Omacetaxine May Have a Role in Chronic Myeloid Leukaemia Eradication Through Downregulation of Mcl-1 and Induction of Apoptosis in Stem/Progenitor Cells
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Leukemia (2011) 25, 985–994 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE Omacetaxine may have a role in chronic myeloid leukaemia eradication through downregulation of Mcl-1 and induction of apoptosis in stem/progenitor cells EK Allan1,2, TL Holyoake1, AR Craig3 and HG Jørgensen1 1Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK; 2Scottish National Blood Transfusion Service, Gartnavel General Hospital, Glasgow, UK and 3ChemGenex Pharmaceuticals Inc., Menlo Park, CA, USA Chronic myeloid leukaemia (CML) is maintained by a rare Therefore this product has, for a second time, become a population of tyrosine kinase inhibitor (TKI)-insensitive malig- valuable option in the treatment of resistant disease. Moreover, nant stem cells. Our long-term aim is to find a BcrAbl- the demonstration that omacetaxine can kill leukaemic stem independent drug that can be combined with a TKI to improve 4 overall disease response in chronic-phase CML. Omacetaxine cells in murine models has allowed the drug to be considered mepesuccinate, a first in class cetaxine, has been evaluated by as a therapeutic option for both persistent and resistant disease. clinical trials in TKI-insensitive/resistant CML. Omacetaxine Homoharringtonine (from here is referred to as omacetaxine) inhibits synthesis of anti-apoptotic proteins of the Bcl-2 family, is derived from various Cephalotaxus species (Chinese yew tree) including (myeloid cell leukaemia) Mcl-1, leading to cell death. and was first discovered by the Chinese to have natural anti- Omacetaxine effectively induced apoptosis in primary CML tumour and anti-leukaemic properties in the 1970s.5,6 Recent stem cells (CD34 þ 38lo) by downregulation of Mcl-1 protein. In work has elucidated a mechanism of action targeted to the contrast to our previous findings with TKIs, omacetaxine did 7 not accumulate undivided cells in vitro. Furthermore, the A-site cleft of eukaryotic ribosomes resulting in transient functionality of surviving stem cells following omacetaxine inhibition of protein synthesis of short-lived proteins, such as exposure was significantly reduced in a dose-dependant myeloid cell leukaemia (Mcl-1) protein (a highly expressed manner, as determined by colony forming cell and the more member of the Bcl-2 family of anti-apoptotic proteins), leading stringent long-term culture initiating cell colony assays. This to apoptosis.8 Further, it has been shown that Mcl-1 has a stem cell-directed activity was not limited to CML stem cells as þ critical role in the survival of leukaemia cells and its expression both normal and non-CML CD34 cells were sensitive to 9,10 inhibition. Thus, although omacetaxine is not leukaemia stem is upregulated with respect to normal. As such, omacetaxine cell specific, its ability to induce apoptosis of leukaemic stem acts independently of BcrAbl kinase activity, the causative cells distinguishes it from TKIs and creates the potential for a oncoprotein in CML. Taken together with a recently reported curative strategy for persistent disease. ability to kill stem cells,4 omacetaxine could therefore be an Leukemia (2011) 25, 985–994; doi:10.1038/leu.2011.55; interesting therapeutic modality in minimal residual disease in published online 5 April 2011 Keywords: omacetaxine; leukaemic stem cells; CML; BcrAbl; Mcl-1 CML, where the continued detection of BcrAbl transcripts is thought to originate from the stem cell compartment,11 and leukaemic stem cell survival may be BcrAbl independent.12 Therefore, the aim of our study was to assess the activity of omacetaxine against CML stem/progenitor cells in vitro in order Introduction to identify potential ways of eradicating persistent disease. Sequential clinical samples from newly diagnosed cases were Omacetaxine mepesuccinate (ChemGenex Pharmaceuticals selected for assay and were not subject to mutation analysis. We Inc., Menlo Park, CA, USA), a first in class cetaxine, is a have previously shown CML CD34 þ cells to be insensitive to TKI semisynthetic alkaloid that has demonstrable activity in clinical by virtue of their quiescence and primitive phenotype. From our trials in acute myeloid leukaemia, myelodysplastic syndrome, as on-going work and that of others, it is our belief that CML stem well as in chronic myeloid leukaemia (CML).1 However, with cells may not be oncogene addicted (that is, dependant on BcrAbl the continued success of tyrosine kinase inhibitors (TKIs), such for their survival), and thus any novel agents or novel indications as imatinib, nilotinib and dasatinib, as first- and second-line for existing molecules with targeting potential at the (stem) cell treatment for CML, the role of omacetaxine has been restricted level to complement TKI modalities is of significant interest. to niche indications, including failure of two or more TKI or with the TKI-insensitive mutation, T315I, against which all currently licensed TKI are ineffective. Omacetaxine is a semisynthetic, Materials and methods subcutaneously bioavailable form of homoharringtonine. Homoharringtonine was used in second-line therapy for those Reagents CML patients in the pre-TKI era of the 1990s who failed Omacetaxine mepesuccinate, supplied by ChemGenex 2,3 interferon-a therapy and were ineligible for a transplant Pharmaceuticals , was stored lyophilised at room temperature; once reconstituted in saline to 10 mM, the stock solution was Correspondence: Professor TL Holyoake, Paul O’Gorman Leukaemia stored at 4 1C for up to 3 months. Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Gartnavel General Hospital, 1053 Great Western Road, Glasgow G12 0YN, UK. E-mail: tessa.holyoake@ glasgow.ac.uk Cell culture Received 19 August 2010; revised 28 January 2011; accepted Primary CML cells were obtained with informed consent from 14 February 2011; published online 5 April 2011 leukapheresis samples of newly diagnosed patients with Omacetaxine induces stem cell apoptosis EK Allan et al 986 chronic-phase CML or PhÀ haematological disorders (for significant reduction in total cells (undivided plus proliferating example, lymphoma) as controls. CD34 þ cells enriched by cells), the absolute number of undivided cells (product of % positive selection (CliniMACS, Miltenyi Biotec Ltd., Bisley, Â total cell number) may remain unchanged. Surrey, UK) according to the manufacturer’s instructions to 495% purity were cryopreserved in 10% (v/v) dimethylsulph- oxide in 4% (w/v) ALBA (human albumin solution, Scottish Primitive subsets National Blood Transfusion Service, Edinburgh, UK) and stored The CD34 þ -enriched cells were stained with anti-CD34-APC in the vapour phase of liquid nitrogen until required. CD34 þ and anti-CD38-PE antibodies before cell sorting using an FACS cells were cultured in serum-free media comprising Iscove’s Aria (Becton Dickinson) into three populations: CD34lo38 þ modified Dulbecco’s medium with BIT (bovine serum albumin, (mature), CD34 þ 38 þ (progenitor) and CD34 þ 38lo (primitive). insulin, transferrin; Stem Cell Technologies, Vancouver, BC, The CD34 þ 38lo fraction approximates the most primitive Canada), 1 mmol/l glutamine, 1 mmol/l streptomycin/penicillin, quiescent stem cell pool (o 5% total CD34 þ cells). Cells were 40 ng/ml low-density lipoprotein and 0.1 mmol/l 2-mercapto- analysed using a BD FACS Canto instrument with Diva software. ethanol (all, Invitrogen Ltd, Paisley, UK), further supplemented with a 5-growth factor cocktail as previously described.13 The BcrAbl-positive human myeloid cell lines, K562 and Colony forming cell (CFC) and long-term KCL22 were maintained in RPMI 1640 (Sigma–Aldrich, Poole, culture-initiating cell (LTC-IC) Dorset, UK) supplemented with 10% (v/v) foetal calf serum After culture in omacetaxine for 24 or 72 h, CD34 þ cells containing 1 mmol/l each of streptomycin, penicillin and remaining were set up in CFC and LTC-IC assays to assess the glutamine (RPMI þ ). Total cell counts and viability were proliferative potential of cell populations remaining after drug assessed using the trypan blue dye exclusion method. treatment. For CFC assays, 2.5 Â 103 cells were added to 1.5 ml Methocult (Stem cell Technologies) in duplicate 35 mm culture dishes and cultured for 12–14 days at 37 1Cina Flow cytometry 5% CO2-humidified incubator. Apoptosis. To assess apoptosis after drug treatment, an LTC-IC assays were set up as previously described.15 Briefly, aliquot of cell suspension containing 1–2 Â 105 cells was M2-10B4 cells and SL/SL fibroblasts were established as feeder removed for staining with Annexin V-fluorescein isothiocyanate layers then irradiated at 80 Gy. CML cells remaining in culture and Viaprobe (BD Biosciences, Oxford, UK) as per the after drug treatment were washed three times and then plated in manufacturer’s instructions and analysed by flow cytometry duplicate on the irradiated feeder layers in Myelocult medium using a BD FACS Canto with Diva software. Cells undergoing supplemented with hydrocortisone at a final concentration of apoptosis initially become Annexin V positive (early apoptosis), 1 mM (Stem Cell Technologies). Cultures were maintained for and as apoptosis progresses, become positive for both Annexin V 5 weeks with weekly half medium changes. After 5 weeks, cells and Viaprobe (late apoptosis). were counted and 2.5 Â 104 cells were transferred to CFC assays and maintained in culture for a further 2 weeks in Methocult medium before the colonies were counted. Cell division tracking Upon recovery from liquid nitrogen, CML CD34 þ cells were stained with 1 mM carboxy-fluorescein diacetate succinimidyl Western blotting diester (CFSE; Molecular Probes, Invitrogen Ltd, Paisley, UK), as Following treatment with drugs, K562 and primary CML cells described previously in detail.14 Cells were then cultured in (2–5 Â 106) were pelleted by centrifugation, washed twice with serum-free media þ 5-growth factor in the presence or absence ice-cold phosphate-buffered saline, and then lysed in buffer of omacetaxine for 24 or 72 h.