Oncogenic Insult In Bone Marrow Endothelial Cells Causes Hematopoietic Abnormalities

Bone marrow endothelial cells (BMECs), the cells forming bone marrow vasculature, have been implicated as a critical constituent of bone marrow microenvironment essential for the maintenance of normal and malignant hematopoietic stem cells. They are a part of BM niche regulating the self-renewal and differentiation of hematopoietic stem cells (HSCs) and the homing and progression of pre-leukemia and leukemia cells. It has been realized that pathological angiogenesis such as increased bone marrow microvessel density is associated with a spectrum of human diseases including MDS, MPD and leukemia. Recent discovery of oncogenic mutations of BCR-ABL and JAK2V617F in BMECs and spleen ECs primes the hypothesis that pathological angiogenesis from oncogenic insults may promote or facilitate abnormal hematopoiesis leading to the development and maintenance of blood malignancies. Clinical studies have linked germline mutations of Ras signaling components (e.g. NF1, SHP2, CBL and K-RAS) with JMML, a subgroup of MPD/MDS that is difficult to cure and for which bone marrow transplantation often results in relapses and graft failure. Studies in animal models have shown that activation of oncogenic K-Ras in hematopoietic stem cells could partially recapitulate the symptoms of these MPD/MDS patients, suggesting a HSC intrinsic role of oncogenic transformation. It remains to be seen if deregulated K-Ras signaling in the BM microenvironment, BMECs in particular, may also contribute to the hematologic abnormalities and disease progression, and whether pathologicial BMECs provide an addition therapeutic opportunity to limit transplant-associated relapse. To this end, we recently established a new mouse model that allows endothelial lineage-specific, inducible genetic manipulation of BMECs in adult animals in vivo and a cell culture system to select, expand and functional assay freshly isolated murine BMEC activities in vitro. A strain of Tie2-CreER transgenic mice crossed with loxP-stop-loxP fluorescence reporters (EGFP or Td-tomato) is sensitive to Tamoxifen induction in specifically turning on Cre activity in endothelial cells in 4-8 week old animals with over 80% efficiency in BMECs. Our culture conditions select and expand mouse primary BMECs in a large scale without immortalization or genetic manipulation, and the resulting BMECs retain endothelial lineage markers and are fully functional in 2-demensional and 3-demensional capillary formation assays as well as Matrigel implant plug assay. Using this inducible model, we generated Tie2CreER/LSL-K-Ras G12D mice, to specifically turn on K-Ras G12D in endothelial cells to test if an oncogenic K-Ras insult in EC compartment can affect hematopoiesis. Three consecutive Tamoxifen injections to the mice readily activated K-Ras G12D expression in the BMECs, but not in the blood lineages. The mice bearing endothelial K-Ras G12D died 4-6 months after Tamoxifen induction. An analysis of the K-Ras G12D mice revealed that the oncogenic insult caused a significant change in BM vascular structure and abnormal hematopoiesis. K-Ras G12D mutation in the ECs led to pathological angiogenesis in the bone marrow as manifested in the K-Ras G12D mice an increased colony-forming unit EPC activity, drastically increased vessel permeability, and increased BMEC proliferation. The pathological angiogenesis in the bone marrow was associated with abnormal blood phenotypes in K-Ras G12D mice, showing a significant increase of WBC and lymphocyte counts in peripheral blood, splenomegaly, severe anemia, and a significant increase of CFU-C activity in the blood and spleen. Ongoing transplant experiments will address whether the oncogenic K-Ras expression in endothelial cells is sufficient to fully promote blood cell transformation. In parallel, the oncogenic K-Ras expressing BMECs in primary culture are being examined to determine whether abnormal Ras signaling including RAF-MEK and/or PI3K-AKT pathways are involved in mediating BMEC proliferation and angiogenesis. Our study suggests a novel concept that pathologic bone marrow microenvironment, BMECs in particular, can play an important role in blood cell transformation, and implies that rational targeting of oncogenic pathways in the BMECs may provide an additional therapeutic avenue for blood malignancies, besides direct suppression of abnormal hematopoietic cells, to restore normal hematopoiesis.