

Previous studies have demonstrated resistance to naphthalene-induced injury in proximal airways of mice with lung epithelial-specific deletion of the tumor-suppressor gene Pten, attributed to increased proliferation of airway progenitors. We tested effects of Pten loss following bleomycin injury, a model typically used to study distal lung epithelial injury, in conditional Pten SFTPC-cre knockout mice. Pten-deficient airway epithelium exhibited marked hyperplasia, particularly in small bronchioles and at bronchoalveolar duct junctions, with reduced E-cadherin and β-catenin expression between cells toward the luminal aspect of the hyperplastic epithelium. Bronchiolar epithelial and alveolar epithelial type II (AT2) cells in Pten SFTPC-cre mice showed decreased expression of epithelial markers and increased expression of mesenchymal markers, suggesting at least partial epithelial-mesenchymal transition at baseline. Surprisingly, and in contrast to previous studies, mutant mice were exquisitely sensitive to bleomycin, manifesting rapid weight loss, respiratory distress, increased early mortality (by day 5), and reduced dynamic lung compliance. This was accompanied by sloughing of the hyperplastic airway epithelium with occlusion of small bronchioles by cellular debris, without evidence of increased parenchymal lung injury. Increased airway epithelial cell apoptosis due to loss of antioxidant defenses, reflected by decreased expression of superoxide dismutase 3, in combination with deficient intercellular adhesion, likely predisposed to airway sloughing in knockout mice. These findings demonstrate an important role for Pten in maintenance of airway epithelial phenotype integrity and indicate that responses to Pten deletion in respiratory epithelium following acute lung injury are highly context-dependent and region-specific.

Granulocyte macrophage-colony-stimulating factor (GM-CSF) signaling regulates hematopoiesis and immune responses. CSF2RA , the gene encoding the α-subunit for GM-CSF, is significantly downregulated in t(8;21) (RUNX1-ETO or RE) leukemia patients, suggesting that it may serve as a tumor suppressor. We previously reported that GM-CSF signaling is inhibitory to RE leukemogenesis. Here we conducted gene expression profiling of primary RE hematopoietic stem/progenitor cells (HSPCs) treated with GM-CSF to elucidate the mechanisms mediating the negative effects of GM on RE leukemogenicity. We observed that GM treatment of RE HSPCs resulted in a unique gene expression profile that resembles primary human cells undergoing myelopoiesis, which was not observed in control HSPCs. Additionally, we discovered that GM-CSF signaling attenuates MYC-associated gene signatures in RE HSPCs. In agreement with this, a functional screen of a subset of GM-CSF-responsive genes demonstrated that a MYC inhibitor, MXI1 (Max interactor 1), reduced the leukemic potential of RE HSPCs and t(8;21) acute myeloid leukemia (AML) cells. Furthermore, MYC knockdown and treatment with the BET (bromodomain and extra terminal domain) inhibitor JQ1 reduced the leukemic potential of t(8;21) cell lines. Altogether, we discovered a novel molecular mechanism mediating the GM-CSF-induced reduction in leukemic potential of RE cells, and our findings support MYC inhibition as an effective strategy for reducing the leukemogenicity of t(8;21) AML.

Epidermal growth factor (EGF) receptor (EGFR) has been implicated in tumor development and invasion. Dimerization and autophosphorylation of EGFR are the critical events for EGFR activation. However, the regulation of EGF-dependent and EGF-independent dimerization and phosphorylation of EGFR has not been fully understood. Here, we report that cytoplasmic protein plakophilin-2 (PKP2) is a novel positive regulator of EGFR signaling. PKP2 specifically interacts with EGFR via its N-terminal head domain. Increased PKP2 expression enhances EGF-dependent and EGF-independent EGFR dimerization and phosphorylation. Moreover, PKP2 knockdown reduces EGFR phosphorylation and attenuates EGFR-mediated signal activation, resulting in a significant decrease in proliferation and migration of cancer cells and tumor development. Our results indicate that PKP2 is a novel activator of the EGFR signaling pathway and a potential new drug target for inhibiting tumor growth.

Mounting evidences indicate that leukemic cells in patients with acute myeloid leukemia (AML) are derived from leukemia stem cells (LSC). In analogy to normal hematopoietic stem cells (HSC), LSC remain mostly dormant and are hence resistant to conventional chemotherapy. Residual, physiological HSC exist alongside with LSC, with heterogeneous dominance of LSC over HSC in individual patients. We have devised a flow cytometric method for the identification and separation of these two stem cell populations based on surface antigen markers such as CD34, CD38, lineage aberrant markers, and aldehyde dehydrogenase (ALDH) enzyme activity.

RUNX1-ETO (also known as AML1-ETO and AML1-MTG8 ) is a fusion gene generated from t(8;21), which is a common chromosome translocation in acute myeloid leukemia (AML). It has been shown that t(8;21) requires additional aberrations to induce leukemia. Interestingly, 32-59% of t(8;21) patients also display loss of a sex chromosome (LOS) in their leukemia cells. Therefore, loss of the genes located on the sex chromosomes, especially in the pseudoautosomal regions (PARs) that are shared between the X and Y chromosomes, may contribute to RUNX1-ETO leukemia development. One gene of interest in the PARs is CSF2RA , which encodes the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. When the GM-CSF receptor is bound to its ligand, downstream signaling events promote various functional outcomes including proliferation, differentiation, self-renewal, and survival of myeloid cells. Thus, GM-CSF signaling has the potential to regulate both normal and malignant hematopoiesis. We previously reported that mice expressing RUNX1-ETO in GM-CSF deficient hematopoietic cells displayed higher incidence of leukemia (Matsuura S et al. 2012 Blood 119:3155). This result suggests that GM-CSF signaling is inhibitory to RUNX1-ETO dependent leukemogenesis. Furthermore, GM-CSF treatment reduces the self-renewal potential of RUNX1-ETO expressing cells and promotes myeloid differentiation in replating assays. We therefore hypothesize that the negative effect of GM-CSF on RUNX1-ETO induced leukemia development is due to the activation of selected GM-CSF downstream signaling pathway(s) that diminish self-renewal capacity and promote myeloid differentiation. To understand the molecular mechanism of the negative effect of GM-CSF on t(8;21) leukemogenesis, in the current report, we conducted a gene expression profiling assay to examine the effect of GM-CSF on RUNX1-ETO cells. MigR1 vector control or MigR1-RUNX1-ETO retrovirus transduced lineage negative/c-Kit positive (Lin - /c-Kit + ) murine hematopoietic stem/progenitor cells (HSPCs) were cultured with or without GM-CSF for 24 hours. Then, Lin - /c-Kit + /GFP + HSPCs were isolated for the profiling study. We observed little response to GM-CSF in control HSPCs, with only 4 genes being differentially expressed after a 2-fold cutoff. Conversely, 122 genes were differentially expressed in RUNX1-ETO cells treated with GM-CSF. These results clearly indicate that RUNX1-ETO specifically enhances GM-CSF responsiveness in HSPCs. Gene Set Enrichment Analysis (GSEA) of the differentially expressed genes in RUNX1-ETO cells reveals that this response resembles that of GM-CSF-induced myeloid differentiation. Furthermore, pathway analysis of these differentially expressed genes predicts MEK1/2 and ERK1/2 to be activated after GM-CSF treatment in RE cells. We previously reported that ERK1/2, downstream targets of MEK1/2, are hyper-phosphorylated after GM-CSF treatment of RUNX1-ETO cells, and MEK-ERK activation has been shown to regulate cell proliferation and myelopoiesis. Other GM-CSF induced genes are predicted targets of MYD88. MYD88 is upregulated during myeloid differentiation. Its in vivo knockout has been reported to result in an increase of hematopoietic stem cells (HSCs) and reduction of mature granulocytes. Most interestingly, a subset of genes upregulated in GM-CSF treated RUNX1-ETO cells are predicted to be activated by CEBPβ. CEBPβ can heterodimerize with CEBPα and is induced during myelopoiesis, critical for macrophage differentiation, capable of promoting granulopoiesis, and involved in regulating granulopoiesis in vivo . In conclusion, our data suggest that RUNX1-ETO expression results in hyper-responsiveness to GM-CSF. Such enhanced GM-CSF signaling activates the expression of a specific group of genes and results in the reduced self-renewal capacity and increased myeloid differentiation of HSPCs. These GM-CSF effects are likely involved in reducing the leukemogenic potential of RUNX1-ETO and may be considered for specific therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with β-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.

Leukemia stem cell candidates (LSCC) can be enriched from patients with acute myeloid leukemia by high aldehyde dehydrogenase (ALDH) activity and CD34 expression. We have previously demonstrated the leukemia-initiating activity of ALDH(bright) cells in xenograft transplantation models, as well as in vitro. Applying single-cell long-term culture-initiating cell assays, we have correlated the functional properties of individual cells within this LSCC population and the respective phenotypes. To define their biologic significance, we also analyzed the relationship between LSCC at diagnosis to long-term clinical outcomes. The median percentage of ALDH(bright) cells among 101 acute myeloid leukemia patients was 0.51% (range, 0.01-12.90%). Single-cell long-term culture-initiating cell assays, followed by genetic analysis of the progeny cells, showed that the leukemia-initiating activity was found in the ALDH(bright)/CD34(high) subset and, to a lesser extent, in ALDH(bright)/CD34(low) or ALDH(bright)/CD34(-) subsets. Nevertheless, the frequency of ALDH(bright) cells at diagnosis correlated significantly with the persistence of leukemia after induction chemotherapy (n = 84, Spearman R = 0.3261; p < 0.0025). In the multivariate model, frequency of ALDH(bright) cells was the strongest prognostic marker (p = 0.0095) affecting overall survival (hazard ratio = 9.107). LSCC are heterogeneous and best reflected by ALDH activity. The frequency of ALDH(bright) cells at diagnosis is a significant prognostic marker for acute myeloid leukemia. (C) 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Objective. Leukemia-initiating cells can retrospectively be defined by tumorigenicity in immunodeficient mice and be characterized by surface markers. The latter still being discussed for acute myeloid leukemia (AML), nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were used to evaluate long-time reconstitution and expansion of AML subpopulations.Materials and Methods. Bone marrow cells from patients with AML were separated according to CD34 expression, aldehyde dehydrogenase (ALDH) activity, and divisional kinetics in comparison to cord blood - derived CD34(+) hematopoietic stem cells, evaluating survival and expansion in NOD/SCID mice. The AML long-term surviving capacity of subpopulations recovered from NOD/SCID mice was confirmed by ex vivo survival.Results. AML mononuclear cells were detected in bone marrow and spleen of NOD/SCID mice 12 weeks after transplantation. The majority of recovered cells were CD34(+) and significantly more CD34(+) cells were recovered after application of ALDH(bright) (high ALDH activity), CD34(+), or slowly dividing (PKHbright) than after ALDH(dim), CD34(-), or fast dividing (PKHdim) cell application. CD123(+), CD63(+), and CD44v7(+) cells were also more abundant after the transfer of ALDH(bright) or CD34(+) AML mononuclear cells. In the spleen, large AML cell clusters were only recovered after ALDH(bright), CD34(+), or PKHbright cell transfer. Importantly, in secondary long-term in vitro cultures, quite exclusively CD34(+) AML mononuclear cells survived and expanded.Conclusions. Separation of ALDH(bright), CD34(+), or PKHbright cells enriches for AML long-term surviving capacity, which reside in the CD34(+) subpopulation, as rather exclusively CD34(+) cells survived and expanded in vivo and ex vivo. Long-term survival capacity may be supported by CD44v7 expression. (C) 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Objective:To analyze the clinical features of patients with hemophagocytic syndrome (HPS)and enhance understanding of the disease further. Methods: Clinical data of 13 patients diagnosed with HPS were analyzed retrospectively. Results: Thirteen patients showed persistent or intermittent high fever,splenomegaly,two cases with massive splenomegaly,nine cases had hepatomegaly at the same time. Four cases had the symptom of gastrointestinal system; 13 cases showed less for the blood cells,as three or two less; 7 cases with hypertrigliceridemia,4 cases with hypofibrinogenemia. Ferritin increased in 8 cases,and 4 above 2000 μg/L,4 cases developed to multiple organ failure; four cases were examined for the peripheral blood flow type,their NK cells were significantly lower than normal; increased histiocytosis in bone marrow were found in all patients,ratio from 2.0% to 18.0%,phagocytosis of platelets and red blood cells in the histiocytes were common; 13 cases were examined EBV-related antibodies,12 cases were IgG,1 case was IgM and IgG antibodies positive; 6 cases were treated with the chemotherapy,of which 5 cases used the VP regimen,one case used CHOP with 6 courses,then got remission; 2 patients had no chemotherapy,and improved too. Conclusions: HPS often has the dynamic development process,easily misdiagnosed in early stages of disease. The examinations of bone marrow cytology,flow immunophenotyping and sL-2R level should be carried out actively,and try to search for the causes and incentives. Some diagnostic criteria may become apparent during the development of the disease. Early treatment can help improve patiats' prognosis.

As more efficient agents for stem cell mobilization are being developed, there is an urgent need to define which patient population might benefit from these novel drugs. For a precise and prospective definition of "poor mobilization" (PM), we have analyzed the efficiency of mobilization in patients intended to receive autologous transplantation at our center in the past 6 years. Between January 2003, and December 2008, 840 patients with the following diagnoses were scheduled to undergo leukapheresis: multiple myeloma (MM, n = 602) and non-Hodgkin lymphoma (NHL, n= 238). Most patients mobilized readily: close to 85% of the patients had a level of 20/microL to >500/microL of CD34(+) cells at the peak of stimulation. Of the 840 patients, 129 (15.3%) were considered to be PMs, defined as patients who had a peak concentration of <20/microL of CD34(+) cells upon stimulation with granulocyte-colony stimulating factor (G-CSF) subsequent to induction chemotherapy appropriate for the respective disease. Among them, 38 (4.5%) patients had CD34(+) levels between 11 and 19/microL at maximum stimulation, defined as "borderline" PM, 49 (5.8%) patients had CD34(+) levels between 6 and 10/microL, defined as "relative" PM, and 42 patients (5%) with levels of <5/microL, defined as "absolute" PM. There was no difference in the incidence of PM between patients with MM versus those with NHL. Sex, age, body weight (b.w.) and previous irradiation therapy did not make any significant difference. Only the total number of cycles of previous chemotherapy (P = .0034), and previous treatment with melphalan (Mel; P = .0078) had a significant impact on the ability to mobilize. For the good mobilizers, the median time to recovery of the white blood cells (WBCs) to 1.0/nL or more was 13 days with a range of 7 to 22 days, whereas for the PM group it was 14 days with a range of 8 to 37 days. This difference was statistically not significant. The median time to recovery of the platelets counts to an unmaintained level of >20/nL was 11 days with a range of 6 to 17 days for the good mobilizers, whereas for the PM it was 11 days with a range of 7 to 32 days. Again, this difference was not significant. The majority of the patients today intended for autologous transplantations were able to mobilize readily. As long as > or =2.0 x 10(6) of CD34(+) cells/kg b.w. have been collected, PM was not associated with inferior engraftment.

Abstract Abstract 2160 We have shown that leukemia stem cells candidates (LSCC) can be prospectively identified by high activity of aldehyde dehydrogenase (ALDHbr) and expression of CD34 among the leukemia blasts from the marrow of patients with AML. In this study we have examined the relationship between the frequency of LSCC at diagnosis with persistence of leukemia blasts after induction chemotherapy as well as with long-term clinical outcome. Using single cell sorting, we have further separated subsets among the LSCC and correlated their individual functional properties with the respective marker constellation. The percentage of LSCC in 101 patients ranged from 0.01% to 12.90% with a median of 0.51%. Frequencies of LSCC among the leukemia blasts at diagnosis correlated significantly with the persistence of leukemia after the first induction chemotherapy (n=79, Spearman R=0.7797, P<0.0001). During the observation period of 24 months, 21 of 60 patients with high levels of LSCC died as compared to 7 of 41 patients with low levels of LSCC (p=0.029). The overall survival (OS) probability for the patients with high levels of LSCC was significantly worse (p=0.05) than in those with low LSCC. Characterization of these LSCC at a single cell level showed that a varying proportion, i.e. 15% to 78% of their progeny cells demonstrated the same chromosomal aberrations as the original leukemia population, indicating the presence of residual normal HSC. Thus high frequencies of LSCC at the time of diagnosis predict persistence of leukemia blasts, failure to achieve CR within the first cycle and poor overall clinical outcome. Our prospective separation of LSCC permits precise characterization of the biological properties of the subsets of stem cell candidates in human AML. Disclosures: Ho: Genzyme: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees.