Tcf3/Pbx1 Fusion Molecule Induces the Transcription of Prox-1 and Vascular Endothelial Growth Factor Receptor Type-3

Abstract 1487

Background and Aims: It is now known that, in some hematological malignancies, hematopoietic cytokines and their specific receptors work on the progression of leukemia with an autocrine stimulation of immature leukemic blasts. Vascular endothelial growth factor (VEGF)-C and VEGF receptor type-3 (VEGFR-3), which play an important role in creation of systematic lymphatic vessel systems, are also reported to work on the progression of malignant solid tumors and some of acute leukemias. We recently reported that in two cases of acute lymphocytic leukemia (ALL) with TCF3/PBX1 fusion, VEGF-C autocrine stimulation played an important role in the proliferation of ALL (Shirasaki, et al. International Journal of Hematology 2011). To make clear its pathogenesis molecularly, we made a transformant that expressed full-length cDNA of Tcf3/Pbx1 fusion transcript, and its protein-expression was analyzed. We also transplanted leukemic cells with Tcf3/Pbx1 fusion to non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice, and analyzed in vivo.

Materials and Methods: Full-length cDNA fragment of Tcf3/Pbx1 was prepared with polymerase-chain reaction, in which the primers were selected according to GeneBank data and our patients' cDNA sequences of leukemic cells. A plasmid that expressed full-length cDNA for Tcf3/Pbx1 was transfected to K562 human myeloid cell line, and after G418 selection, cells were analyzed. Patient-derived ALL cells and the Tcf3/Pbx1-transfected transformants were injected to 2 gray-irradiated NOD/SCID mice intra-vascularly, and intraperitoneally. Mice were fed in pathogen-free room with antibiotics-containing water, and were monitored with Tcf3/Pbx1 in blood cells. When Tcf3/Pbx1 was positive in blood, they were sacrificed and characterized.

Results and Discussion: Tcf3/Pbx1-introduced transformants expressed Prox-1, which is a master molecule for lymphatic duct neogenesis, and also VEGFR-3. LYVE1, a marker for lymphatic vascular endothelial cell, was not induced to be expressed. VEGF-C production was also demonstrated in the transformants. When de-novo ALL blasts with t(1;19) from our experienced cases, or Tcf3/Pbx1-introduced transformants were injected to NOD/SCID mice, leukemia was demonstrated after 60 days, in which human VEGF-C was elevated in blood, and VEGFR-3 was also expressed in the leukemic cells. Tcf3 is a trans-activator of transcription factors, and induces immature cells to shift to the B-cell lineage and activate. Pbx1 is able to associate with most members of the Hox protein family; however, no expression has been reported in B cells. When TCF3 is fused with PBX1, the constitutive activation of transcription is observed, and it is possible that the fused Pbx1 is expressed in the B-cell lineage to activate various kinds of protein that are not normally expressed in B-cell progenitors. Our observations indicate that the Tcf3-Pbx1 fusion molecule regulates Prox-1. It is very important to determine the precise molecular mechanism of the TCF3/PBX1 on the transcription of Prox-1.