Neoangiogenesis has been shown to play an important role in the pathogenesis of acute myeloid leukemia (AML). Autocrine and paracrine secretion of angiogenic and hematopoietic growth factors such as vascular endothelial growth factor (VEGF) and stem cell factor (SCF) in the bone marrow microenvironment may promote proliferation and survival of leukemic blasts. This concept represented the rationale for the initiation of a multicenter phase 2 trial of SU5416, a small molecule inhibitor of phosphorylation of VEGF receptors 1 and 2, c-kit, the SCF receptor, and fms-like tyrosine kinase-3 (FLT3) in patients with advanced AML. Entered into the study were 43 patients with refractory AML or elderly patients not judged medically fit for intensive induction chemotherapy; 42 patients received at least one dose of study drug. Treatment was generally well tolerated, with nausea, headache, and bone pain the most frequent treatment-related side effects. One patient had a morphologic remission (French-American-British [FAB] criteria of complete response without normalization of blood neutrophil and platelet counts) lasting for 2 months. There were 7 patients who achieved a partial response (reduction of blasts by at least 50% in bone marrow and peripheral blood) lasting 1 to 5 months. Patients with AML blasts expressing high levels of VEGF mRNA by quantitative polymerase chain reaction (PCR) had a significantly higher response rate and reduction of bone marrow microvessel density than patients with low VEGF expression consistent with the antiangiogenic effects of SU5416.

In acute myeloid leukemia (AML), autocrine or paracrine activation of receptor tyrosine kinases such as c-kit and FLT3 contributes to proliferation and apoptosis resistance of leukemic blasts. This provided the rationale for a multicenter clinical trial in patients with refractory AML with SU5416, a small molecule kinase inhibitor which blocks phosphorylation of c-kit, FLT3, VEGFR-1, VEGFR-2 (KDR) and VEGFR-3. The levels of VEGF mRNA expression were investigated in peripheral blood leukemic blasts taken from AML patients before and during treatment with SU5416. Rapid down regulation of VEGF was observed in AML blasts from 72% (13 of 18) of patients analysed. Patients initially expressing high VEGF-levels had a stronger downregulation and a higher clinical response rate (mean 865-fold, n = 10, P = 0,01) than patients initially expressing low VEGF-levels (mean four-fold, n = 8). These results suggest that abnormal high VEGF expression is downregulated by SU5416 treatment, and furthermore that decreases in VEGF mRNA levels may provide an early marker of therapeutic response with anti-angiogenic therapy. Additionally, protein expression of STAT5 and AKT was assessed by western blotting in these patient samples, as well as in the leukemia cell line, M-07e, treated in vitro with SU5416 as a model system. In the AML patient samples, parallel downregulation of both STAT5 and AKT was observed in several cases (STAT5 in four of 15; AKT in three of six examined patients). These effects were confirmed with the cell line M-07e after incubation with SU5416 in vitro using concentrations that are achievable in patients. In summary, our data show suppression of the expression of VEGF and key signal transduction intermediates in AML blasts during treatment with SU5416.

Autocrine or paracrine activation of receptor tyrosine kinases are important pathogenetic factors in AML. Signal transduction via c-kit and FLT3 increase proliferation and apoptosis resistance of leukemic blasts. Additionally bone marrow angiogenesis plays a significant role in disease progression. This provided the rationale for a multicenter clinical trial in patients with refractory AML with SU5416, a small molecule kinase inhibitor which blocks phosphorylation of c-kit, FLT3, VEGFR-1, VEGFR-2 (KDR) and VEGFR-3. In the study presented here, we analysed expression levels of VEGF and the downstream signal transduction intermediates STAT5 and Akt in patients before and during treatment with SU5416. The levels of VEGF mRNA expression were investigated in peripheral blood leukemic blasts by means of quantitative Real-Time PCR. 74% (14 of 19) of analyzed patients had rapid and persistent down regulation of VEGF during therapy. Patients initially expressing high VEGF-levels had a stronger down regulation and a higher clinical response rate (mean 865-fold, n=9) than patients initially expressing low VEGF-levels (mean 4-fold, n=8). These results suggest that downregulation of high pretherapeutic VEGF expression by SU5416 treatment may serve as an early surrogate marker of therapeutic response to anti-angiogenetic therapy. Additionally, protein expression of STAT5 and Akt was assessed by Western blotting. Incubation of the leukemic cell line M-07e in-vitro with SU5416 using concentrations which are achievable in patients was used as a model system. In the AML patient samples parallel downregulation of both STAT5 and Akt was observed in several cases (STAT5 in 4 of 15, Akt in 3 of 6 examined patients). Decreased intracellular levels of STAT5 and Akt were also found in the line M-07e after incubation with SU5416 in vitro. In summary, our data provide evidence for suppression of expression of VEGF and of the key signal transduction intermediates STAT5 and Akt in AML blasts in-vivo during treatment with SU5416.