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Abnormalities in nestin-expressing bone marrow stromal cells in patients with myelodysplastic syndrome
S86
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3140 - DELINEATION OF NICHES WHICH SUPPORT HEMATOPOIESIS IN SPLEEN Hong K. Lim1, Pravin Periasamy1, Sawang Petvises1, Terence O’Neill2, and Helen O’Neill1,2 1 Australian National University, Canberra, Australian Capital Territory, Australia; 2 Bond University, Gold Coast, Queensland, Australia Hematopoietic stem cell (HSC) niches in bone marrow have been described in terms of several distinct cell types including osteoblastic, endothelial and perivascular reticular cells. However, niches which support hematopoiesis in other sites like spleen remain to be elucidated. Previous studies in this lab have described splenic stromal cells which support hematopoiesis in vitro from purified HSC and multipotential progenitors (MPP) leading to the production of specific myeloid cell types. Stromal cells were originally derived from longterm cultures of splenocytes, and later as freshly isolated stromal cell subsets prepared by enzymatic digestion and sorting. Competent and non-competent stromal cell lines have been derived. Transcriptome analysis has revealed that the 5G3 and 3B5 stroma express many genes in parallel with perivascular reticular cells described in bone marrow, although only 5G3 supports in vitro hematopoiesis. Both 5G3 and 3B5 share a common mesenchymal lineage with perivascular reticular cells in bone marrow, as well as mesenchymal stem/progenitor cells and CXCL12-abundant reticular cells described in humans. 5G3 and 3B5 express many cell surface markers common to these cell types including CD105, CD29, VCAM1, CD51, CD140a and CD90. A number of new markers and genes have been identified which either delineate this cell type or determine ability to support hematopoiesis. Initial studies to determine the molecular signals which support hematopoiesis have involved use of inhibitors to block stromal cell function and interaction with HSC, as well as gene knockdown in stroma to identify regulators of hematopoiesis in vitro.
3141 - THE DYNAMIC INTERPLAY OF GATA ZINC-FINGERS IN BLOOD DEVELOPMENT AND DISEASE Salima Nurmohamed1,2 1 University of Oxford, Oxford, United Kingdom; 2University College London, London, United Kingdom Acute myeloid leukaemia (AML) is the most common type of blood cancer in adults. AML is highly malignant and most patients eventually die from the disease in spite of aggressive therapy. GATA2 is a protein essential to blood-cell development; it is a master-regulator of stem and progenitor cell development. GATA2 has two main functions – it binds DNA to switch genes on and off, and simultaneously makes physical contacts with other proteins. Interruption of these interactions alters the behaviour of blood cells and ultimately causes leukaemia. How the GATA2 mutations identified in AML patients alter its function is unknown. The structure-function approach presented here sheds light on the dynamic interplay between the zinc-fingers and their protein/DNA binding-partners to elucidates the molecular mechanism of how GATA2 may function in haematopoiesis and disease.
3142 - CHLOROQUINE INHIBITS AUTOPHAGY AND INDUCES THE APOPTOSIS OF PRIMARY EFFUSION LYMPHOMA VIA ER STRESS Md. Masud Alam, Ryusho Kariya, Kouki Matsuda, and Seiji Okada Center for AIDS Research, Kumamoto University, Kumamoto, Kumamoto, Japan Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin B cell lymphomas that occurred predominantly in patients with advanced AIDS. Chloroquine is an anti-malarial drug but unclear in the treatment of hematological malignancies. Therefore, we examined the effect of chloroquine on PEL cells in vitro and in vivo. AnnexinV assay revealed that PEL cells were efficaciously killed by chloroquine in vitro. Chloroquine was found to inhibit autophagolysosome formation by increasing the accumulation of LC3-II protein on autophagosome. Chloroquine stimulated the agglomeration of unfolded protein within aggresomes that induced endoplasmic reticulum (ER) stress mediated caspase dependent apoptosis in PEL. Chloroquine induced CHOP, an ER stress marker and induction of cleaved caspase 3, an apoptotic marker in PEL. Additionally, caspase 3 and ER stress inhibitor rescued PEL cells from chloroquine induced apoptosis. In a PEL xenograft mouse model, GTO cells were intraperitoneally inoculated into Nude-Rag2/Jak3 double deficient mice, and treated with chloroquine or PBS once a day for 21 days. Chloroquine reduced the amount of ascites without significant systemic toxicity in mice model. These results suggest that chloroquine has given a promising reagent for the treatment and prevention of PEL in the HIV/AIDS patients.
3143 - ABNORMALITIES IN NESTIN-EXPRESSING BONE MARROW STROMAL CELLS IN PATIENTS WITH MYELODYSPLASTIC SYNDROME Naoshi Obara, Shigeru Chiba, Takayasu Kato, Tatsuhiro Sakamoto, Luan Cao, Sachie Suzuki, Naoki Kurita, Yasuhisa Yokoyama, Mamiko Sakata, and Yuichi Hasegawa University of Tsukuba, Tsukuba, Japan Background: Bone marrow stromal cells expressing nestin are proposed to play a fundamental role in hematopoiesis. Nestin-positive cells as well as Schwann cells were reported to be decreased in the bone marrow of patients with myeloproliferative neoplasms (MPN) and in JAK2-V617F transgenic mice, and damage to these cells was found to lead to MPN in mice. However, a correlation between blood cancers other than MPN and nestin-expressing stromal cells has not yet been established. Moreover, the molecular mechanism that operates in these cells to support hematopoiesis is still unclear. We investigated the integrity of nestin-expressing cells in the bone marrow of patients with myelodysplastic syndromes (MDS), and established mice conditionally lacking the RBPjk gene that encodes an essential nuclear protein for Notch signaling in the nestin-expressing cells. Methods: Bone marrow biopsy specimens from patients who were diagnosed with MDS between 2010 and 2014 were immunostained for nestin, together with other stromal cell markers. The structural and numerical characteristics of nestin-expressing cells were evaluated microscopically. Then, the bone marrow and hematopoietic phenotypes of mice with an Rbpjk f/f genotype and a nestin-CreERT2 transgene were analyzed. Results: In the bone marrow of MDS patients, the majority of these were dissociated from the vascular structures, whereas the number of nestin-positive cells was increased. The mice lacking Rbpjk gene in nestin-expressing cells showed marked splenomegaly and mild anemia. We observed that erythroblast maturation was impaired in the bone marrow of these mice. Discussion: Our observations suggest that abnormalities of nestin-expressing stromal cells are associated with impaired hematopoiesis in MDS. Functional abnormalities in these cells may be attributed to cellular signaling disturbances, such as that of Notch signaling. Further investigation is required to clarify whether the impaired Notch signaling in the nestin-expressing bone marrow stromal cells, at least in part, accounts for the blocked normal differentiation in the bone marrow of MDS patients.
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3140 - DELINEATION OF NICHES WHICH SUPPORT HEMATOPOIESIS IN SPLEEN Hong K. Lim1, Pravin Periasamy1, Sawang Petvises1, Terence O’Neill2, and Helen O’Neill1,2 1 Australian National University, Canberra, Australian Capital Territory, Australia; 2 Bond University, Gold Coast, Queensland, Australia Hematopoietic stem cell (HSC) niches in bone marrow have been described in terms of several distinct cell types including osteoblastic, endothelial and perivascular reticular cells. However, niches which support hematopoiesis in other sites like spleen remain to be elucidated. Previous studies in this lab have described splenic stromal cells which support hematopoiesis in vitro from purified HSC and multipotential progenitors (MPP) leading to the production of specific myeloid cell types. Stromal cells were originally derived from longterm cultures of splenocytes, and later as freshly isolated stromal cell subsets prepared by enzymatic digestion and sorting. Competent and non-competent stromal cell lines have been derived. Transcriptome analysis has revealed that the 5G3 and 3B5 stroma express many genes in parallel with perivascular reticular cells described in bone marrow, although only 5G3 supports in vitro hematopoiesis. Both 5G3 and 3B5 share a common mesenchymal lineage with perivascular reticular cells in bone marrow, as well as mesenchymal stem/progenitor cells and CXCL12-abundant reticular cells described in humans. 5G3 and 3B5 express many cell surface markers common to these cell types including CD105, CD29, VCAM1, CD51, CD140a and CD90. A number of new markers and genes have been identified which either delineate this cell type or determine ability to support hematopoiesis. Initial studies to determine the molecular signals which support hematopoiesis have involved use of inhibitors to block stromal cell function and interaction with HSC, as well as gene knockdown in stroma to identify regulators of hematopoiesis in vitro.
3141 - THE DYNAMIC INTERPLAY OF GATA ZINC-FINGERS IN BLOOD DEVELOPMENT AND DISEASE Salima Nurmohamed1,2 1 University of Oxford, Oxford, United Kingdom; 2University College London, London, United Kingdom Acute myeloid leukaemia (AML) is the most common type of blood cancer in adults. AML is highly malignant and most patients eventually die from the disease in spite of aggressive therapy. GATA2 is a protein essential to blood-cell development; it is a master-regulator of stem and progenitor cell development. GATA2 has two main functions – it binds DNA to switch genes on and off, and simultaneously makes physical contacts with other proteins. Interruption of these interactions alters the behaviour of blood cells and ultimately causes leukaemia. How the GATA2 mutations identified in AML patients alter its function is unknown. The structure-function approach presented here sheds light on the dynamic interplay between the zinc-fingers and their protein/DNA binding-partners to elucidates the molecular mechanism of how GATA2 may function in haematopoiesis and disease.
3142 - CHLOROQUINE INHIBITS AUTOPHAGY AND INDUCES THE APOPTOSIS OF PRIMARY EFFUSION LYMPHOMA VIA ER STRESS Md. Masud Alam, Ryusho Kariya, Kouki Matsuda, and Seiji Okada Center for AIDS Research, Kumamoto University, Kumamoto, Kumamoto, Japan Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin B cell lymphomas that occurred predominantly in patients with advanced AIDS. Chloroquine is an anti-malarial drug but unclear in the treatment of hematological malignancies. Therefore, we examined the effect of chloroquine on PEL cells in vitro and in vivo. AnnexinV assay revealed that PEL cells were efficaciously killed by chloroquine in vitro. Chloroquine was found to inhibit autophagolysosome formation by increasing the accumulation of LC3-II protein on autophagosome. Chloroquine stimulated the agglomeration of unfolded protein within aggresomes that induced endoplasmic reticulum (ER) stress mediated caspase dependent apoptosis in PEL. Chloroquine induced CHOP, an ER stress marker and induction of cleaved caspase 3, an apoptotic marker in PEL. Additionally, caspase 3 and ER stress inhibitor rescued PEL cells from chloroquine induced apoptosis. In a PEL xenograft mouse model, GTO cells were intraperitoneally inoculated into Nude-Rag2/Jak3 double deficient mice, and treated with chloroquine or PBS once a day for 21 days. Chloroquine reduced the amount of ascites without significant systemic toxicity in mice model. These results suggest that chloroquine has given a promising reagent for the treatment and prevention of PEL in the HIV/AIDS patients.
3143 - ABNORMALITIES IN NESTIN-EXPRESSING BONE MARROW STROMAL CELLS IN PATIENTS WITH MYELODYSPLASTIC SYNDROME Naoshi Obara, Shigeru Chiba, Takayasu Kato, Tatsuhiro Sakamoto, Luan Cao, Sachie Suzuki, Naoki Kurita, Yasuhisa Yokoyama, Mamiko Sakata, and Yuichi Hasegawa University of Tsukuba, Tsukuba, Japan Background: Bone marrow stromal cells expressing nestin are proposed to play a fundamental role in hematopoiesis. Nestin-positive cells as well as Schwann cells were reported to be decreased in the bone marrow of patients with myeloproliferative neoplasms (MPN) and in JAK2-V617F transgenic mice, and damage to these cells was found to lead to MPN in mice. However, a correlation between blood cancers other than MPN and nestin-expressing stromal cells has not yet been established. Moreover, the molecular mechanism that operates in these cells to support hematopoiesis is still unclear. We investigated the integrity of nestin-expressing cells in the bone marrow of patients with myelodysplastic syndromes (MDS), and established mice conditionally lacking the RBPjk gene that encodes an essential nuclear protein for Notch signaling in the nestin-expressing cells. Methods: Bone marrow biopsy specimens from patients who were diagnosed with MDS between 2010 and 2014 were immunostained for nestin, together with other stromal cell markers. The structural and numerical characteristics of nestin-expressing cells were evaluated microscopically. Then, the bone marrow and hematopoietic phenotypes of mice with an Rbpjk f/f genotype and a nestin-CreERT2 transgene were analyzed. Results: In the bone marrow of MDS patients, the majority of these were dissociated from the vascular structures, whereas the number of nestin-positive cells was increased. The mice lacking Rbpjk gene in nestin-expressing cells showed marked splenomegaly and mild anemia. We observed that erythroblast maturation was impaired in the bone marrow of these mice. Discussion: Our observations suggest that abnormalities of nestin-expressing stromal cells are associated with impaired hematopoiesis in MDS. Functional abnormalities in these cells may be attributed to cellular signaling disturbances, such as that of Notch signaling. Further investigation is required to clarify whether the impaired Notch signaling in the nestin-expressing bone marrow stromal cells, at least in part, accounts for the blocked normal differentiation in the bone marrow of MDS patients.