FLT4 Expressing CD34⁺CD38^- Leukemic Stem Cells in Bone Marrow of Refractory Patients are Protected by Receptor Internalization under Abundant VEGF-C Condition

So far, the cause of relapse or refractoriness in acute myeloid leukemia (AML) is regarded as the persistence of chemoresistant leukemic stem cells (LSCs). Even VEGF-C/FLT4 axis mediates blast proliferation and survival, understanding of the dynamic behavior of LSCs in bone marrow remains elusive. Especially, FLT4 endocytosis by VEGF-C is related to various biologic functions including signal activation. Based on our previous study, which addressed the potential of FLT4 as a marker for LSCs, we showed that surface FLT4 can be internalized by VEGF-C exposure in bone marrow (BM) derived LSCs. It resulted in resistant to chemotherapy, suggesting LSCs protection. In addition, data for apoptosis revealed that inhibition of FLT4 using MAZ51 with cytosine arabinoside (Ara-C) can effectually increase cell death under abundant VEGF-C in refractory patients. It indicated that targeting FLT4 allows investigators to establish advanced therapeutic strategy with conventional Ara-C in refractory patients with high VEGF-C. In clinic, low level of surface FLT4 in de novo AML-BM LSCs, but not PB cells were inclined to undergo refractory status after induction chemotherapy. To investigate whether FLT4 expressing CD34⁺CD38^- LSCs can be protected by VEGF-C via internalization, FACS analysis, western blotting and immunocytochemistry were performed. Meanwhile, total 66 newly diagnosed (ND) AML patients were used to analyze for clinical correlation. (BM, n=37; PB, n=19). FACS analysis showed that surface FLT4 expressing CD34⁺CD38^- LSCs in BM, but not PB, were significantly decreased by VEGF-C (In BM; without VEGF-C & with VEGF-C, 38.7±8.1% & 16.0±5.1%). Similar with surface FLT4 in PB cells, both intracellular FLT4 in BM and PB was highly sustained, regardless of VEGF-C (In BM; without VEGF-C & with VEGF-C, 62.9±17.2% & 69.7±14.9%, In PB; without VEGF-C & with VEGF-C, 77.4±15.6% & 70.4±21.8%). Immunocytochemistry and western blotting also showed internalization of surface FLT4 by VEGF-C treatment. FLT4⁺CD34⁺CD38^- cells both in BM and PB were significantly higher in refractory patients than in post allogeneic stem cell transplantation (SCT) group, implying clinical correlation of FLT4 with refractory patients. Consistent with findings in ND-AML and refractory groups, FLT4⁺CD34⁺CD38^- cells were highly sustained in complete remission group, showing drug resistant population (ND-AML, n=30; refractory AML, n=9; Complete remission AML, n=18; post-allogeneic stem cell transplantation, n=9). High level of VEGF-C was detected in refractory patients, compared to that of normal donors (Refractory AML, n=9, normal donors, n=7). Apoptosis results showed that high number of apoptotic CD45^dimCD34⁺CD38^- cells in MAZ51 (FLT4 antagonist) and Ara-C dual treatment under VEGF-C exposure, compared to no treatment of both MAZ51 and Ara-C (without VEGF-C; 0.5-1.3 folds, with VEGF-C; 2.7-43.3 folds) or single exposure either to MAZ51 or Ara-C (In Ara-C only; without VEGF-C; 1.0-9.5 folds, with VEGF-C; 0.1- 1.3 folds, In MAZ51 only; without VEGF-C; 1.1-1.7 folds, with VEGF-C; 1.3-39.3 folds), suggesting that Ara-C induced-blast apoptosis can be augmented by FLT4 inhibition even in the presence of VEGF-C in a sub-group of either refractory or relapsed AML patients. VEGF-C/FLT4 axis in AML is involved in PI3K and AKT pathway.

Collectively, we demonstrated that FLT4 internalization under VEGF-C in BM leads to protection of FLT4 expressing LSCs and is clinically relevant to refractory subgroup. This data could suggest some clues to develop therapeutic strategies in targeting FLT4 expressing refractory LSCs.