Characterisation of Histone Deacetylase (HDAC) Expression Profiles in Acute Myeloid Leukaemia: A Basis for the Development of Targeted Therapy Using Histone Deacetylase Inhibitors.

Post-translational modifications of chromatin structure are recognised as having a potential role in the pathogenesis of acute myeloid leukaemia (AML). Histone deacetylases play a central role in determining the acetylation status of histones and are emerging as novel targets in AML therapy. Histone deacetylase inhibitors (HDIs) inhibit growth of primary AML blasts in vitro and demonstrate clinical activity in patients with relapsed AML. To date, three major classes of HDACs have been characterised which differ in their susceptibility to HDIs. However, little is known of the pattern of HDAC expression in AML and this limits the rational use of HDIs in this disease.

We have analysed the pattern of class I, II and III HDAC expression in AML cell lines, primary AML blasts and CD34+ selected progenitors from cord blood and normal donors by real time quantitative PCR(RT-PCR). RT-PCR analyses demonstrated consistently increased expression in AML blasts of two HDACs compared with proliferating CD34^+ve cells from cord blood (n=5) . HDAC2 (class I HDAC) was more highly expressed in 3/3 myeloid cell lines and 20/24 primary AML samples, compared to cord blood CD 34+ve cells. SIRT1 (class III HDAC) was also more highly expressed in 3/3 myeloid cell lines and 24/24 primary AMLs. In contrast, no marked differences were detected in expression of HDAC1, 3, 4, 5, 6, 7, 9, 10, 11 and SIRT 2–6.

We therefore studied the impact of sodium valproate (SV), an HDI with reported activity in AML, on HDAC activity and expression. SV treatment resulted in time and dose dependent increases in histone acetylation and specific methylation at H3K4, in both AML cell lines and primary AML cells. We have shown that the mechanism of the increased methylation at H3K4 is partly a result of the preference of the methyltransferase enzyme for acetylated histones. Using quantitative RT-PCR we found that SV treatment of HL-60 cells resulted in increased expression of the gene for MLL, an enzyme known to be capable of methylating H3K4. These changes in chromatin were associated with dose dependent cell killing. Significant (p<0.001, n=10) in vitro killing of primary AML blasts was observed at 1 mM SV and killing was enhanced by the addition of ATRA. However, histone modifications were observed in HL-60 cells at doses that had little effect upon cell viability or proliferation. We have immunoprecipitated complexes containing HDACs 1, 2 and 3 and assayed their enzymatic activity using a tritium labelled H4 peptide substrate. By this method, 1mM SV was potent at inhibiting HDACs 1, 2 and 3 activity. We have also studied the effects of SV on HDAC expression (all three classes) by RT-PCR in HL60 cells at 15 mins, 1h, 4h and 8h. A small number of HDACs were consistently and significantly upregulated by treatment with SV, TSA, Butyrate and SAHA. In contrast, the expression levels of HDAC2 and SIRT1 were relatively unchanged. These changes in HDAC expression may contribute to HDI resistance with continuous therapy. These data provide information which may be of value in determining choice of HDACis in Phase I/II studies in AML. They also identify increased histone methylation as a potentially important outcome of HDI treatment.