Activation of the Canonical Notch Signaling Pathway in the Hematopoietic Microenvironment Is Required to Maintain Normal Hematopoiesis

Abstract 723

Recent studies demonstrated that the causes of Myeloproliferative syndromes (MPS) is not limited to factors intrinsic to hematopoietic cells, but include also non-cell autonomous mechanisms. Here, we demonstrate that loss of CSL-dependent Notch signaling in the hematopoietic microenvironment leads to a lethal myeloproliferative disorder. Following pIpC induction, Mx1-Cre+/RBP-J^lox/lox mice showed a significantly increase in granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, spleen and liver and died by week 16–20. Survival at 5 months was 12% for the RBPJ KO mice compared to 100% for the control mice. Transplant studies revealed that the myeloproliferative disorder occurred rapidly when WT-hematopoietic cells were transplanted into MX1-Cre+/RBP-J^lox/lox-recipients, whereas WT-recipients of MX1-Cre+/RBP-J^lox/lox hematopoietic cells did not show onset of diseases at 6 months of follow-up. Furthermore, the disease was not transplantable in WT-recipients, indicating that abnormal Notch signaling in the microenvironment is critical for the development and sustainment of MPS. Transplants conducted with Lys/GFP HSC as WT donor cells, showed that transplant of myeloid progenitors (LK) are sufficient for the development of disease, as documented by rapid expansion of LK-Lys/GFP cells into MX1-Cre+/RBP-J^lox/lox recipients by intravital microscopy of the BM niche. Levels of hematopoietic cytokines were not significantly different in MX1-Cre+/RBP-J^lox/lox recipients prior to transplantation. Histological analysis of BM and spleen showed increased microvascular density and increased CD31+ EC by IHC. To dissect the mechanisms, we analyzed different cellular components of the microenvironment. Mx1Cre driven deletion of RBP-J occurred in osteoblasts, endothelial progenitors and mesenchymal stem cells. Co-culture experiments of HSC with these different cell types suggested cooperative effects in altering proliferation and differentiation of myeloid cells. Crosses of RBP-J^lox/lox mice with mice bearing cell type promoter specific Cre are ongoing to determine the cellular type(s) critical for the myeloid expansion and to identify downstream molecular mediators. These studies will provide further insights on the role of the microenvironment in contributing to myeloid proliferative diseases and leukemia.