BCR-ABL-induced expression of angiogenic factors: Evaluation of biochemical mechanisms and pathopysiologic significance

Recent data suggest that angiogenic molecules like VEGF, play a crucial role in the pathogenesis of various neoplasms. These factors have not only been implicated in tumor-associated angiogenesis but also as potential autocrine growth factors produced in leukemic cells. Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by increased production and accumulation of clonal myeloid cells carrying the BCR/ABL oncogene. BCR/ABL exhibits increased tyrosine kinase activity as compared to ABL, and activates a number of signaling cascades including the p21Ras-, PI-3-kinase-, and STAT5 pathways. Recent studies have shown that in patients with CML, increased levels of angiogenic factors (VEGF, bFGF, HGF, PDGF) as well as an increase in bone marrow angiogenesis is found. However, so far, it is unknown whether the BCR/ABL oncogene is directly involved in the generation of angiogenic factors, and whether angiogenic cytokines are critical factors promoting bone marrow angiogenesis, autocrine growth of CML cells, or disease progression. In recent experiments, we have shown that doxycycline-inducible expression of BCR/ABL in Ba/F3 cells is associated with expression of VEGF, and that BCR/ABL-induced VEGF-expression depends on signaling through PI3-kinase and mTOR. To our knowledge, this is the first time that expression of a leukemia-specific oncogene can be linked to induction of an angiogenic molecule in neoplastic cells. Based on these observations we now plan to analyze BCR/ABL-dependent expression of other angiogenic factors (VEGF, bFGF, HGF, PDGF) in cell line models, murine in vivo models, and patients with CML. One major question to be answered will be whether angiogenic cytokines can also act as autocrine growth factors for CML cells. Another important question to be solved would be whether oncogene- dependent expression of angiogenic molecules is a causative phenomenon directly involved in the malignant transformation of BCR/ABL-expressing myeloid cells. It will also be of substantial importance to learn whether expression of angiogenic factors correlates with the stage of disease, progression, and survival in CML patients. Finally, we plan to study signal transduction events in detail and to identify suitable pharmacologic inhibitors of BCR/ABL-dependent expression of angiogenic factors. The results of our project should thus provide new insight into the pathophysiology of CML and give answers to important questions raised in leukemia research in general. In addition, results from studying signal transduction events and respective inhibitors may potentially lead to the development of new pharmacologic concepts and new therapeutic strategies.

The constant supply of nutritive factors through larger and smaller blood vessels is essential for normal physiologic organ function. Similarly, the proliferation and growth of endothelial cells and the consecutive formation of new blood vessels (neovascularization, angiogenesis) are essential for the growth and metastasis of solid tumors. More recent studies suggest that angiogenesis is also of crucial importance in various myeloid leukemias. Likewise, in several myeloid leukemias, the numbers of microvessels were found to be significantly increased compared to normal bone marrow. In addition, several studies have shown that the microvessel density in the bone marrow is a prognostic factor and correlates with survival. The current project examined the expression, regulation, and functional role of various angiogenic growth factors in neoplastic cells in chronic myeloid leukemia (CML), a disease that is driven by the BCR/ABL oncogene. A number of angiogenic growth factors have been identified in the past. Of these, the vascular endothelial growth (VEGF) has been identified as a major regulator of angiogenesis in the bone marrow. The results of the FWF project show that primary CML cells express VEGF mRNA as well as the VEGF protein in a constitutive manner and that the CML-related oncoprotein BCR/ABL promotes expression of VEGF in neoplastic cells. Correspondingly, the BCR/ABL-inhibitor STI571 (imatinib) was found to counteract VEGF expression in CML cells. In consecutive experiments, it was found that the BCR/ABL-induced expression of VEGF in leukemic cells is mediated through a signaling pathway that involves the PI3-kinase as well as the mammalian target of rapamycin (mTOR). Based on this observation, the mTOR-inhibitor rapamycin was applied and found to counteract growth along with VEGF expression in neoplastic cells in patients with CML. This effect was seen in chronic phase CML as well as in imatinib-resistant CML cells. Based on these interesting data, several clinical trials using mTOR inhibitors (rapamycin or others) alone or in combination with imatinib have now been initiated (by various companies as well as by the PI). In other parts of the project, CML cells were found to express several VEGF receptors including neuropilin-1 and neuropilin-2, and to express several additional angiogenic factors including hepatocyte growth factor (HGF), as well as important survival molecules including heme oxygenase-1 (HO-1). The respective targeted drugs were found to inhibit growth of CML cells in vitro. These compounds will be examined for their value as antileukemic agents in clinical trials in the near future.