Effective Targeting of Quiescent CML Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate.

Abstract 190

The BCR-ABL tyrosine kinase inhibitor imatinib mesylate (IM) is highly effective in inducing remissions and improving survival in CML patients but fails to eliminate leukemia stem cells, which remain a potential source of relapse. Quiescent leukemia stem cells resist apoptosis following BCR-ABL kinase inhibition, and other strategies are required for their elimination. Histone deacetylase inhibitors (HDACi) have shown promise in the treatment of several cancers, and it is of particular interest that reports suggest that they are also capable of inducing apoptosis in non-proliferating cells. It is known that the potent pan-HDACi LAQ824 (LAQ) and LBH589 (LBH) can induce apoptosis in CML cell lines and blast crisis CML cells. However, the effect of HDACi on quiescent leukemia stem cells from chronic phase CML patients is not known. Here we investigated the effects of LAQ and LBH, alone and in combination with IM, on CML stem and progenitor cells. CML and normal CD34+ cells were cultured with LAQ (10–100nM) or LBH (25–50nM) alone, IM (1mM) alone, and LAQ or LBH with IM for 96 hours. HDACi treatment effectively enhanced Histone H3 and H4 acetylation in CML CD34+ cells. HDACi treatment by itself induced less apoptosis in CML compared to normal CD34+CD38- primitive and CD34+CD38+ committed progenitors, but was highly effective in inducing apoptosis in CML progenitors when combined with IM. In addition, the combination induced significantly higher apoptosis in CML compared with normal CD34+ cells, and unlike IM, also induced apoptosis in non-dividing cells. Combination treatment also inhibited the proliferation of CML progenitor cells as measured by CFSE and growth of CML CFC in methylcellulose progenitor assays. Treatment of CML CD34+ cells with IM resulted in modest reduction in levels of engraftment in NSG mice. In contrast treatment with LAQ824 plus IM resulted in abrogation of engraftment of BCR-ABL+ CML cells (p<0.001). Significantly less inhibition of normal compared to CML cell engraftment was seen following LAQ824 and IM treatment (p=0.006). We used a transgenic Scl-tTa-BCR-ABL mouse model to investigate the effect of HDACi treatment on CML stem cells in vivo. SCLtTA/BCR-ABL transgenic mice were crossed with GFP transgenic mice to allow tracking of transplanted cells. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Mice were treated with IM (200mg/kg daily by gavage), LBH (30 mg/kg IP 3 times a week), LBH with IM, or vehicle alone (control) for 4 weeks. LBH combined with IM resulted in greater reduction in WBC, neutrophils and GFP+ cells than LBH or IM alone. Significantly increased apoptosis and a profound reduction of Lin- Sca-1+ Kit+ (LSK) stem cells were seen in mice treated with IM plus LBH (p<0.001) but not IM or LBH alone. LBH plus IM treatment did not significantly inhibit LSK cells in normal mice. BCR-ABL-transgenic mice treated with IM plus LBH demonstrated prolonged leukemia-free survival after discontinuing treatment compared with mice treated with IM or LBH alone (p<0.05). Combined IM and LAQ824 treatment resulted in marked inhibition of the anti-apoptotic protein MCL-1 in CML CD34+ cells (p<0.001). RNAi mediated inhibition of MCL-1 expression resulted in increased apoptosis in CML compared with normal CD34+ cells that was further enhanced by IM treatment. Our results indicate that treatment with HDACi plus IM effectively and selectively targets leukemia stem cells in CML, suggest a potential role for MCL-1 inhibition in HDACi induced apoptosis, and support ongoing clinical trials of LBH combined with IM to eliminate residual leukemia stem cells in IM-treated CML patients.