Abstract

Imatinib mesylate (IM) is highly effective in the treatment of chronic myeloid leukemia (CML). However IM does not eliminate quiescent, primitive CML hematopoietic cells, which persist through treatment and remain a potential source of relapse. Therefore additional interventions are required to more effectively target primitive CML cells. The hydroxamic acid analogue histone deacetylase (HDAC) inhibitors, LAQ824, and LBH589, are reported to induce apoptosis and inhibit cell cycle in CML cell lines and blast crisis (BC) CML cells. Although most pro-apoptotic agents preferentially target dividing cells, HDAC inhibitors are a rare class of drugs reported to induce apoptosis in non-dividing cells. We were therefore interested in investigating whether the HDAC inhibitor LAQ824 (LAQ) could induce apoptosis in CD34+ cells from chronic-phase (CP) CML patients. CML and normal CD34+ cells were cultured with LAQ (10–100nM) alone and in combination with IM (1μM) for 96 hours in growth factor-supplemented medium. Treatment with LAQ (25–100 nM) effectively enhanced Histone H3 acetylation in CML and normal CD34+ cells as detected by Western blotting with an anti-Histone H3 (Lys9/Lys14) antibody. Although LAQ treatment by itself induced significantly less apoptosis in CML compared to normal CD34+ cells, LAQ in combination with IM was highly effective in inducing apoptosis in CML CD34+ cells (11+/−3%, apoptosis with IM alone, 18+/−3% with IM+10 nM LAQ, 22+/−2% with IM+25 nM LAQ, and 32+/−3% with IM+50 nM LAQ824, n=5). Significantly higher levels of apoptosis were seen in CML compared with normal CD34+ cells at lower doses of LAQ (p=0.03 at 10 nM). LAQ combined with IM also resulted in enhanced apoptosis of non-dividing CML CD34+ cells, identified using CFSE labeling, with 43% apoptosis observed in cells treated with 50 nM LAQ [n=5]. Combined LAQ and IM treatment also resulted in significant inhibition of CML progenitor proliferation measured in CFSE assays [n=4] and in the number of CML progenitors measured in methylcellulose progenitor assays [n=5]. In contrast to what has been described for BCR-ABL expressing cell lines and BC CML cells, LAQ treatment did not result in significant change in BCR-ABL levels in CML CD34+ cells on Western blotting. Significant down-regulation of MCL-1, a critical apoptosis regulator in early hematopoietic cells, was observed on Western blotting of CML cells treated with the LAQ and IM combination [n=5]. Levels of other anti-apoptotic proteins BCL-xL and BCL-2 were not affected. MCL-1 inhibition was not observed in normal CD34+ cells [n=4]. In contrast, normal cells treated with LAQ or LAQ and IM demonstrated enhanced levels of the pro-apoptotic protein Bim, but Bim levels were not increased in CML CD34+ cells. Q-PCR analysis revealed changes in MCL-1 and BIM mRNA levels in CML and normal CD34+ cells with LAQ and IM treatment that were consistent with the results of Western blotting. We conclude that treatment with the HDAC inhibitor LAQ in combination with imatinib results in significantly enhanced apoptosis of CML CD34+ cells, including non-dividing cells, compared with IM alone. Different mechanisms appear to be responsible for HDAC induced apoptosis in CML and normal CD34+ cells. Our results provide a strong rationale for future clinical application of HDAC inhibitors to enhance elimination of CML progenitors in combination with BCR-ABL tyrosine kinase inhibitors.

Author notes

Disclosure: No relevant conflicts of interest to declare.