SOX7-enforced expression promotes the expansion of adult blood progenitors and blocks B-cell development

During embryogenesis, the three SOXF transcription factors, SOX7, SOX17 and SOX18, regulate the specification of the cardiovascular system and are also involved in the development of haematopoiesis. The ectopic expression of SOX17 in both embryonic and adult blood cells enhances self-renewal. Likewise, the enforced expression of SOX7 during embryonic development promotes the proliferation of early blood progenitors and blocks lineage commitment. However, whether SOX7 expression can also affect the self-renewal of adult blood progenitors has never been explored. In this study, we demonstrate using an inducible transgenic mouse model that the enforced expression of Sox7 ex vivo in bone marrow/stroma cell co-culture promotes the proliferation of blood progenitors which retain multi-lineage short-term engrafting capacity. Furthermore, SOX7 expression induces a profound block in the generation of B lymphocytes. Correspondingly, the ectopic expression of SOX7 in vivo results in dramatic alterations of the haematopoietic system, inducing the proliferation of blood progenitors in the bone marrow while blocking B lymphopoiesis. In addition, SOX7 expression induces extra-medullary haematopoiesis in the spleen and liver. Together, these data demonstrate that the uncontrolled expression of the transcription factor SOX7 in adult haematopoietic cells has dramatic consequences on blood homeostasis.

Wnt Signaling Is Associated with Anti-Apoptosis in the Interaction Between Acute Myeloid Leukemia Cells and Stromal Cells

Abstract 1366 Recent studies have revealed that strength of the Wnt signaling pathway regulates normal hematopoiesis including hematopoietic stem cells, and aberrant activation of the pathway is involved in the development of several types of leukemias. In the bone marrow microenvironment, stromal cells are rich sources of cytokines and some secreted cytokines have been observed to block induction of cell death in myeloid leukemia cells exposed to chemotherapy. Here, we examined the role of the Wnt signaling pathway on cell-adhesion, proliferation and survival of the stroma-dependent human AML cell line, TRL-01 cells, which we previously established (Ninomiya, et al. Cancer Gen Cyto, 2006). TRL-01 cells were maintained in the co-culturing with the hTERT-transduced human bone marrow stroma cell line, HTS cells, and cell death of TRL-01 cells was induced after removal of HTS cells. Treatment with the Wnt-receptor competitor, secreted Frizzled related protein (sFRP)-1, or the Rho kinase inhibitor, Y29632 (previously reported as an inhibitor of the non-canonical Wnt downstream pathway), but not with the specific inhibitor of the canonical Wnt pathway (DKK-1) induced apoptosis in dose-dependent manners in TRL-01 cells co-cultured with HTS cells. These results suggested that the non-canonical pathway of Wnt signaling might regulate survival of TRL-01 on the stromal cells. Next, we comprehensively investigated transcripts of the Wnt pathway components (10 Frizzleds, 2 LRPs, and 18 Wnts) in TRL-01 cells and HTS cells using RT-PCR. Transcripts of Wnt5A and Wnt9A were expressed in TRL-01 cells, but not in HTS cells. Moreover, the cell death of TRL-01 cells after removal of HTS cells was partially prevented by additional treatment with Wnt5A or Wnt9A not by other Wnt molecules such as Wnt5B. On the other hand, treatment with Wnt3A induced activated nuclear beta-catenin using Western-blotting, however, did not contribute to the survival of TRL-01 cells without the stromal co-culturing. Moreover, we are investigating regulation of the anti-apoptotic downstream pathway molecules in TRL-01 cells as well as effects of other inhibitors targeted for the Wnt signaling. We are also examining expressions of Wnt pathway components in primary AML bone marrow cells and other human bone marrow stroma cell lines such as HS-5 cells. These results imply association of Wnt5A and Wnt9A with anti-apoptosis in the interaction between AML cells and stromal cells, and a possible therapeutic target of AML for overcoming the resistance to chemotherapy in the bone marrow microenvironment. Disclosures: Naoe: Chugai Pharma: Research Funding; Novartis Pharma: Research Funding; Kyowa-Hakko Kirin: Research Funding.

The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell–induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)

Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.