The generation of B cells is a complex process requiring several cellular transitions, including cell commitment and differentiation. Proper transcriptional control to establish the genetic programs characteristic of each cellular stage is essential for the correct development of B lymphocytes. Deregulation of these particular transcriptional programs may result in a block in B-cell maturation, contributing to the development of hematological malignancies such as leukemia and lymphoma. However, very little is currently known about the role of transcriptional repressors in normal and aberrant B lymphopoiesis. Here we report that histone deacetylase 7 (HDAC7) is underexpressed in pro-B acute lymphoblastic leukemia (pro-B-ALL) and Burkitt lymphoma. Ectopic expression of HDAC7 induces apoptosis, leads to the downregulation of c-Myc and inhibits the oncogenic potential of cells in vivo, in a xenograft model. Most significantly, we have observed low levels of HDAC7 expression in B-ALL patient samples, which is correlated with the increased levels of c-Myc. From a mechanistic angle, we show that ectopically expressed HDAC7 localizes to the nucleus and interacts with the transcription factor myocyte enhancer factor C (MEF2C) and the corepressors HDAC3 and SMRT. Accordingly, both the HDAC7–MEF2C interaction domain as well as its catalytic domain are involved in the reduced cell viability induced by HDAC7. We conclude that HDAC7 has a potent anti-oncogenic effect on specific B-cell malignancies, indicating that its deregulation may contribute to the pathogenesis of the disease.

FLT3 abnormalities are negative prognostic markers in acute leukemia. Infant leukemias are a subgroup with frequent MLL (KMT2A) rearrangements, FLT3 overexpression and high sensitivity to cytarabine, but dismal prognosis. Cytarabine is transported into cells by Human Equilibrative Nucleoside Transporter-1 (hENT1, SLC29A1), but the mechanisms that regulate hENT1 in acute leukemia have been scarcely studied.We explored the expression and functional link between FLT3 and main cytarabine transporters in 50 pediatric patients diagnosed with acute lymphoblastic leukemia and MLL rearrangement (ALL-MLL+) and other subtypes of leukemia, and in leukemia cell lines.A significant positive correlation was found between FLT3 and hENT1 expression in patients. Cytarabine uptake into cells was mediated mainly by hENT1, hENT2 and hCNT1. hENT1-mediated uptake of cytarabine was transiently abolished by the FLT3 inhibitor PKC412, and this effect was associated with decreased hENT1 mRNA and protein levels. Noticeably, the cytotoxicity of cytarabine was lower when cells were first exposed to FLT3 inhibitors (PKC412 or AC220), probably due to decreased hENT1 activity, but we observed a higher cytotoxic effect if FLT3 inhibitors were administered after cytarabine.FLT3 regulates hENT1 activity and thereby affects cytarabine cytotoxicity. The sequence of administration of cytarabine and FLT3 inhibitors is important to maintain their efficacy.

Histone deacetylase inhibitors (HDACi) had emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. There was a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles identified specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with KMT2A (MLL) rearrangement, with high HDAC9 and MEF2D expression, regardless of age, KMT2A partner and lineage. Moreover, we observed an adverse prognostic value of HDAC9 overexpression, regardless of KMT2A rearrangement. Our results provide useful knowledge on the complex picture of HDAC expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Introduction: Infant acute leukemias (those diagnosed at age <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 (>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.