Omacetaxine May Have a Role in Chronic Myeloid Leukaemia Eradication Through Downregulation of Mcl-1 and Induction of Apoptosis in Stem/Progenitor Cells

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. After 3 days, cells were containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet harvested, total cell viability assessed, and an aliquot of cells P-40, 1 mM EDTA, plus protease inhibitors (complete mini from each treatment stained with anti-CD34-PE antibody (BD protease inhibitor cocktail tablets; Roche Diagnostics, Burgess Pharmingen) for flow cytometry analysis. Cells cultured with Hill, UK). Protein lysates were separated on 4–15% Tris-HCl 100 ng/ml Colcemid (Invitrogen), which arrests cell-cycle gradient gels (Bio-Rad Laboratories, Hemel Hempstead, progression, were used to identify the undivided (CFSEmax) cell Hertfordshire, UK), transferred onto nitrocellulose membrane population. (Bio-Rad Laboratories), and blocked with 5% bovine serum To measure the overall effect of the treatments on cell survival albumin in Tris-buffered saline (0.1%) Tween 20 for 1 h at room and the number of undivided cells remaining after culture with temperature. Membranes were incubated with primary antibody omacetaxine, the percentage recovery of CD34 þ cells in the overnight at 4 1C: poly(ADP-ribose) polymerase, Mcl-1, GAPDH undivided CFSEmax peak was calculated with respect to the (Cell Signalling, New England Biolabs (UK) Ltd, Hertfordshire, input as previously described13 and expressed as a proportion of UK), abl, Bcl-2, Bcl-2-interacting mediator of cell death (BD the no drug control (NDC). Briefly, the number of CD34 þ cells Biosciences) at 1:1000 and anti-heat-shock protein 90 (R&D used to establish each culture was recorded. After the 3-day Systems, Abingdon, UK) at 1:10 000 in 1% bovine serum culture period, the total number of viable cells harvested from albumin/Tris-buffered saline Tween 20. Membranes were then each culture condition was recorded, as were the percentages of washed five times with Tris-buffered saline Tween 20 and then viable CD34 þ cells found in the undivided fraction and in each incubated with horseradish peroxidase-labelled secondary anti- division peak from the CFSE/CD34 þ flow cytometry plots. body at 1:3000 in 1% bovine serum albumin/Tris-buffered saline Percentage recovery of input cells in each peak could then be Tween 20 for 1 h at room temperature. Antibody detection calculated by dividing the absolute number of CD34 þ viable was performed using enhanced chemiluminescence reagent cells in each peak on day 3, corrected for cell division by the (Immun-Star Western C Kit, Bio-Rad) and visualised in a total number of input CD34 þ cells and multiplying by 100. ChemiDoc illuminator (Bio-Rad Laboratories) with Quantity 1 Note, although the percentage in the CFSEmaxCD34 þ peak may image capture software (Bio-Rad Laboratories, Hercules, increase with omacetaxine treatment, because of a very CA, USA).

Leukemia Omacetaxine induces stem cell apoptosis EK Allan et al 987 þ Results V/Viaprobe. Treatment of CML CD34 cells with 100 nM omacetaxine for only 6 h reduced viable cell numbers at 72 h Omacetaxine potently inhibits growth of K562, primary to 50% of NDC. However a minimum of 24-h exposure to either þ þ CML CD34 and non-CML CD34 cells in vitro 10 or 100 nM omacetaxine was required to signiﬁcantly reduce The drug concentration required to reduce cell numbers to 50% viable cell numbers to greater than 50% with respect to 15-min of the NDC (IC50) was determined by viable cell counts for drug treatment (Figure 2a; Po0.001; n ¼ 3) (as NDC had been K562, CML CD34 þ and non-CML CD34 þ cells at 24-h normalised to 100% in each experiment, there was no deviation intervals over 3 days (Figure 1). The mean IC50 values for and therefore statistics were generated with respect to the next omacetaxine-treated K562 in three independent experiments time point). Omacetaxine (100 nM) effectively induced apopto- þ were 41000, 35 and 35 nM at 24, 48 and 72 h, respectively sis in CML CD34 cells after just 24-h treatment in 58% of cells (Figure 1a). Primary CML CD34 þ cells were more sensitive to as measured by the total cells staining positive for Annexin V at omacetaxine treatment than K562 with IC50 values of 40, 4 and 72 h; however, prolonged 72-h drug treatment at this concen- 2.8 nM at 24, 48 and 72 h, respectively (n ¼ 3; Figure 1b). The tration resulted in almost all cells (90%) undergoing apoptosis omacetaxine effect appeared to be stem/progenitor cell directed (Figure 2b). CD34 þ cells from a myeloma patient (non-CML as it produced similar IC50 values of 20, 4.5 and 3.5 nM at 24, 48 control) that had been treated with 10 nM omacetaxine for 24 h and 72 h, respectively, with non-CML CD34 þ cells (n ¼ 2; reduced in viable cell count to only 17% of the NDC (Figure 2c) Figure 1c). and to a similar degree to CML CD34 þ cells, again suggesting a stem/progenitor cell-directed effect of the drug. Treatment of non-CML CD34 þ cells induced similar levels of apoptosis as for The minimum exposure to omacetaxine required to CML (Figure 2d). inhibit cell growth by 450% and induce apoptosis in CD34 þ cells is 24h CML CD34 þ cells were cultured with and without omacetaxine Omacetaxine does not accumulate cells in the in serum-free media þ 5-growth factor for various time points undivided fraction from 15 min to 72 h (Figure 2), after which cells were washed Although omacetaxine was clearly inducing apoptosis in CML free of drug and re-seeded in fresh serum-free media þ 5-growth CD34 þ cells in vitro, an effect not observed with TKIs,13,16,17 factor. Omacetaxine was used in this experiment at 10 and we next sought to determine whether omacetaxine was anti- 100 nM, being in the approximate range of the calculated IC50 proliferative against the stem/progenitor cell compartment, values (Figure 1) and clinically achievable concentrations leading to arrest of cell-cycle progression or block of cell (personal communication with ChemGenex). Cells were division in a proportion of cells without killing. This was done counted after a total of 72 h in culture for all samples and using ﬂow cytometry with CFSE staining to facilitate analysis stained with Annexin V/Viaprobe to measure apoptosis. Total of cell-division history.14 Although there was an increase in viable cells were expressed as trypan blue cell counts multiplied the percentage of cells remaining alive in the undivided fraction by the percentage cells, which were negative for Annexin (for example, 30% with 100 nM omacetaxine; Figure 3a)

K562 (n=3) 125 24h 48h 100 IC50 (nM) 24h 48h 72h 72h 75 K562 >1000 34 32 50 CML 40 4 2.8 Non-CML 20 4.5 3.5 25 Counts as % of NDC 0 0.001 0.01 0.1 1 10 Omacetaxine (µM)

CML CD34+ (n=3) Non-CML CD34+ (n=2) 125 125 24h 24h 100 48h 100 48h 72h 72h 75 75

50 50

25 25 Counts as % of NDC Counts as % of NDC 0 0 0.001 0.01 0.11 10 0.001 0.01 0.11 10 Omacetaxine (µM) Omacetaxine (µM)

þ þ Figure 1 The inhibitory effect of omacetaxine on growth of K562, CML CD34 and non-CML CD34 in vitro. The IC50 values for omacetaxine, as indicated by the horizontal dotted lines on the line graphs, were determined by counting viable cells by trypan dye exclusion in the BcrAbl blast crisis cell line K562 (a), primary CML CD34 þ cells (b), and non-CML CD34 þ cells (c) at 24-h intervals over 3 days. Results represent the mean of three separate experiments (±s.e.m.) using either the K562 cell line or CD34 þ cells from CML and non-CML patients (Ewing’s sarcoma and multiple myeloma). All counts were done in duplicate.

Leukemia Omacetaxine induces stem cell apoptosis EK Allan et al 988 with increasing omacetaxine concentration, at 24 h, there was a Omacetaxine and stem/progenitor cell functionality dose-dependant significant reduction in the total number of Although, encouragingly, it appeared that omacetaxine activity CD34 þ cells recovered with respect to the NDC (Figure 3ci, in vitro was stem/progenitor cell directed in that the drug reduced *Po0.05, ***Po0.001). The fold change in recovery of input total CD34 þ cell numbers, we were interested to know the stem/ of undivided CD34 þ cells is calculated as the product of progenitor cell function of any remaining apparently viable CD34 þ the percentage in the undivided gate (which has increased) cells following treatment. This was assessed in CFC and LTC-IC times the total CD34 þ cell number (which has decreased assays. CFC assays demonstrated that omacetaxine significantly through apoptosis). There was no statistically significant change reduced the number of CML and non-CML CFC, in a dose dependant in CML CD34 þ cells recovered in the undivided fraction with manner with respect to the NDCs (Figure 4a, ***Po0.001). We were respect to NDC with omacetaxine at any of the concentrations also able to confirm in a single sample of normal granulocyte-colony- tested (Figure 3di). Although only assayed once in normal stimulating factor mobilised donor CD34 þ cells that omacetaxine at granulocyte-colony-stimulating factor -mobilised donor cells, high concentrations (50 and 100 nM) was cytotoxic for CFC activity to more CD34 þ cells were recovered in all divisions as a a similar degree as for CML cells. percentage of the NDC in comparison with CML with 10 or CFC assays give an indication of committed progenitor cell 50 nM, but not 100 nM omacetaxine (Figure 3cii); moreover, survival, but for stem cell enumeration, LTC-IC assays are more fewer cells were recovered in the undivided fraction in a dose- appropriate. Mirroring the CFC result, omacetaxine reduced the dependant manner relative to NDC (Figure 3dii) as compared LTC-IC number in CML, non-CML and normal donor CD34 þ with CML (Figure 3di). cells after 24-h exposure (Figure 4b).

CML CD34+ (n=3) CML CD34+ (n=3)

120 100 10nM OMA 10nM OMA 100 100nM OMA 80 100nM OMA 80 60 60 40 40

20 20 Total live cells as % of NDC 0 0 % cells in late apoptosis [LR+UR] 0 0.25 2 6 24 48 72 0.25 2 6 24 48 72 Time (h) Time (h)

Non-CML (n=1) Non-CML (n=1)

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20 20 Total live cells as % of NDC 0 0 % cells in late apoptosis [LR+UR] 0 0.25 2 6 24 48 72 0.25 2 6 24 48 72 Time (h) Time (h)

Figure 2 Determination of the minimum exposure time to omacetaxine required to inhibit cell growth and induce apoptosis in CML CD34 þ þ cells. CML CD34 cells were cultured with and without omacetaxine at 10 nM (grey bars) or 100 nM (white bars) in serum-free media (SFM) þ 5- growth factor (5GF) for the time points indicated, after which cells were washed free of drug, re-seeded in fresh SFM þ 5GF and counted at 72 h. Total viable cells were determined as trypan blue cell counts multiplied by the percentage of cells that were negative for Annexin V/Viaprobe, and expressed as a percentage of the no drug control (NDC) (a and c). Percentage of cells undergoing apoptosis (sum of Annexin V þ in lower right (LR) quadrant of FACS plot plus Annexin V/Viaprobe double positive in upper right (UR) quadrant) are also displayed for CML (b) and non-CML (multiple myeloma) (d). Results in (a) and (b) are the mean of three independent experiments using three individual CML patient samples (±s.e.m.).

Figure 3 Effect of omacetaxine on dividing versus non-dividing cells. Representative histogram plots for CD34 þ cells from one CML patient (a) and one normal granulocyte-colony-stimulating factor-mobilised donor (b) showing CFSE fluorescence in cells remaining at 72 h after treatment in the first 24 h with (i) 5-growth factor (5GF) alone, (ii) 10 nM omacetaxine or (iii) 100 nM omacetaxine, and the overlay of all three plots in panel (iv). Dot plots below each histogram illustrate CFSE (X axis) with CD34 þ (Y axis) expression in the same samples. The viable cells that remain undivided (CD34 þ CFSEmax) in each treatment are indicated by the box. (c) Total recovery of CD34 þ cells in all divisions with respect to input cells for (i) CML (n ¼ 4) and (ii) normal (n ¼ 1) CD34 þ cells treated with omacetaxine for 24 h and analysed by flow cytometry at 72 h. Data are expressed as a percentage of NDC. Omacetaxine treatment significantly reduced the total CD34 þ cells recovered in a dose-dependant manner with respect to the NDC (*Po0.05, ***Po0.001). (d) The fold change in recovery of undivided (CD34 þ CFSEmax) cells after 24-h treatment with omacetaxine was calculated. The calculated percentage recovery in the NDC was normalised to 1 arbitrary unit and each drug treatment scaled accordingly to give fold change in (i) CML (presented as mean±s.e.m.) and (ii) normal CD34 þ cells treated with omacetaxine. There was no significant increase in the number of CML CD34 þ cells recovered in the undivided fraction following omacetaxine treatment of any concentration relative to input.

Leukemia Omacetaxine induces stem cell apoptosis EK Allan et al 989 Scheduled combination of omacetaxine with imatinib 24 h significantly reduced cell viability with respect to improves cell kill in Ph þ cell lines omacetaxine alone, when omacetaxine was administered before The Ph þ blast crisis cell line, KCL22, was significantly more imatinib (Po0.05; Figure 5ai). Addition of imatinib first did not sensitive to omacetaxine than to imatinib (Po0.05; Figure 5ai). achieve increased cell kill with respect to omacetaxine alone. Scheduled combination of omacetaxine (20 nM, approximate Simultaneous treatment with omacetaxine and imatinib for 72 h IC50) with imatinib (0.5 mM) with or without drug washout at was no more effective than omacetaxine alone.

0nM10nM 100nM overlay 100 100 100 100 80 80 80 80 60 60 60 40 40 40 60 Count 20 20 20 40 0 0 0 100 101 102 103 100 101 102 103 100 101 102 103 20

0 0 1 2 3 103 103 103 10 10 10 10 + 102 102 102

CD34 101 101 101 + 100 100 100 CML CD34 100 101 102 103 100 101 102 103 100 101 102 103

CFSE

0nM 10nM 100nM overlay 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 Count 20 20 20 40 0 0 0 20 100 101 102 103 100 101 102 103 100 101 102 103 0 100 101 102 103 103 103 103 +

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CD34 101 101 101 Normal CD34+ 100 100 100 100 101 102 103 100 101 102 103 100 101 102 103

CFSE

CML CD34+ (n=4) Normal CD34+ (n=1) in all in all 100

+ 100 +

75 75 *

50 50 *** *** 25 25 divisions as a % of NDC divisions as a % of NDC Total recovery of CD34 Total recovery of CD34 0 0 0 10 50 100 0 10 50 100 Omacetaxine (nM) Omacetaxine (nM)

CML CD34+ (n=4) Normal CD34+ (n=1)

2.0 2.0

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Fold change recovery in undivided 0 10 50 100 0 10 50 100 Fold change recovery in undivided Omacetaxine (nM) Omacetaxine (nM)

Leukemia Omacetaxine induces stem cell apoptosis EK Allan et al 990 CFC CML CD34+ (n=4) Non-CML CD34+ (n=2) Normal CD34+ (n=1) 100 100 100

75 75 75

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0 Colonies as % of NDC 0 10 50 100 Colonies as % of NDC 0 10 50 100 0 10 50 100 Omacetaxine (nM) Omacetaxine (nM) Omacetaxine (nM)

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0 0 0 Colonies as % of NDC Colonies as % of NDC 0 10 50 100 Colonies as % of NDC 0 10 50 100 0 10 50 100 Omacetaxine (nM) Omacetaxine (nM) Omacetaxine (nM)

Figure 4 The clonogenic and stem cell capacity of cells remaining after omacetaxine treatment. (a) CFC: CD34 þ cells were cultured ± omacetaxine in serum-free media (SFM) þ 5-growth factor (5GF) as indicated after which time cells were washed free of drug and added to semisolid culture media to assess colony forming capacity as described in the materials and methods. Colonies were counted and are expressed as a percentage of the NDC for (i) CML (n ¼ 4), (ii) non-CML (n ¼ 2: multiple myeloma and mantle cell lymphoma) and (iii) normal granulocyte- colony-stimulating factor-mobilised donor (n ¼ 1). Omacetaxine signiﬁcantly reduced the colony forming potential of CML and non-CML stem cells, in a dose- and time-dependent manner with respect to the NDC (***Po 0.001) and also reduced colony formation in normal stem cells. (b) LTC-IC: CD34 þ cells remaining after omacetaxine treatment as in (a) were cultured for 5 weeks on stroma then plated out in CFC as described in the materials and methods. Total colonies were counted for (i) CML (n ¼ 2), (ii) non-CML (n ¼ 2) and (iii) normal CD34 þ (n ¼ 1).

In order to determine any combination effect in primary Omacetaxine induces apoptosis of primitive CML CD34 CD34 þ cells, we selected the lowest concentration of cell subsets with reduction in Mcl-1 expression omacetaxine (10 nM) as this had shown lesser toxicity by CFC It was of interest to determine the activity of omacetaxine in in the normal CD34 þ sample tested previously (Figure 4a). CML more primitive subsets of CD34 þ cells. To achieve this, total þ þ CD34 cells were treated with omacetaxine or imatinib (5 mM) CML CD34 cells were sorted by flow cytometry into three cell or both in combination. When administered together, all of the fractions (mature CD34lo38 þ , CD34 þ 38 þ progenitors and the combinations tested were significantly more effective than more primitive CD34 þ 38lo population). As can be seen in treatment with either imatinib (Po0.001) or omacetaxine alone Figure 7, even the more primitive CD34 þ 38lo cells that we have (Po0.05; Figure 5bi). In contrast to KCL22, treatment of primary previously shown to be insensitive to TKI like imatinib were not CD34 þ cells with omacetaxine and imatinib for 72 h was spared by omacetaxine, with levels of apoptosis in the same equally effective whether administered simultaneously or in a order as mature and progenitor cells (Figure 7a). Apoptosis was scheduled combination (Figure 5bii and iii). By cell count, confirmed by cleavage of poly(ADP-ribose) polymerase apoptosis and colony forming potential, normal donor CD34 þ (Figure 7b) and was mediated in the same way as in bulk cells were as sensitive as CML CD34 þ cells to treatment with CD34 þ by Mcl-1 downregulation (cf Figure 6). omacetaxine alone or in combination with imatinib.

Mechanism of action of omacetaxine in Ph þ cells Discussion Increasing concentrations of omacetaxine induced apoptosis in K562 and CML stem/progenitor cells as conﬁrmed by poly(ADP- Our interests in CML leukaemic stem cell survival in the face of ribose) polymerase cleavage and loss of the anti-apoptotic complete BcrAbl kinase inhibition have led us to investigate protein, Mcl-1 (Figure 6a). K562 cells are known to have other agents that exhibit activity at the stem/progenitor cell level extremely low expression of Bcl-218 and indeed we did not and have a mechanism of action that is BcrAbl independent. We detect this protein by western blotting of lysates from this cell considered omacetaxine to be worthy of further investigation line, but it was found to be unchanged in primary CML CD34 þ with its BcrAbl-independent mechanism of action, acceptable cells with omacetaxine treatment (Figure 6b). Likewise, both the safety in humans and potential for stem cell activity.4 pro-apoptotic Bcl-2-interacting mediator of cell death protein The semisynthetic orthologue of the natural phytochemical, and the molecular chaperone, heat-shock protein 90, were homoharringtonine, omacetaxine was awarded ‘Fast Track’ unaffected by omacetaxine treatment as determined by densito- status by the Food and Drug Administration in late 2006 for metric analysis of western blots with respect to the loading those CML patients who fail imatinib or have demonstrable control (GAPDH). heat-shock protein 90 had previously been T315I BcrAbl mutation in which the drug has, rather uniquely, shown to be lost on omacetaxine treatment in Ph þ cell lines4 proven activity. A number of Phase I/II clinical trials or case together with loss of BcrAbl protein, which we did not observe reports have emerged describing disappearance of T315I-BcrAbl (Supplementary Figures 1 and 2). transcripts with omacetaxine therapy,19,20 although as might be

Leukemia Omacetaxine induces stem cell apoptosis EK Allan et al 991 KCL22 Counts Apoptosis 100 100

75 75

* 50 50

25 NS 25 *

Cell counts as % NDC * % cells AnnexinV positive 0 0

NDC NDC IM 72h IM 72h OMA 72h OMA 72h IM+OMA 72h IM+OMA 72h IM 24h OMA 48h 0 drug 24h IM 48h 0 drug 24h IM 48h 0 drug 24h OMAIM 24h 48h then OMAOMA 48h 24h then IM 48h 0 drug 24h OMA 48hIM 24h OMAW/O OMA24hOMA then 48h 24h IM W/O 48h IM 48h

IM 24h W/O thenOMA OMA 24h 48h W/O then IM 48h

CML CD34+ 100 75 100

75 75 50 50 50 ***

baseline 25 25 25 * % apoptosis above Colonies as % NDC

Cell counts as % NDC 0 0 0

IM 72h IM 72h IM 72h No drugOMA 72h OMA 72h No drugOMA 72h OMA +IM 72h OMA 24h w/o OMA +IM 72h OMA 24h w/o OMA +IM 72h OMA 24h w/o

OMA +IM 24h W/O0 24h then IM 48h OMA +IM 24h W/O0 24h then IM 48h OMA +IM 24h W/O0 24h then IM 48h OMA 24h then IM 48h OMA 24h then IM 48h OMA 24h then IM 48h OMA 24h w/o then IM 48h OMA 24h w/o then IM 48h OMA 24h w/o then IM 48h

Normal CD34+

100 75 100

75 75 50 50 50

baseline 25 25 25 % apoptosis above Colonies as % NDC Cell counts as % NDC 0 0 0

IM 72h IM 72h IM 72h No drug No drug OMA 72h OMA 72h OMA 72h OMA +IM 72h OMA 24h w/o OMA +IM 72h OMA 24h w/o OMA +IM 72h OMA 24h w/o OMA +IM 24 W/O OMA +IM 24h W/O0 24h then IM 48h OMA +IM 24h W/O0 24h then IM 48h 0 24h then IM 48h OMA 24h then IM 48h OMA 24h then IM 48h OMA 24h then IM 48h OMA 24h w/o then IM 48h OMA 24h w/o then IM 48h OMA 24h w/o then IM 48h

Figure 5 Effect of scheduled combination of omacetaxine with imatinib in Ph þ cell lines and primary CML CD34 þ cells (a) The number of viable KCL22 cells remaining after culture for a total of 72 h with omacetaxine (20 nM)±imatinib (0.5 mM) was determined by trypan blue staining. (i) KCL22 cells were significantly more sensitive to treatment with omacetaxine than to imatinib (*Po0.05). All combinations were better than imatinib alone. When omacetaxine was administered before imatinib, cell number was significantly reduced with respect to treatment with omacetaxine alone (*Po0.05). (ii) The percentage of cells undergoing apoptosis is also displayed for the various treatment arms. Results are the þ mean of three independent experiments (±s.e.m.). (b) CML CD34 cells were treated with omacetaxine (10 nM)±imatinib (5 mM)orin combination as illustrated. (i) All of the combinations tested were significantly more toxic than treatment with either imatinib (***Po0.001) or omacetaxine alone (*Po0.05). (ii) Treated cells were stained with Annexin V to measure apoptosis or (iii) washed free of drug and added to semisolid culture media for measurement of colony formation as described in the materials and methods. Colonies are expressed as a percentage of the NDC. Omacetaxine þ imatinib was equally effective whether administered simultaneously or with omacetaxine introduced 24 h before imatinib for 48 h. Results are the mean of three independent experiments using three individual CML patient samples (±SEM) except for the CFC assay where n ¼ 2. (c) Normal donor CD34 þ cells (n ¼ 1) were cultured and treated with omacetaxine and/or imatinib as for CML CD34 þ cells. (i) Cell counts (ii) apoptosis and (iii) colony forming potential of cells remaining after treatment showed that normal CD34 þ cells were as sensitive to treatment with omacetaxine alone or in combination with imatinib as CML CD34 þ for all of the treatment arms tested. expected, cytogenetic responses were poorer in comparison survival and therefore sensitive to the activity of omacetaxine.9 with the efficacy of TKI in newly diagnosed patients.20,21 We confirmed the downregulation of Mcl-1 in K562 cells and Omacetaxine is targeted to the A-site cleft of eukaryotic moreover primary CML CD34 þ cells in response to omacetaxine, ribosomes.7 Following exposure to this translation elongation which precipitated caspase-dependant apoptosis as demon- inhibitor, transient inhibition of protein synthesis of short-lived strated by poly(ADP-ribose) polymerase cleavage. Furthermore, proteins, such as Mcl-1, leads to apoptosis. Leukaemia cells, and omacetaxine induced apoptosis in primitive CD34 þ subsets specifically CML cells, may be more Mcl-1 dependant for (CD34 þ 38lo), an effect not observed with TKI. In addition,

Leukemia Omacetaxine induces stem cell apoptosis EK Allan et al 992 01050 100 200 01050 100 200 Omacetaxine (nM) PARP Cleaved PARP Mcl-1

Bcl-2

GAPDH K562 CML CD34+

CML 1 CML 2 0 10 50 100 200 0 10 50 100 0 10 50 100 Omacetaxine (nM ) HSP90

Mcl-1

BcrAbl

Bim

GAPDH K562 CML CD34+

Figure 6 Apoptotic protein expression after 24-h omacetaxine treatment. (a) Exposure to omacetaxine for 24 h induced apoptotic signalling in (i) K562 cells and (ii) CML CD34 þ cells, as shown by cleavage of poly(ADP-ribose) polymerase (PARP) and reduction in Mcl-1 protein levels in a dose-dependant manner. Levels of anti-apoptotic protein Bcl-2 remained unchanged following omacetaxine treatment of CML stem cells but were absent in the CML cell line K562. Expression levels were normalised against GAPDH loading control by densitometric analysis. (b) 24-h treatment with omacetaxine did not change expression of heat-shock protein 90, BcrAbl or Bcl-2-interacting mediator of cell death (Bim) proteins in (i) K562 or (ii) two CML CD34 þ samples.

Apoptosis Sorted viable cell counts

lo + 100 60 CD34 38 CD34lo38+ CD34+38+ CD34+38+ 50 75 CD34+38lo CD34+38lo 40 30 50 20 25 10

0 Viable cells as % of NDC 0 % cells annexin V/VP positive 0 OMA 10nM OMA 100nM OMA 0 OMA 10nM OMA 100nM OMA

10nM Omacetaxine (h) 10nM Omacetaxine (h) 024240 2424 PARP Cleaved PARP Mcl-1

GAPDH CD34+38+ CD34+38lo Figure 7 Apoptotic protein expression in primary CML CD34 þ subpopulations after omacetaxine treatment. (a) (i) Measurement of apoptosis by Annexin V/Viaprobe staining in sorted cell fractions (CD34lo38 þ (white bars), CD34 þ 38 þ (grey bars) and more primitive CD34 þ 38lo (black bars)) after 24-h omacetaxine uptake expressed as the total percentage of Annexin V þ cells. (a) (ii) Viable cells remaining after 24-h omacetaxine treatment of sorted CML cell fractions were calculated as the product of the percentage Annexin V þ cells times the live (trypan blue negative) count and expressed as a percentage of the NDC. (b) Analysis of protein lysates from sorted (i) CD34 þ 38 þ and more primitive (ii) CD34 þ 38lo CML cell populations treated with omacetaxine at indicated time points demonstrated poly(ADP-ribose) polymerase (PARP) cleavage and reduced expression of Mcl-1 protein in both cell populations after 24 h. GAPDH was used a loading control for all gels ((i) Mcl1:GAPDH ratio by densitometry was 0.881 and 0.248 for 0 and 24 h, respectively; (ii) Mcl1:GAPDH was 0.528 and 0.272 at 0 and 24 h, respectively).

primary CML cells were found to be more sensitive to significant result that needs verification in primary material omacetaxine than K562 blast crisis cells, with a significantly derived from CML patients as cellular context of BcrAbl lower IC50 in the former. expression (de novo expression in human stem cells versus Despite the evidence of efficacy from clinical trials and the retroviral transduction of bone marrow cells from 5-fluorouracil- known molecular mode of action of omacetaxine, it was only treated donor mice) may be significant, given the survival reported very recently4 that the drug could eliminate 490% of mechanisms that may be invoked by stem cells programmed to leukaemia stem cells in murine models of BcrAbl þ CML and stay alive. B-acute lymphoblastic leukemia, whereas TKI (imatinib or In an in vitro drug combination study in CML cell lines,22 it dasatinib) achieved less than 25% kill. Interestingly, the kill had been suggested that cytosine arabinoside or omacetaxine was seen for both wild-type and T315I mutant BcrAbl. This is a might act synergistically with imatinib in the case of resistance

Leukemia Omacetaxine induces stem cell apoptosis EK Allan et al 993 to imatinib. However, we16 demonstrated at the stem cell level its clinical activity and allow omacetaxine to be considered in vitro that cytosine arabinoside was anti-proliferative in as an option in the management of minimal residual disease combination with imatinib, resulting in accumulation of stem in CML. cells. In our current study, we did not assay omacetaxine in combination in primary CML CD34 þ , but uniquely demonstrated apoptosis in the primitive (CD34 þ 38lo) leukaemic cell Conflict of interest subpopulation. Thus, the prevention of colony formation by omacetaxine observed by Tipping et al. is most likely the result The authors declare that E Allan, Dr Jørgensen and of CFC loss through death, as opposed to cell-cycle arrest. Of Prof Holyoake received research support from ChemGenex note, if omacetaxine was to be combined with imatinib then Pharmaceuticals; Dr A Craig, an employee of ChemGenex there is evidence to suggest that the sequence of exposure would Pharmaceuticals, originated and participated in the concept be critical. In one previous study, antagonism was seen with the discussion that led to the experimental work. simultaneous combination23 in clonogenic assays. However this could be reverted to a cooperative effect, if omacetaxine was Acknowledgements pulsed for 24 h before imatinib. Indeed, we have shown in a 24 relatively less imatinib-sensitive cell line (KCL22) to corrobo- HGJ was supported by William Thyne Centenary Fellowship, rate this latter finding, suggesting that when introduced into the Dr Rhona Reid Charitable Trust, and Louis and Marion Ferrar culture before imatinib, omacetaxine is indeed more lethal than Trust. We are grateful to Kara Melchizedek for her contribution to the reverse sequence. As omacetaxine is likely to block cell-line data through her summer vacation project sponsored by accumulation of Bcl-2-interacting mediator of cell death, which the McGuigan family and the Pathological Society. is critical to imatinib’s induction of apoptosis,25 this may explain the antagonism seen with simultaneous addition of the two drugs.23 However, the loss of the Bcl-2-interacting mediator of References cell death-negative regulator, Mcl-1, on omacetaxine treatment may sensitise the cells to the combined effect of both drugs. 1 Kantarjian HM, Talpaz M, Santini V, Murgo A, Cheson B, Both simultaneous and scheduled permutations of the combina- O’Brien SM. Homoharringtonine: history, current research, and future direction. Cancer 2001; 92: 1591–1605. tion showed parity in toxicity between CML and normal donor þ 2 de Lavallade H, Khorashad JS, Davis HP, Milojkovic D, Kaeda JS, CD34 cells (Figure 5) at the concentrations selected to be Goldman JM et al. Interferon-alpha or homoharringtonine tested. Thus, our results support the BcrAbl-independent stem as salvage treatment for chronic myeloid leukemia patients cell-directed effect of omacetaxine, but predict toxicity in vivo who acquire the T315I BCR-ABL mutation. Blood 2007; 110: owing to the non-selective killing of both normal and leukaemic 2779–2780. cells, which is unlikely to be abrogated through scheduled 3 Quintas-Cardama A, Cortes J. Homoharringtonine for the treatment of chronic myelogenous leukemia. Expert Opin Pharmacother dosing regimens. 2008; 9: 1029–1037. Combinations aside, it is clear from this study that omacetaxine 4 Chen Y, Hu Y, Michaels S, Segal D, Brown D, Li S. Inhibitory has activity at the primary CML leukaemia stem/progenitor effects of omacetaxine on leukemic stem cells and BCR-ABL- cell level in vitro. Samples were consecutive newly diagnosed induced chronic myeloid leukemia and acute lymphoblastic cases of CML in chronic phase, in which TKI-resistant mutations leukemia in mice. Leukemia 2009; 23: 1446–1454. are extremely rare and never dominant in the stem cell 5 Quintas-Cardama A, Kantarjian H, Cortes J. Homoharringtonine, omacetaxine mepesuccinate, and chronic myeloid leukemia circa compartment before evolution under TKI-selection pressure. 2009. Cancer 2009; 115: 5382–5393. This may mean that omacetaxine has a potential role in 6 Powell RG, Weisleder D, Smith Jr CR. Antitumor alkaloids for eradicating minimal residual disease achieved in response to Cephalataxus harringtonia: structure and activity. J Pharm Sci TKI, but maintained by persistent, as opposed to overtly resistant 1972; 61: 1227–1230. leukaemia stem cells. The shortcoming for this agent is toxicity 7 Gurel G, Blaha G, Moore PB, Steitz TA. U2504 determines the to normal stem cells, which could be predicted from on-going species specificity of the A-site cleft antibiotics: the structures of tiamulin, homoharringtonine, and bruceantin bound to the clinical trials in which myelosuppression is a significant ribosome. J Mol Biol 2009; 389: 146–156. À issue. Indeed, in vitro toxicity of omacetaxine to Ph HSC is 8 Tang R, Faussat AM, Majdak P, Marzac C, Dubrulle S, reported here. The paucity of results in normal donor stem Marjanovic Z et al. Semisynthetic homoharringtonine induces cells is in keeping with the ethical concerns surrounding apoptosis via inhibition of protein synthesis and triggers rapid prolonging stem cell collections for the purposes of research myeloid cell leukemia-1 down-regulation in myeloid leukemia from healthy individuals over and above the required clinical cells. Mol Cancer Ther 2006; 5: 723–731. 9 Aichberger KJ, Mayerhofer M, Krauth MT, Skvara H, Florian S, dose, hence the decision to assay more readily available Sonneck K et al. Identification of mcl-1 as a BCR/ABL-dependent À þ Ph non-CML CD34 cells from diseases in which the HSC is target in chronic myeloid leukemia (CML): evidence for coopera- not implicated. tive antileukemic effects of imatinib and mcl-1 antisense oligonu- A number of reports have been made recently demonstrating cleotides. Blood 2005; 105: 3303–3311. stem cell activity of novel agents for use in CML (for example, 10 Akgul C. Mcl-1 is a potential therapeutic target in multiple types of farnesyl transferase inhibitors (for example, BMS-214662),15 cancer. Cell Mol Life Sci 2009; 66: 1326–1336. 26 11 Chu S, Xu H, Shah NP, Snyder DS, Forman SJ, Sawyers CL et al. histone deacetylase inhibitors (for example, LBH589), auto- Detection of BCR-ABL kinase mutations in CD34+ cells from 27 phagy inhibitors (for example, chloroquine), proteasome chronic myelogenous leukemia patients in complete cytogenetic inhibitors (for example, bortezomib28). Clearly, there is con- remission on imatinib mesylate treatment. Blood 2005; 105: siderable interest in the field to uncover a suitable partner to 2093–2098. kinase inhibition to think in terms of cure for CML. Omacetaxine, 12 Sharma SV, Gajowniczek P, Way IP, Lee DY, Jiang J, Yuza Y et al. however, is the most advanced in its clinical development. A common signaling cascade may underlie ‘addiction’ to the Src, BCR-ABL, and EGF receptor oncogenes. 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