Abstract C46: Implication of FLT3 in human equilibrative nucleoside transporter 1 (hENT1)-mediated uptake of Ara-C in pediatric acute leukemia.
Authors: Fernández-Calotti Paula , Català Albert , Pastor-Anglada MarÇal , Malatesta Roberta , Rives Susana , Camos Mireia 
Published: August 2014

Introduction: Infant acute leukemias (those diagnosed at age &lt;1 year) are characterized by high FMS-like tyrosine Kinase 3 (FLT3) expression and high sensitivity to the nucleoside analogue Ara-C. FLT3 is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Ara-C is known to be transported by hENT1 undergoing metabolic activation within target cells. In fact, high hENT1 expression levels have been reported to determine Ara-C sensitivity in patients with acute leukemia. However, the mechanisms that regulate the expression of hENT1 and its activity, as well as the putative relationship between FLT3 and Ara-C transport and metabolism are poorly known.

Aim: To study the role of FLT3 in the regulation of the expression and activity of the main Ara-C transporters and metabolizing enzymes (ME) in pediatric acute leukemia.

Experimental Procedures: Bone marrow samples from 56 pediatric patients diagnosed with acute leukemia in Hospital San Joan de Deu of Barcelona and 3 acute leukemia cell lines (MV4-11, SEM, K562) were used in this study. In all cases an informed consent was signed. The mRNA amounts of FLT3, the putative Ara-C nucleoside transporters (NT), hENT1, hENT2, hCNT1, hCNT3, and the main Ara-C metabolic enzymes, DCK and CNII were quantified by real-time PCR. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters was addressed by exposure of cells to the FLT3 inhibitor PKC412.

Results: A significant positive correlation was found between FLT3 and hENT1 mRNA levels when analysing the whole cohort of patients. DCK and CNII also showed a positive correlation. As expected, FLT3 expression was higher in cases with acute lymphoblastic leukemia (ALL) and mixed lineage leukemia (MLL) rearranged genes and, to a lesser extent, in ALL with hyperdiploidy (&gt;50 chromosomes), as these are the subgroups with highest expression of FLT3 reported in the literature.

Ara-C uptake into cells was mediated mainly by hENT1, hENT2, and hCNT1. The hENT1-mediated uptake of Ara-C was transiently abolished by the FLT3 inhibitor PKC412 reaching its minimum activity after 16 hours with the inhibitor. This down-regulation of hENT1 activity seemed to be dependent on the ability of PKC412 to inhibit hENT1 gene (SLC29A1) expression.

Conclusions: The positive correlation between the FLT3 and hENT1 expression levels in leukemic cells from patients, along with the in vitro evidence that FLT3 inhibition represses hENT1 expression and down-regulates Ara-C uptake in leukemic derived cell lines, supports the idea that FLT3 modulates hENT1. This evidence should be taken into account at the therapy schedule level when therapeutic combinations including both AraC and FLT3 inhibitors are designed.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C46.

Citation Format: Paula Fernández-Calotti, Albert Català, MarÇal Pastor-Anglada, Roberta Malatesta, Susana Rives, Mireia Camos. Implication of FLT3 in human equilibrative nucleoside transporter 1 (hENT1)-mediated uptake of Ara-C in pediatric acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C46.