Analysis of the Crosstalk Between TGF-β–VEGF-Angiogenesis in an In Vivo Model of Acute Promyelocytic Leukemia

Abstract 1845

Acute Promyelocytic Leukemia (APL) patients present increased bone marrow microvascular density (MVD) compared to normal bone marrow, which has been associated with the aberrant secretion of the proangiogenic factor VEGF by leukemic cells. The APL associated fusion protein PML-RARα is thought to deregulate the TGF-β pathway, through its dominant negative action on cytoplasmic PML, thus down regulating SMAD2/3 signaling, and VEGF transcription. However, PML-RARα expression was associated with increased TGF-β gene transcription and secretion. We used the low molecular weight quinazolinone alkaloid Halofuginone (HF), which has been shown to be a potent TGF-β inhibitor, to test the association between TGF-β/VEGF/angiogenesis. HF inhibited the VEGF secretion by NB4 (an APL cell lineage) and cell proliferation. To determine the effects of HF in vivo, irradiated NOD/SCID mice were transplanted with leukemic cell from hCG-PML-RARα transgenic mice. Twenty-four hours after transplantation, mice were treated with 150μg/kg of HF by intraperitoneal injections for 21 days. All recipients developed leukemia, however the leukemic infiltration of bone marrow and WBC were significantly lower in animals treated HF (4.2 ± 3.89 vs. 20.6 ± 21.9, p <0.0001) and hemoglobin and platelet counts higher in this treated group (Hb: 12.0 ± 1.40 vs. 9.6 ± 1.67, p <0.0001; and Platelets: 932.0 ± 122.5 vs. 552.0 ± 83.2, p <0.001). Accordingly, a lower leukemic infiltration of spleen was detected (ratio spleen/body of 0.006 vs. 0.012 in treated and untreated groups respectively, p=0.0415). Furthermore, the differential count and immunophenotyping of bone marrow showed a lower percentage of immature myeloid cells (27 ± 9.3 vs. 66.3 ± 17.9; p=0.0037 and 16.88 ± 6.27 vs. 44.06 ± 27.06, respectively). HF treatment did not induce molecular remission nor increased the survival of leukemic animals. However, the lack of difference in survival could be atributted to HF hepatoxicity, as a significant rise in AST and ALT serum levels was observed (661.2 ± 262.6/177.9 ± 107.4 vs. 89.88 ± 11.65/27.86 ± 4.47 U/L, prespectively). Phosphorilated SMAD2 levels were determined by ELISA in NB4 cells and a significant dose dependent decrease was observed in samples treated with HF at the doses of 50, 100 and 200 ng/mL. Gene expression analysis showed that the HF treatment inhibited the expression of TgfB, Smad3, Smad4, Myc, Vegf and Egf and the immunohistochemistry analysis of BM sections revealed a significant decrease of VEGF staining (30 vs. 80%, p =0.0227), but there was no decrease in the microvascular density. Taken together, these results showed that angiogenesis is an important therapeutic target in APL, and despite the toxicity, HF has antileukemic potential due to its antiproliferative and proangiogenic factors inhibitory capabilities.