Several leukemias including CML are propagated by small populations of LSC, eradication of which may be required to achieve long-term remission and cure. Populations of cancer stem cells have also been identified in several solid tumors (Clarke et al., 2006
). Treatment with BCR-ABL TKI including IM, nilotinib and dasatinib reverses the proliferative advantage of CML progenitors resulting in remission induction but does not eliminate LSC, which continue to be detectable in patients despite prolonged IM treatment. Persistent LSC represent a reservoir of disease and potential source of relapse. CML patients have a high likelihood of disease relapse on discontinuation of treatment and it is currently recommended that patients should be treated with IM indefinitely to prevent relapse. Concerns regarding the lack of disease cure with IM alone, the potential risk of side effects with long-term treatment, issues of non-compliance, and the high financial burden associated with drug treatment provide a strong impetus to develop strategies to target residual LSC.
In the current study we found that treatment with HDACi in combination with IM was significantly more effective in inducing apoptosis in CP CML progenitor cells compared with IM alone. The combination of HDACi and IM induced apoptosis in quiescent CML progenitor cells that are highly resistant to elimination following treatment with IM alone. HDACi in combination with IM also inhibited CML progenitor proliferation and CML committed progenitor growth in colony assays. In contrast to reports that HDACi could induce apoptosis in CML cell lines and BC CML cells (Fiskus et al., 2006a
; Fiskus et al., 2006b
; Nimmanapalli et al., 2003a
; Nimmanapalli et al., 2003b
), we found that HDACi by themselves had minimal effect on apoptosis of CP CML cells. CML progenitor proliferation and CFC frequency were reduced only at the highest HDACi concentrations. Therefore targeting of primitive CP CML cells by HDACi is potentiated by concomitant inhibition of BCR-ABL tyrosine kinase activity. Importantly treatment with the combination of HDACi and IM markedly depleted CML LSC capable of long-term, multilineage engraftment in immunodeficient mice. Furthermore administration of the HDACi and IM in combination resulted in profound depletion of LSC with secondary repopulating capacity in the in vivo
setting in a transgenic BCR-ABL mouse model of CML, and prevented leukemia relapse after discontinuation of treatment. Therefore our data consistently show that the combination of IM with an HDAC inhibitor effectively targets primitive CML LSC and is superior to either agent alone. On the other hand this combination may be less suitable for treating patients harboring IM resistance-conferring BCR-ABL mutations, since the mutant kinase would be inhibited poorly by IM, thus eliminating its required contribution to the effect of the combination.
The effects of HDACi are complex, and involve multiple genes and pathways. HDACi-induced reduction in BCR-ABL protein expression and kinase activity could potentiate the effects of IM on CML LSC. We also observed that combination with IM enhanced HDACi-induced gene expression changes, compared with HDACi alone. Although the mechanisms underlying this effect are still unknown, novel nuclear functions of cytoplasmic tyrosine kinases related to histone and transcription factor modulation have been recognized. Potential mechanisms involved in CML LSC inhibition by the IM and HDACi combination, compared with IM or HDACi alone, suggested by gene expression analyses included reduced expression of genes related to the primitive HSC state such as downregulation of HOX, MYC and WNT related genes; reduced expression of E2F regulated genes, which may play an important role in protecting non-proliferating cells from stress-induced apoptosis (Moon et al., 2005
); and increased G-protein coupled receptor expression, possibly influencing microenvironmental interactions of CML progenitors (). These observations although preliminary, indicate promising avenues for further investigation of molecular mechanisms underlying the effects of the HDACi and IM combination.
Our studies indicate significant activity of HDACi against normal hematopoietic progenitors with increased levels of apoptosis, inhibition of proliferation, and inhibition of CFC and SRC growth. In vivo administration of LBH was associated with moderate inhibition of normal blood cell counts and BM stem and progenitor populations and reduced weight gain. These inhibitory effects on normal progenitors are consistent with clinical observations of thrombocytopenia and myelosuppression in clinical trials of LBH (Bruserud et al., 2007
). On the other hand the HDACi and IM combination resulted in significantly less apoptosis of normal compared with CML progenitors, and significantly less inhibition of normal SRC compared with CML SRC. In addition, in vivo
administration of the combination resulted in significantly less inhibition of normal blood cell counts, and BM stem and progenitor populations, compared with near complete elimination of CML LSC. These observations suggest a therapeutic window for HDACi and IM effects on CML LSC compared with normal stem cells that could be exploited clinically. However, the toxicity of LBH to normal progenitors indicates a need for continued exploration of mechanisms underlying activity of the LBH and IM combination on CML LSC to aid development of more selective, non-toxic approaches for targeting LSC in future.
There is considerable interest in devising improved approaches to target CML LSC. The therapeutic application of these studies will be towards using a combination of LBH and IM to achieve elimination of residual LSC in IM-treated and responsive CML patients. We have recently shown that a farnesyltransferase inhibitor BMS-214662 can selectively kill quiescent primitive CML progenitor cells (Copland et al., 2008
). However this agent is not being developed for clinical testing in CML. In contrast, several HDACi are currently in clinical trials for hematological malignancies as well as solid tumors (Glaser, 2007
). The role of HDACi in targeting cancer stem cells has not been previously described. Based on our observation that LAQ and LBH combined with IM can eliminate CML LSC, we have developed and initiated a clinical trial to determine the safety and tolerability of LBH in combination with IM in CML patients in cytogenetic remission with evidence of residual BCR-ABL+ cells. The ultimate measure of success for these studies in achieving elimination of residual LSC will be the ability of patients to maintain long-term remission after discontinuation of IM treatment. Historically, studies in CML have greatly enhanced our understanding of chromosomal translocations and oncogenes in cancer biology and have led the way in successful application of targeted therapies. It remains to be determined whether the current studies of targeting of LSC in CML using HDACi will have broader application to targeting of primitive, quiescent cancer-initiating cells in other leukemias and solid tumors.