Histone Deacetylase Inhibitors Are Capable to Modify Leukaemia-Specific Phenotype of TEL/AML1-Positive Leukaemic Cells.

Abstract

The cryptic translocation t(12;21), resulting in TEL/AML1 fusion, is the single most frequent non-random genetic aberration in childhood ALLs (up to 25%). Both partner genes are important for the development and maintenance of normal haematopoiesis. TEL part of the fusion protein contains domains interacting with the mSin3, N-CoR and HDAC-3 corepressors. Part of AML1 gene involved in the fusion carries a DNA binding domain that controls the interaction with the cis-regulatory elements of the target genes. TEL/AML1 is supposed to negatively regulate the expression of the genes that, under normal conditions, are transactivated by AML1. TEL/AML1 chimaeric protein seems to affect genes responsible for the differentiation during haematopoiesis by chromatin remodelling via association with histone deacetylases (HDAC). We used HDAC inhibitors (HDACi) to overcome the presumptive differentiation block mediated by HDAC recruited by TEL/AML1. Trichostatin A (TSA) and valproic acid (VPA), common antiepileptic drug with attributed HDACi activity, were used as HDACi. We tested different concentrations of TSA (120nM, 240nM and 600nM) and VPA (0.5mM, 1.0mM and 2.0mM; concentrations close to VPA plasmatic level used in the treatment of epilepsy). We determined the cell cycle and the state of differentiation using flow cytometry 24 and 48 hours after HDACi administration. We compared TEL/AML1[+] (REH) and TEL/AML1[-] (Nalm-6) leukaemic cell lines to the non-leukaemic lymphoid cell line (NC-NC). Under HDACi treatment, leukaemic cells were arrested in proliferation and entered apoptosis. In REH cell line, the apoptosis rate was 11% using 1mM of VPA, and 30% using 240nM of TSA in contrast to 3.7% in control. Similarly, Nalm-6 apoptosis rate reached 16% with VPA, and 39% with TSA compared to 0.6% in control. In contrast, NC-NC cell line showed only negligible shift in cell cycle. Nalm-6 and REH cell lines reduced their proliferation activity by 36% and 19%, respectively. Furthermore, after both TSA and VPA treatment, surviving TEL/AML1[+] cells showed drift in expression of CD10 and CD20 surface antigens and in intracellular level of RAG-1 and TdT proteins. The CD 10 decreased to 88% and 64%, TdT decreased to 46% and 29%, RAG-1 decreased to 57% and 38% compared to the levels in untreated controls, after 48-hour treatment with 1.0mM VPA and 240nM TSA, respectively (all p<0.01). Moreover, the expression of CD20 antigen increased to the 150% and 227% of the level in untreated controls, after 24-hour treatment with 1.0mM VPA (p<0.05) and 240nM TSA (p<0.01), respectively. In other concentrations and time-points similar dose-dependent maturation drift was observed and the pattern of this drift corresponded to the changes observed during the maturation in physiological lymphopoesis. Our results support the hypothesis that HDACi is generally able to affect maturation stage and viability of leukaemic cells. We suppose that the differentiation effect of HDACi on TEL/AML1[+] cells is directly related to the function of TEL/AML1 protein. The valproate has two advantages for the potential treatment use: (i) it has been already tested for the clinical use in the therapy of epilepsy and doses we used were close to those applied in anti-epilepsy treatment (ii) it should specifically interact with Class I HDACi (HDAC 3 that binds to TEL/AML1 co-repressor complex belongs to Class I). Supported by grants: 7433 IGA MZ, 301/D0189 GACR, 75/2004C GAUK.