research paper

The investigational agent MLN2238 induces apoptosis and is cytotoxic to CLL cells in vitro, as a single agent and in combination with other drugs

Aneel Paulus,1,2* Aisha Masood,3,4* Kena C. Miller,2 A. N. M. Nazmul H. Khan,3,5 Drusilla Akhtar,3 Pooja Advani,2 James Foran,2 Candido Rivera,2 Vivek Roy,2 Gerardo Colon-Otero,2 Kasyapa Chitta1 and Asher Chanan-Khan2 1

Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, 2Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, 3

Department of Medicine, Roswell Park Cancer

Institute, Buffalo, NY, 4Department of Internal Medicine, State University of New York at Stony Brook University, Stony Brook, NY, and 5

Department of Infectious Diseases, Roswell Park

Cancer Institute, Buffalo, NY, USA Received 17 July 2013; accepted for publication 13 October 2013 Correspondence: Dr Asher Chanan-Khan, Division of Hematology & Oncology, Mayo Clinic Cancer Center, 4500 San Pablo Road, Jacksonville, FL 32224, USA. E-mail: [email protected] *Contributed equally to the study.

Summary Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in the U.S. The course of the disease has been shown to be negatively impacted by increased levels of BCL2. Strategies to downregulate BCL2 and shift the balance towards cellular demise are actively being explored. Therefore, we examined whether the investigational agent MLN2238 could inhibit the proteasomal machinery and induce CLL cell death while also downregulating BCL2. MLN2238-induced cell death was studied in peripheral blood mononuclear cells from 28 CLL patients. MLN2238 produced a dose-dependent reduction in BCL2 and CLL cell viability with maximum cell death observed at a 50 nmol/l concentration by 48 h. Annexin-V staining, PARP1 and caspase-3 cleavage along with an increase in mitochondrial membrane permeability were noted after cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan-caspase inhibitor z-VAD.fmk. Furthermore, we observed enhanced anti-CLL effects in tumour cells treated with either a combination of MLN2238 and the BH3 mimetic AT-101 or MLN2238 and fludarabine. Together, our data suggest the potential for proteasome inhibitor based therapy in CLL and the rationale design of drug combination strategies based on CLL biology. Keywords: MLN2238, MLN9708, Ixazomib, CLL, AT-101.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western hemisphere.(Dores et al, 2007) CLL manifests as a clinically heterogeneous cancer as some patients never require therapy, while those with prominent genetic defects respond poorly to standard chemoimmunotherapeutic agents and often develop relapsed/refractory disease (Rai et al, 1975; Hallek & Pflug, 2010; Wierda et al, 2010; Advani et al, 2011). In order to improve disease outcome, the accurate identification and rational targeting of pro- and anti-apoptotic signalling pathways is crucial, remaining a priority of translational research in CLL. Defects in apoptosis are known to result in aggressive disease behaviour and confer a poor prognosis in most cancers, including CLL (Reed et al, 2002). Importantly, a shift between the pro-apoptotic and anti-apoptotic BCL2 family of proteins is associated with a dysfunctional programmed CLL cell death response (Schena et al, 1993; Thomas et al, 1996; Reed, 1997). The underlying mechanisms that influence the expression of BCL2 proteins are complex and remain an investigational First published online 27 January 2014 doi: 10.1111/bjh.12731

priority for therapeutic exploit. The ubiquitin-proteasome pathway is an integral system involved in governing BCL2 family members. This pathway also controls the degradation of various intracellular proteins (Fennell et al, 2008). Its central component, the proteasome, is responsible for the recycling and destruction of transcription factors, such as TP53 (p53) and NFKB1 (nuclear factor-jB, NF-jB), and cyclin-dependent kinase inhibitors (Adams, 2003). The ubiquitin-proteasome pathway also plays an important role in the regulation and stabilization of pro-apoptotic molecules (particularly the BH3 domain proteins BIK, PMAIP1 [NOXA] and BCL2L11 [BIM]) and as such, links the apoptotic machinery to the protein disposal systems of the proteasome. Inhibition of this pathway via proteasome inhibitors (PIs) has been shown to cause accumulation of TP53, p27 and proapoptotic BCL2 proteins, which results in activation of the mitochondria- mediated cell death pathway (Fennell et al, 2008). Thus, identification and correction of defects that affect apoptosis may offer a therapeutic opportunity to reset and ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

MLN2238 Induces Apoptosis and is Cytotoxic to CLL Cells engage cell death pathways in CLL. Bortezomib (VELCADE, Millennium Pharmaceuticals, Inc., The Takeda Oncology Company,Cambridge, MA, USA) was the first in class PI and was the first PI to be approved for the treatment of multiple myeloma (MM) and relapsed mantle cell lymphoma. As investigated in CLL cells in a preclinical setting, cytotoxic activity of bortezomib is associated with alteration of mitochondrial outer membrane permeability (MOMP) and caspase activation along with up regulation of PMAIP1 and BBC3 [PUMA] in vitro (Pahler et al, 2003; Pei et al, 2003). However, this biological activity did not wholly translate into an observable clinical benefit when the efficacy of single agent bortezomib was examined in fludarabine-refractory CLL patients. This was speculated to be a result of the single agent use of bortezomib in a heavily pretreated patient population (median lines of prior therapy = four, range 2–11)(Faderl et al, 2006). Moreover, the presence of flavonoids, such as quercetin and myricetin, in the plasma have been attributed to the prevention of CLL cell death and are linked to the chemical reaction between quercetin and the boronic acid group in bortezomib (Liu et al, 2008; Wickremasinghe, 2008). Thus, PIs that do not contain a boron moiety and the partnership of these compounds with additional drugs whose activity complements theirs may be necessary to achieve clinical remission in heavily pretreated CLL patients. As such, we sought to determine the anti-leukaemic effects of the investigational PI MLN2238 (ixazomib), which has different pharmacokinetic and structural properties to bortezomib. The investigational drug MLN9708 (ixazomib citrate), which is an orally available small molecule PI, converts into the biologically active form, MLN2238, upon hydrolysis. Compared with bortezomib in preclinical models, MLN2238 demonstrates a faster dissociation rate from the proteasome and improved pharmacokinetic, pharmacodynamic and antitumour properties in preclinical models (Kupperman et al, 2010; Chauhan et al, 2011). Pre-clinical data suggest a synergistic effect when PIs are coupled with BCL2 inhibitors (Pei et al, 2003; Perez-Galan et al, 2007), prompting us to explore this combination with MLN2238. In the current study, we examined the anti-CLL effect of MLN2238. We aimed to identify the best combination-based therapeutic strategy in vitro using CLL patient cells. We found that treatment with MLN2238 leads to induction of apoptosis in primary CLL cells and, when combined with the pan-BCL2 inhibitor AT-101, results in increased tumour kill. Similarly, the combination of MLN2238 plus conventional cytotoxic agents (fludarabine or dexamethasone) also resulted in increased CLL cell death as compared to when each agent was used alone.

provided written informed consent to participate in the study. This study and the consent form were approved by the Roswell Park Cancer Institute institutional review board in accordance with the Declaration of Helsinki. Only patients with a high total white blood cell count in the peripheral blood that also had more than a 90% CD19+ B cell population (hereafter referred to as CLL cells) were included in this study.

Cell isolation, culture and drug treatment Heparinized peripheral blood was obtained from patients (n = 29) with CLL. PBMCs were separated on a Ficoll gradient, washed twice in phosphate-buffered saline (PBS), and resuspended in culture medium (RPMI-1640 containing 10% fetal bovine serum [FBS] and 1% penicillin-streptomycin). Cell viability was determined using a Vi-Cell-XR cell viability ysanalyser (Beckman Coulter, Brea, CA, USA). Experiments were done with cell concentrations of 5 9 106/ml. MLN2238 was from Millennium Pharmaceuticals Inc. (Cambridge, MA, USA), AT-101 was provided as a gift from Ascenta Therapeutics Inc. (Malvern, PA, USA), fludarabine and lenalidomide were purchased from Sellekhem (Houston, TX, USA), and dexamethasone was purchased from Sigma–Aldrich (St. Louis, MO, USA).

Proteasomal activity assay Proteasomal activity was determined by using synthetic fluorogenic peptide substrates. Briefly, the cells were washed twice with cold PBS and the total cell extracts were made in lysis buffer containing 25 mmol/l HEPES, pH 7
5, 500 lmol/l EDTA, 0
05% Nonidet P-40, and 0
001% sodium dodecyl sulfate (SDS) (w/v) without protease inhibitors, to a final concentration of 4 9 106 cells/ml. The reaction mixture containing 10 ll of the lysate (40 000 cells) was incubated at 37°C for 30 min with the PI, followed by the addition of 50 lmol/l fluorogenic peptides (Suc-Leu-Leu-Val-Tyr-AMC (LLVY; for chymotrypsin-like activity), Ac- Leu-Arg-ArgAMC (LRR; for tryspin-like activity), or Z-Leu-Leu-GluAMC (LLE; caspase-like activity) and further incubated for 60 min at 37°C. Release of the fluorometric reporter, aminomethylcoumarin (AMC) as a result of the activity of the respective enzymes was quantified in a BioTek FL800 plate reader using 360 nmol/l excitation and 460 nmol/l emission wavelengths. Enzymatic activity represents the mean fluorescence values of triplicate independent assays.

Apoptosis assay Materials and methods Patients Chronic lymphocytic leukaemia cells were obtained from patients with a confirmed diagnosis of CLL. All patients ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

Apoptosis was measured by the annexin V binding assay kit from Pharmingen (San Diego, CA, USA) according to manufacturer’s instructions. Briefly, cells were washed with PBS and 1 9 106 cells were resuspended in 100 ll of 1 9 binding buffer. Fluorescein isothiocyanate (FITC)-labelled Annexin 79

A. Paulus et al V (5 ll) and propidium iodide (10 ll) were added to each sample and incubated in the dark for 15 min at room temperature. Subsequently, cells were analysed by flow cytometry. Data from 10 000 events per sample were collected and processed using CELL QUEST software (Becton Dickinson, Franklin Lakes, NJ, USA).

Determination of Mitochondrial Outer Membrane Permeability (MOMP) Chronic lymphocytic leukaemia cells treated with MLN2238 were tested for MOMP using tetramethylrhodamine methyl ester [TMRM] (Invitrogen, Carlsbad, CA, USA). The cells were washed twice with PBS, incubated in PBS containing 20 nmol/l TMRM for 15 min and analysed for fluorescence on a FACScaliber flow cytometer (FL2). Data from at least 10 000 events per sample were collected and analysed using the CELL QUEST software (Becton Dickinson). TMRM-negative (%) cells were calculated to determine (%) MOMP.

cells. Proteasomal activity of CLL cells was determined as described in materials and methods using fluorogenic peptide substrates. Variable basal activity was detected in CLL cells from all patients investigated for the three major catalytic components of the proteasome that confer chymotrypsinlike, trypsin-like and caspase-like enzymes. Chymotrypsinlike activity from eight representative patient samples is shown in Fig 1A. The effect of MLN2238 on proteasomal

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Immunoblot analysis Total protein extracts were made using radioimmunoprecipitation assay (RIPA) lysis buffer (50 mmol/l Tris containing 150 mmol/l NaCl, 0
1% SDS, 1% TritonX-100, 1% sodium deoxycholate, pH 7
2) with 0
2% protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA) on ice for 40 min, vortexing for 5 s every 10 min. Following centrifugation at 18 400 g for 20 min, the supernatant was collected and used for Western blot analyses. Protein content in the extracts was measured by the Bradford method using BioRad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Aliquots of total protein (30 lg) and 25 lg of nuclear/cytoplasmic protein were boiled in Laemmli sample buffer and subjected to 10% SDS-polyacrylamide gel electophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked for 1 h in Tris-buffered saline/Tween 20 [TTBS] containing 1% nonfat dried milk and 1% BSA. Incubation with primary antibodies was done overnight at 4°C, followed by washing three times with TTBS and incubation for 1 h with horseradish peroxidase-conjugated secondary antibody. The blots were developed using chemiluminescence (Thermo Scientific, Waltham, MA, USA).

Results MLN2238 inhibits proteasomal activity in CLL cells MLN2238 is the biologically active form of the investigational proteasome inhibitor MLN9708. It has a shorter proteasome dissociation half-life and improved pharmacokinetics and pharmacodynamics compared to bortezomib in preclinical models (Kupperman et al, 2010). We conducted in vitro studies to understand the effect of MLN2238 in CLL 80

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Fig 1. Chymotrypsin-like activity in the proteasomes of B-CLL cells is inhibited by MLN2238 in vitro: Chymotrypsin-like, caspase-like and tryspin-like activities (using their respective fluorogenic peptides as described in Materials and methods) were measured in primary B-CLL cells at 4 9 104 cells/reaction in triplicates. (A) Chymotrypsin-like activity from eight representative patients samples shows variation in baseline activity. (B) Chymotrypsin-like activity in the cells was most significantly inhibited (P < 0
005) by 10 nmol/l MLN2238 in all samples tested (n = 28). Data from four patients is presented. (C) Western blot analysis of protein extracts from Chronic lymphocytic leukaemia (CLL) cells treated with 10 nmol/l MLN2238 for 24 h for the presence of PSMB5 showed that PSMB5 protein levels are not altered in in presence of MLN2238. b-actin was used as control for equal protein loading. ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

MLN2238 Induces Apoptosis and is Cytotoxic to CLL Cells enzyme activity was measured by pre-incubating the cell extracts with 10 nmol/l of MLN2238 for 30 min followed by incubation with specific fluorogenic substrate for another 60 min. MLN2238 inhibited the chymotrypsin-like activity by more than 90% (P < 0
005) (Fig 1B). A moderate to minimal inhibitory effect on caspase-like and trypsin-like activities, respectively, was also noted (data not shown). As chymotrypsin-like activity is mediated by the b5 subunit of the proteasome (PSMB5 gene), the effect of MLN2238 on PSMB5 protein levels was evaluated by Western blot analysis. PSMB5, a 23 kDa protein, was detectable in CLL cells showing measurable chymotrypsin-like activity (data from three representative patients is presented in Fig 1C) and was not detectable in patients with low levels of enzyme activity (one sample from Fig 1C), which suggests a low threshold expression in these cells. Further, treatment of CLL cells for 24 h with 25 nmol/l MLN2238 did not inhibit PSMB5 protein levels, which suggests that MLN2238 inhibits the catalytic activity of the proteasome without changing PSMB5 protein levels. Collectively, these results indicate that MLN2238 potently inhibits chymotrypsin-like proteasomal activity in CLL cells without affecting protein expression levels of PSMB5.

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Exposure to MLN2238 results in the loss of cell viability and induces apoptosis in CLL cells Sustained proteasomal activity is indispensable for cellular protein homeostasis, viable maintenance and growth. Thus, we hypothesized that CLL cells are dependent on proteasomal activity for their survival and that MLN2238 treatment would induce death in CLL cells. CLL cells from 18 representative patients were treated with different concentrations of MLN2238 for 24 h and cell viability was determined at 48 h by the trypan blue exclusion assay. Cells showed a concentration-dependent decrease in viability with 42% cell death noted at a 50 nmol/l concentration of MLN2238 (Fig 2A). Next, we sought to delineate the mechanism of death that occurred in response to MLN2238 exposure via staining with annexin V and propidium iodide. MLN2238-treated CLL cells underwent apoptosis in a dose-dependent manner with a maximal effect at a 50 nmol/l concentration (Fig 2B). Cell death was observed in a median of 43% of cells at 25 nmol/l (range 10–54%) and 60% cells at 50 nmol/l (range, 25–73%). Apoptotic effects of MLN2238 were not associated with clinical stage of the disease or number of previous treatments that the patients had been exposed to. Additionally, status of ZAP70, immunoglobulin expression, IGHV mutation status, TP53 expression, ATM mutation or chromosomal trisomy did not alter the extent of apoptosis induced by MLN2238 (Table I). Induction of apoptosis was confirmed by PARP cleavage, which occurred in a dosedependent manner (Fig 2C). The results indicated that MLN2238 induces a dose-dependent induction of apoptosis in CLL cells. ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

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Fig 2. Loss of Chronic lymphocytic leukaemia (CLL) cell viability and induction of apoptosis in primary CLL cells by MLN2238 in vitro: (A) B-CLL cells from 18 representative patients were treated with 12
5, 25, 50 nmol/l concentrations of MLN2238 for 24 h and cell viability was determined at 48 h. Cells showed a concentrationdependent decrease in viability with 58% of cells remaining viable at a concentration of 50 nmol/l. (B) Apoptosis was assessed by FITC labelled annexin V (5 ll) and propidium iodide. Subsequently, samples were analysed by flow cytometry. Maximum apoptosis occurred at 50 nmol/l of MLN2238 at 24 h. (C) B-CLL cells from Patient 4 were incubated with 0, 25, 50 and 100 nmol/l of MLN2238 for 24 h and induction of PARP1 cleavage was measured by Western blot. ACTB (b-actin) was used as a control.

MLN2238 activates caspases 3 and 9 in CLL cells; however, apoptosis is caspase independent To determine if MLN2238-induced apoptosis in CLL is caspase-mediated, CLL cells were treated with increasing concentrations of MLN2238 for 24 h and the protein extracts were probed for caspases 9, 8 and 3. While untreated cells showed only the full length forms of caspase 9 and 3 at 45 kDa and 35 kDa respectively, treatment with MLN2238 81

A. Paulus et al Table I. Clinical characteristics of patients whose cells were used in study (n = 28). Pt.

Age (years)

Stage

ALC (9 10 (/l)

Prior therapies (n)

IGHV status

b2M

17p

11q

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

41 84 63 52 40 63 63 70 65 56 54 82 75 66 64 59 69 67 72 66 60 62 66 65 87 81 66 61 68

NA IV NA II NA 0 NA IV I II I NA IV I IV IV IV IV I IV NA IV II IV 0 IV I I II

48
63 NA 20
62 71
37 20
67 36
8 20
62 10
35 NA NA 16
24 94
64 1
93 44
29 0
53 3
73 NA NA 0
93 NA NA 40
58 NA NA 34
95 3
81 2
28 51
29 0
55

0 2 2 0 0 0 2 3 NA 1 1 4 1 1 1 8 4 5 1 4 NA 0 1 1 0 2 1 0 4

NA NA NA NA Mutated Mutated NA NA NA NA NA NA NA NA Mutated NA NA NA NA NA Mutated Mutated NA NA Unmutated NA Unmutated NA Unmutated

1
61 1
93 3
32 3
75 2
91 1
71 3
32 4
71 NA 3
93 1
8 6
53 8
43 3
24 5
42 3
48 5
79 2
45 2
16 3
48 2
01 4
97 6
09 2
93 7
84 5
05 3
49 1
39 4
52

NA Normal Normal Normal Normal Normal Normal Normal NA Normal Normal Deletion Normal Normal NA Normal Deletion Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal

Normal Normal Normal Deletion Normal Normal Normal NA Normal Normal Deletion Deletion Normal NA Deletion Deletion Normal Deletion Normal Deletion Normal Normal Deletion Normal Normal Deletion Normal Normal Normal

ALC, absolute lymphocyte count; NA, not available; IGHV, immunoglobulin heavy chain mutation status; b2M, beta 2 microglobulin; Pt., patient; 17p, chromosome 17p; 11q, chromosome 11q.

resulted in an increased appearance of small cleaved proteins in a dose-dependent manner (at 35 kDa for caspase 9 and 19 kDa for caspase 3) indicating activation of these two caspases and the intrinsic apoptotic pathway (Fig 3A). Activation of caspase 8 was not observed in CLL cells treated with MLN2238 [data not shown]. To evaluate the dependence of MLN2238-mediated apoptosis on the activation of caspases, CLL cells were pre-treated for 1 h with 25 lmol/l z-VAD.fmk, a pan caspase inhibitor, followed by 25 nmol/l MLN2238 for 24 h and the extent of apoptosis was ascertained. Treatment with z-VAD.fmk alone did not induce the activation of caspase-3, which was similar to untreated cells. While MLN2238 induced the activation of caspase-3 in these cells, pre-treatment with z-VAD.fmk blocked this activation, which suggests the inhibition of caspase activation in these cells in presence of z-VAD.fmk (Fig 3B). However, cell death by MLN2238, as determined by annexin V staining, was not abrogated by pre-treatment with z-VAD.fmk (Fig 3C). This observation suggests that MLN2238-mediated apoptosis in CLL cells can occur independently of caspase activation, despite their induction in the presence of the drug. 82

MLN2238 induces MOMP in CLL cells Activation of caspases 9 and 3 in CLL cells treated with MLN2238 indicated a possible involvement of the mitochondrial-mediated intrinsic apoptosis pathway. To test our hypothesis, we investigated MOMP in CLL cells treated with MLN2238. CLL cells were incubated alone or with increasing concentrations of MLN2238 for 24 h. MOMP was measured using TMRM by flow cytometry as described in the materials and methods section. Untreated cells that tested positive for TMRM fluorescence became progressively negative for TMRM with increasing concentrations of MLN2238, which suggests that that MLN2238 treatment increased MOMP in a dosedependent manner. All the samples used in the study showed an increase in MOMP. Data from eight representative patients is presented in Fig 3D.

BCL2 expression is decreased in MLN2238 treated CLL cells One of the possible mechanisms of increase in MOMP in CLL cells treated with MLN2238 could be due to a shift in ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

MLN2238 Induces Apoptosis and is Cytotoxic to CLL Cells

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Fig 3. MLN2238 activates the intrinsic apoptotic pathway in B-CLL cells: (A) B-CLL cells were treated with 25, 50 and 100 nmol/l concentrations of MLN2238 for 24 h with subsequent gauging of caspase cleavage by Western blot. Cleavage products of caspases 9 and 3 occured starting at 25 nmol/l in treated cells. (B) Extent of caspase 3 involvement in mediating CLL cell death was determined by pre-treated cells for 1 h with 25 lmol/l z-VAD.fmk, a pan caspase inhibitor, followed by MLN2238 (25 nmol/l) for 24 h. Treatment with z-VAD.fmk alone did not induce activation of caspase-3. While MLN2238 induced cleavage of caspase-3 in these cells, pre-treatment with z-VAD.fmk blocked the cleavage. (C) However, cell death by MLN2238, as determined by annexin V staining, was not abrogated by pre-treatment (2 h) with z-VAD.fmk, which suggests that caspase-independent mediated cell death mechanisms are activated by MLN2238 in spite of caspase induction. (D) Cells were treated with 25 and 50 nmol/l of MLN2238 for 24 h and mitochondrial outer membrane permeability (MOMP) activation was analysed using tetramethylrhodamine methyl ester. Maximum loss of MOMP occurred at a 50 nmol/l concentration of MLN2238. Data from eight representative patient samples is shown.

the balance between pro-and anti-apoptotic proteins of the BCL2 family. To evaluate this hypothesis, CLL cells were treated with increasing concentrations of MLN2238 for 24 h and the protein extracts were analysed for the presence of BCL2 by Western blot analysis. MLN2238-treated cells exhibited reduced expression of pro-survival BCL2 members as compared to untreated CLL cells (data from two representative patients, Fig 4A). Inhibition of BCL2 was observed as early as 12 h after treatment with MLN2238, which suggests that altered BCL2 expression is one of the early events associated with MLN2238-mediated apoptosis of CLL B-cells. These results suggest that the apoptosis-inducing function of MLN2238 is mediated in part by its ability to inhibit expression of the BCL2 family of pro-survival proteins.

The combination of MLN2238 and AT-101 (BH3 mimetic) induces robust CLL cell death We have previously shown that AT-101, which is a BH3 mimetic, downregulates BCL2 in WM (Chitta et al, 2009) and primary CLL cells (Masood et al, 2011). The observation that MLN2238 also targets BCL2 proteins suggests its potential for use in combination with BCL2 modulators. To test this, we investigated the effect of AT-101 in combination with MLN2238 on CLL cells. BCL2 protein expression was measured in response to MLN2238 (Fig 4A, data from two representative patients shown), AT-101 or the combination of the ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

two agents together (Fig 4B). BCL2 expression level in CLL cells was decreased by both agents alone and more notably so in AT-101-treated cells (data from one representative patient shown). We next investigated whether AT-101 could be successfully combined with MLN2238 for enhanced tumour kill. As a single agent, MLN2238 induces robust CLL cell death at 50 nmol/l (~50% loss of cell viability), ergo we rationalized that AT-101 could augment the effects of sub-apoptotic concentrations of MLN2238. We treated CLL cells from three patient samples with MLN2238 (12
5 nmol/l) plus AT-101 (2
5 lmol/l or 5 lmol/l) for 24 h. The cells were subsequently stained with annexin V and propidium iodide followed by flow cytometric analysis of apoptotic cell death. We sought to determine if AT-101 and MLN2238 could be combined for enhanced CLL tumour kill. Single agents MLN2238 (12
5 nmol/l) and AT-101 (2
5 lmol/l) produced an average of 3
23% and 9
3% cell death over control cells. As hypothesized, we observed enhanced tumour cell death (16% over control) when AT-101 (2
5 lmol/l) was combined with MLN2238 (12
5 nmol/l), which indicated that a BCL2 inhibitor could effectively lower the apoptotic threshold of CLL cells, rendering them vulnerable to lower concentrations of MLN2238 (Fig 4C, left bars). Apoptotic cell death at these concentrations was also confirmed by immunoblotting for the cleaved protein product of PARP1 (Fig 4D). Interestingly, although greater percent cell death (average 33%) was noted in CLL cells treated with a higher concentration (5 lmol/l) of 83

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Fig 4. MLN2238 inhibits BCL2 and its anti-leukaemic activity is enhanced by addition of the BH3 mimetic AT-101 (A) B-CLL cells from 3 patient samples were treated with different concentrations of MLN2238 for 24 h and the response in BCL2 protein was assessed by Western Blot. MLN2238 exerted a dose-dependent decrease in BCL2 as shown in 2 representative patient samples. (B) MLN2238 (12
5 nmol/l) and AT-101 (2
5 lmol/l) caused a decrease in BCL2 protein expression alone and in combination with one another. (C) Cell death in MLN2238 (M) 12
5 nmol/l treated cells was significantly enhanced (P < 0
05) with the addition of AT-101 (A) at 2
5 lmol/l (M+A). This effect is more notable at an AT-101 concentration of 5 lmol/l (M+A1). This was also evidenced by cleavage of PARP1 on Western blot (D) In all experiments, cells were treated with either MLN2238, AT-101 or the combination of the two agents for 24 h. The percentage of tumour cell death was calculated by using the formula: % apoptosis of control = Tumour cell death – control cell death and reflects the accurate % of cells that underwent apoptosis in response to the drug(s).

single agent AT-101, we noticed only a marginal benefit following the addition of MLN2238 (Fig 4C, right bars).

MLN2238 enhances the anti-CLL activity of fludarabine or dexamethasone MLN2238 combined with AT-101 demonstrated substantial cytotoxic activity; thus, we investigated the effect of MLN2238 on CLL cells in combination with the standard-of-care anti-CLL agents, fludarabine or dexamethasone, along with the immunomodulatory drug (IMiD) lenalidomide. In these experiments, we aimed to determine the effect of MLN2238 84

as an adjunct to the aforementioned chemoimmunotherapies. Thus we used both an optimal (50 nmol/l) and sup-optimal (25 nmol/l) concentration of MLN2238. With the addition of lenalidomide to MLN2238 pre-treated cells, no cytotoxic effects were observed by 24 h (data not shown). In contrast, the addition of MLN2238 to fludarabine- (1 lmol/l) or dexamethasone- (10 lmol/l) treated CLL cells resulted in increased apoptotic cell death by annexin V and propidium iodide staining. Using the lower concentration (25 nmol/l) of MLN2238 (M) in combination with dexamethasone, we observed no significant change in the percent tumour cell death over that of dexamethasone alone (Fig 5A, bar D+M). However, at a 50 nmol/l concentration (M1), MLN2238 plus dexamethasone induced significant (P < 0
05) apoptosis in 45% of malignant tumour cells, compared to control. The average percent tumour cell death in dexamethasone-treated cells was 15%; in MLN2238 [50 nmol/l]-treated cells, it was 30
66% (Fig 5B, bar D+M1). The effects of the lower concentration of MLN2238 were more notable in the MLN2238– fludarabine combination, which showed increased tumour kill at both the low (20% average tumour kill) and higher doses of MLN2238 (45% average tumour kill) (Fig 5B). The results suggest that MLN2238 can sensitize CLL cells for their effective targeting by chemotherapeutic anti-CLL drugs.

Discussion Proteasome inhibitors are potent compounds with diverse effects on cell signalling circuits, such as inactivation of NFKB1, stabilization of TP53 and pro-apoptotic proteins (BID, BAX, PMAIP1) and downstream activation of stress related pathways (Adams, 2003). In this study, we tested MLN2238, the biologically active form of the investigational PI MLN9708, for its direct proteasome-targeted anti-CLL activity. We examined its effects on the downstream auxiliary leukaemogenic pathways that drive CLL proliferation through derangements in the mitochondrial apoptotic system. MLN2238 actively induced apoptosis in all CLL patient samples irrespective of the patients’ disease stage, genomic aberrations or number of prior therapies. Further, we demonstrated that MLN2238 induced apoptosis in CLL cells by targeting anti-apoptotic proteins of the BCL2 family, thus offering an effective strategy to regulate the balance between cell survival and apoptosis. We observed that MLN2238 induces changes to MOMP, PARP1, and caspase activation. Treating the cells with z-VAD.fmk inhibits induction of caspases; however, cell death is not affected. This is not entirely surprising, because disruption of the proteasome can lead to increases in a myriad of protein substrates that are capable of inducing cellular apoptosis independent of caspase activation. These mechanisms may include inhibition of NFKB1, activation of the endoplasmic reticulum (ER) stress response, which is caused by the accumulation of misfolded proteins and generation of ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

MLN2238 Induces Apoptosis and is Cytotoxic to CLL Cells

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Fig 5. MLN2238 successfully induces tumour cell death when combined with standard anti-CLL agents, such as dexamethasone or fludarabine: (A) B-CLL cells from three patient samples were treated with MLN2238 at 25 nmol/l (M) or at 50 nmol/l (M1), dexamethasone (D, 10 lmol/l) or the combination of the two agents for 24 h. Chronic lymphocytic leukaemia cell death was significant in D+M1 treated cells (P < 0
05) (B) Similarly, cells were treated with MLN2238 (M or M1), fludarabine (F, 1 lmol/l) or in combination. Apoptosis was measured by staining for annexin v and propidium iodide. In both F+M and F+M1 treated cells, we observed a significant increase in tumour cell death (P < 0
05) over control cells. The percentage of tumour cell death was calculated by using the formula: % apoptosis of control= Tumour cell death – control cell death and reflects the accurate% of cells that underwent apoptosis in response to the drug(s).

reactive oxygen species upstream of the caspase cascade (Hideshima et al, 2002; Landowski et al, 2005; Perez-Galan et al, 2006). Together, these PI-mediated mechanisms contribute to tumour cell death, which is highly contextual and dependent upon the cell type on which the PI is acting (Rajkumar et al, 2005). Our experimental results suggest that the indirect pro-apoptotic effects of MLN2238 can be augmented when used in combination with agents designed to disrupt antiapoptotic pathways. Such compounds include obatoclax ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

GX15070, ABT-737, HA14-1 and AT-101, which resemble the BH3-pro-apoptotic BCL2 molecules and whose binding activity to BCL2 proper results in tumour cell death (Konopleva et al, 2008; Tse et al, 2008). The activity of BCL2 inhibitors as single agents in CLL has not made a significant clinical impact, despite preclinical studies highlighting their potential for being combined with standard and novel chemoimmunotherapeutic agents for enhanced tumour kill. Bortezomib has been investigated in vitro in CLL and other cancers in combination with several BCL2 inhibitors, including obatoclax (Perez-Galan et al, 2008), HA14-1 (Pei et al, 2003) and oblimersen sodium (BCL2 anti-sense oligonucleotide) (O’Connor et al, 2006) with favourable results. However, these BCL2 inhibitors are primarily selective for BCL2 only, or only weakly bind other anti-apoptotic BCL2 members, limiting their use in lymphoid malignancies where MCL1 and BCL2L1 (BCL-XL) also play a critical role in tumour cell survival (Beroukhim et al, 2010; Davids & Letai, 2012; Stamelos et al, 2012). Of particular interest is AT-101, which is the R-(-)-enantiomer of gossypol and is a small molecule pan-BCL2 inhibitor that binds to the BH3 domains of anti-apoptotic BCL2 proteins and disrupts their functional activity. AT-101 displays high binding affinity for BCL2, BCL2L1 and MCL1 proteins and, in preclinical studies, has demonstrated impressive cytotoxic activity in a variety of B-cell malignancies including CLL (James et al, 2006) and MM (Kline et al, 2008). In our study, AT-101 showed enhancement of the cytotoxic effects of MLN2238 in vitro. When combined with AT-101, MLN2238 induced cell death at a lower dose (12
5 nmol/l) compared to its activity in single-agent (25–50 nmol/l) form. This observation has clinical implications, signifying that MLN2238’s potential benefit could be enhanced when rationally combined with agents targeting complementary oncogenic pathways. Next, we investigated the in vitro anti-tumour activity of MLN2238 in combination with the traditional anti-CLL cytotoxic agent fludarabine, the widely used glucocorticoid dexamethasone and the IMiD lenalidomide. As expected, the MLN2238 and lenalidomide combination yielded little improvement in anti-tumour effect over MLN2238 alone because of the in vitro nature of the experiment and the requirement of lenalidomide for a host environment to exert its maximal activity (Chanan-Khan et al, 2011; Masood et al, 2011). However, we observed improved anti-leukaemic activity of MLN2238 in combination with fludarabine and moderate effects when combined with dexamethasone. Fludarabine is a purine nucleoside analog, which is metabolically converted to its active metabolite, F-ara-ATP (Plunkett et al, 1993; Ross et al, 1993). In this active form, fludarabine directly impedes the actions of DNA polymerase and ribonucleotide reductase by competing with dATP, in effect inhibiting DNA synthesis (Parker et al, 1988; Plunkett & Saunders, 1991). It is also capable of integrating itself into the DNA as a false purine base, resulting in the termination of DNA synthesis and the activation of the programmed cell death 85

A. Paulus et al response, which it is capable of initiating cellular deconstruction even in the absence of its incorporation into the DNA (Spriggs et al, 1986; Huang et al, 1990; Robertson et al, 1993). Fludarabine has been studied in combination with bortezomib in CLL patient cells in vitro. In this combination, additive anti-tumour activity was shown to be present through increased activation of the apoptotic signalling cascade due to upregulation of BAX and downregulation of inhibitor of apoptosis protein, XIAP (Duechler et al, 2005). Perhaps these molecular shifts in apoptotic signalling proteins also account for the increased tumour kill that we observed with the MLN2238-fludarabine combination. These shifts are being further investigated. Dexamethasone binds to cytoplasmic glucocorticoid receptors in the target cell, resulting in nuclear translocation of the receptor and consequent activation of numerous genes responsible for attenuation of the inflammatory response and regulation of immune function (Gross et al, 2009). In CLL cells, the anti-neoplastic effects of dexamethasone have been shown to be reliant on the expression of the pro-apoptotic BCL2 family member BCL2L11 and its indirect activation of BAK1/BAX, thus exposing the anti-CLL activity of the agent to be partially mediated through the mitochondrial apoptotic pathway (Iglesias-Serret et al, 2007; Melarangi et al, 2012). In treating primary patient CLL cells with sub-optimal (25 nmol/l) concentration of MLN2238 plus dexamethasone, we observed a marginal increase in apoptosis as compared to dexamethasone treatment alone. However, when MLN2238 was used at an optimal (50 nmol/l) concentration in combination with dexamethasone, we noted improved anti-CLL activity. This highlights the potential for these agents to be combined and further explored. Collectively, the data in this report attest to the anti-CLL activity of MLN2238 in primary CLL cells by its induction of caspases, PARP1 cleavage, MOMP alteration and inhibition of BCL2. Further, we have demonstrated that MLN2238 can be successfully combined in vitro with other anti-leukaemic agents, resulting in more potent and effective cell death through alternative/complementary oncogenic pathways. Thus, these data provide the rationale for additional preclinical experiments and future clinical investigation of MLN2238 in CLL patients.

References Adams, J. (2003) The proteasome: structure, function, and role in the cell. Cancer Treatment Reviews, 29, 3–9. Advani, P.P., Paulus, A., Masood, A., Sher, T. & Chanan-Khan, A. (2011) Pharmacokinetic evaluation of oblimersen sodium for the treatment of chronic lymphocytic leukemia. Expert Opinion on Drug Metabolism and Toxicology, 7, 765–774. Beroukhim, R., Mermel, C.H., Porter, D., Wei, G., Raychaudhuri, S., Donovan, J., Barretina, J., Boehm, J.S., Dobson, J., Urashima, M., Mc

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Acknowledgements The experiments and analysis carried out in this study were supported by funding from the Leukemia and Lymphoma Society (A.C.-K. is a Leukemia and Lymphoma Scholar in Clinical Research) and the Daniel Foundation of Alabama (A.C-K). We would also like to thank Kelly Viola for her editorial assistance.

Authors’ contributions AM designed the research; collected, analysed and interpreted the data; drafted the article; approved the final draft. AP collected, analysed, and interpreted the data; created the images; drafted the article; approved the final draft. KCM analysed and interpreted the data; performed critical revision for important intellectual content; approved the final draft. ANK performed experiments, collected, analysed, and interpreted the data, generated the Figs; and approved the final draft. DA performed experiments; generated Figs; approved the final draft. PA analysed and interpreted the data; performed critical revision for important intellectual content; approved the final draft. JF analysed and interpreted the data; performed critical revision for important intellectual content; approved the final draft. CR analysed and interpreted the data; performed critical revision for important intellectual content; approved the final draft. GCO analysed and interpreted the data; performed critical revision for important intellectual content; approved the final draft. VR analysed and interpreted the data; performed critical revision for important intellectual content; approved the final draft. KS performed experiments; collected analysed and interpreted the data and images; drafted the article; approved the final draft. ACK conceived and designed the research; analysed and interpreted the data; drafted and performed critical revision of the article for intellectual content; approved the final draft.

Funding and disclosures The authors do not have anything to disclose.

Henry, K.T., Pinchback, R.M., Ligon, A.H., Cho, Y.J., Haery, L., Greulich, H., Reich, M., Winckler, W., Lawrence, M.S., Weir, B.A., Tanaka, K.E., Chiang, D.Y., Bass, A.J., Loo, A., Hoffman, C., Prensner, J., Liefeld, T., Gao, Q., Yecies, D., Signoretti, S., Maher, E., Kaye, F.J., Sasaki, H., Tepper, J.E., Fletcher, J.A., Tabernero, J., Baselga, J., Tsao, M.S., Demichelis, F., Rubin, M.A., Janne, P.A., Daly, M.J., Nucera, C., Levine, R.L., Ebert, B.L., Gabriel, S., Rustgi, A.K., Antonescu, C.R., Ladanyi, M., Letai, A., Garraway, L.A., Loda, M., Beer, D.G., True, L.D., Okamoto, A., Pomeroy, S.L., Singer, S., Golub, T.R., Lander, E.S., Getz,

G., Sellers, W.R. & Meyerson, M. (2010) The landscape of somatic copy-number alteration across human cancers. Nature, 463, 899–905. Chanan-Khan, A.A., Chitta, K., Ersing, N., Paulus, A., Masood, A., Sher, T., Swaika, A., Wallace, P.K., Mashtare, T.L. Jr, Wilding, G., Lee, K., Czuczman, M.S., Borrello, I. & Bangia, N. (2011) Biological effects and clinical significance of lenalidomide-induced tumour flare reaction in patients with chronic lymphocytic leukaemia: in vivo evidence of immune activation and antitumour response. British Journal of Haematology, 155, 457–467.

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

MLN2238 Induces Apoptosis and is Cytotoxic to CLL Cells Chauhan, D., Tian, Z., Zhou, B., Kuhn, D., Orlowski, R., Raje, N., Richardson, P. & Anderson, K.C. (2011) In vitro and in vivo selective antitumor activity of a novel orally bioavailable proteasome inhibitor MLN9708 against multiple myeloma cells. Clinical Cancer Research, 17, 5311–5321. Chitta, K., Miles, K.M., Ghoshal, P., Stein, L., Coleman, M., Furman, R.R., Craig, H., Hayman, S., Lee, K.P. & Chanan-Khan, A.A. (2009) At-101 induces apoptosis waldenstrom macroglobulinemia cells resistant to bortezomib. Blood (ASH Annual Meeting Abstracts), 114, 2861. Davids, M.S. & Letai, A. (2012) Targeting the B-cell lymphoma/leukemia 2 family in cancer. Journal of Clinical Oncology, 30, 3127–3135. Dores, G.M., Anderson, W.F., Curtis, R.E., Landgren, O., Ostroumova, E., Bluhm, E.C., Rabkin, C.S., Devesa, S.S. & Linet, M.S. (2007) Chronic lymphocytic leukaemia and small lymphocytic lymphoma: overview of the descriptive epidemiology. British Journal of Haematology, 139, 809–819. Duechler, M., Linke, A., Cebula, B., Shehata, M., Schwarzmeier, J.D., Robak, T. & Smolewski, P. (2005) In vitro cytotoxic effect of proteasome inhibitor bortezomib in combination with purine nucleoside analogues on chronic lymphocytic leukaemia cells. European Journal of Haematology, 74, 407–417. Faderl, S., Rai, K., Gribben, J., Byrd, J.C., Flinn, I.W., O’Brien, S., Sheng, S., Esseltine, D.L. & Keating, M.J. (2006) Phase II study of singleagent bortezomib for the treatment of patients with fludarabine-refractory B-cell chronic lymphocytic leukemia. Cancer, 107, 916–924. Fennell, D.A., Chacko, A. & Mutti, L. (2008) BCL-2 family regulation by the 20S proteasome inhibitor bortezomib. Oncogene, 27, 1189–1197. Gross, K.L., Lu, N.Z. & Cidlowski, J.A. (2009) Molecular mechanisms regulating glucocorticoid sensitivity and resistance. Molecular and Cellular Endocrinology, 300, 7–16. Hallek, M. & Pflug, N. (2010) Chronic lymphocytic leukemia. Annals of Oncology, 21(Suppl 7), vii154–vii164. Hideshima, T., Chauhan, D., Richardson, P., Mitsiades, C., Mitsiades, N., Hayashi, T., Munshi, N., Dang, L., Castro, A., Palombella, V., Adams, J. & Anderson, K.C. (2002) NF-kappa B as a therapeutic target in multiple myeloma. Journal of Biological Chemistry, 277, 16639–16647. Huang, P., Chubb, S. & Plunkett, W. (1990) Termination of DNA synthesis by 9-betaD-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity. Journal of Biological Chemistry, 265, 16617–16625. Iglesias-Serret, D., de Frias, M., Santidrian, A.F., Coll-Mulet, L., Cosialls, A.M., Barragan, M., Domingo, A., Gil, J. & Pons, G. (2007) Regulation of the proapoptotic BH3-only protein BIM by glucocorticoids, survival signals and proteasome in chronic lymphocytic leukemia cells. Leukemia, 21, 281–287.

James, D.F., Castro, J.E., Loria, O., Prada, C.E., Aguillon, R.A. & Kipps, T.J. (2006) AT-101, a small molecule Bcl-2 antagonist, in treatment naive CLL patients (pts) with high risk features; Preliminary results from an ongoing phase I trial. Journal of Clinical Oncology (ASCO Meeting Abstracts), 24, 6605. Kline, M.P., Rajkumar, S.V., Timm, M.M., Kimlinger, T.K., Haug, J.L., Lust, J.A., Greipp, P.R. & Kumar, S. (2008) R-(-)-gossypol (AT-101) activates programmed cell death in multiple myeloma cells. Experimental Hematology, 36, 568–576. Konopleva, M., Watt, J., Contractor, R., Tsao, T., Harris, D., Estrov, Z., Bornmann, W., Kantarjian, H., Viallet, J., Samudio, I. & Andreeff, M. (2008) Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax). Cancer Research, 68, 3413–3420. Kupperman, E., Lee, E.C., Cao, Y., Bannerman, B., Fitzgerald, M., Berger, A., Yu, J., Yang, Y., Hales, P., Bruzzese, F., Liu, J., Blank, J., Garcia, K., Tsu, C., Dick, L., Fleming, P., Yu, L., Manfredi, M., Rolfe, M. & Bolen, J. (2010) Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer. Cancer Research, 70, 1970–1980. Landowski, T.H., Megli, C.J., Nullmeyer, K.D., Lynch, R.M. & Dorr, R.T. (2005) Mitochondrial-mediated disregulation of Ca2 + is a critical determinant of Velcade (PS-341/bortezomib) cytotoxicity in myeloma cell lines. Cancer Research, 65, 3828–3836. Liu, F.T., Agrawal, S.G., Movasaghi, Z., Wyatt, P.B., Rehman, I.U., Gribben, J.G., Newland, A.C. & Jia, L. (2008) Dietary flavonoids inhibit the anticancer effects of the proteasome inhibitor bortezomib. Blood, 112, 3835–3846. Masood, A., Chitta, K., Paulus, A., Khan, A.N., Sher, T., Ersing, N., Miller, K.C., Manfredi, D., Ailawadhi, S., Borrelo, I., Lee, K.P. & ChananKhan, A. (2011) Downregulation of BCL2 by AT-101 enhances the antileukaemic effect of lenalidomide both by an immune dependant and independent manner. British Journal of Haematology, 157, 59–66. Melarangi, T., Zhuang, J., Lin, K., Rockliffe, N., Bosanquet, A.G., Oates, M., Slupsky, J.R. & Pettitt, A.R. (2012) Glucocorticoid resistance in chronic lymphocytic leukaemia is associated with a failure of upregulated Bim/Bcl-2 complexes to activate Bax and Bak. Cell Death and Disease, 3, e372. O’Connor, O.A., Smith, E.A., Toner, L.E., TeruyaFeldstein, J., Frankel, S., Rolfe, M., Wei, X., Liu, S., Marcucci, G., Chan, K.K. & Chanan-Khan, A. (2006) The combination of the proteasome inhibitor bortezomib and the bcl-2 antisense molecule oblimersen sensitizes human B-cell lymphomas to cyclophosphamide. Clinical Cancer Research, 12, 2902–2911. Pahler, J.C., Ruiz, S., Niemer, I., Calvert, L.R., Andreeff, M., Keating, M., Faderl, S. & McConkey, D.J. (2003) Effects of the proteasome

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88

inhibitor, bortezomib, on apoptosis in isolated lymphocytes obtained from patients with chronic lymphocytic leukemia. Clinical Cancer Research, 9, 4570–4577. Parker, W.B., Bapat, A.R., Shen, J.X., Townsend, A.J. & Cheng, Y.C. (1988) Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase. Molecular Pharmacology, 34, 485–491. Pei, X.Y., Dai, Y. & Grant, S. (2003) The proteasome inhibitor bortezomib promotes mitochondrial injury and apoptosis induced by the small molecule Bcl-2 inhibitor HA14-1 in multiple myeloma cells. Leukemia, 17, 2036–2045. Perez-Galan, P., Roue, G., Villamor, N., Montserrat, E., Campo, E. & Colomer, D. (2006) The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status. Blood, 107, 257–264. Perez-Galan, P., Roue, G., Villamor, N., Campo, E. & Colomer, D. (2007) The BH3-mimetic GX15070 synergizes with bortezomib in mantle cell lymphoma by enhancing Noxa-mediated activation of Bak. Blood, 109, 4441–4449. Perez-Galan, P., Roue, G., Lopez-Guerra, M., Nguyen, M., Villamor, N., Montserrat, E., Shore, G.C., Campo, E. & Colomer, D. (2008) BCL2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its enhanced interaction with bortezomib in chronic lymphocytic leukemia cells. Leukemia, 22, 1712–1720. Plunkett, W. & Saunders, P.P. (1991) Metabolism and action of purine nucleoside analogs. Pharmacology & Therapeutics, 49, 239–268. Plunkett, W., Gandhi, V., Huang, P., Robertson, L.E., Yang, L.Y., Gregoire, V., Estey, E. & Keating, M.J. (1993) Fludarabine: pharmacokinetics, mechanisms of action, and rationales for combination therapies. Seminars in Oncology, 20, 2–12. Rai, K.R., Sawitsky, A., Cronkite, E.P., Chanana, A.D., Levy, R.N. & Pasternack, B.S. (1975) Clinical staging of chronic lymphocytic leukemia. Blood, 46, 219–234. Rajkumar, S.V., Richardson, P.G., Hideshima, T. & Anderson, K.C. (2005) Proteasome inhibition as a novel therapeutic target in human cancer. Journal of Clinical Oncology, 23, 630–639. Reed, J.C. (1997) Bcl-2 family proteins: regulators of apoptosis and chemoresistance in hematologic malignancies. Seminars in Hematology, 34, 9–19. Reed, J.C., Kitada, S., Kim, Y. & Byrd, J. (2002) Modulating apoptosis pathways in low-grade B-cell malignancies using biological response modifiers. Seminars in Oncology, 29, 10–24. Robertson, L.E., Chubb, S., Meyn, R.E., Story, M., Ford, R., Hittelman, W.N. & Plunkett, W. (1993) Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2’deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine. Blood, 81, 143–150.

87

A. Paulus et al Ross, S.R., McTavish, D. & Faulds, D. (1993) Fludarabine. A review of its pharmacological properties and therapeutic potential in malignancy. Drugs, 45, 737–759. Schena, M., Gottardi, D., Ghia, P., Larsson, L.G., Carlsson, M., Nilsson, K. & Caligaris-Cappio, F. (1993) The role of Bcl-2 in the pathogenesis of B chronic lymphocytic leukemia. Leukaemia & Lymphoma, 11, 173–179. Spriggs, D., Robbins, G., Mitchell, T. & Kufe, D. (1986) Incorporation of 9-beta-D-arabinofuranosyl-2-fluoroadenine into HL-60 cellular RNA and DNA. Biochemical Pharmacology, 35, 247–252.

88

Stamelos, V.A., Redman, C.W. & Richardson, A. (2012) Understanding sensitivity to BH3 mimetics: ABT-737 as a case study to foresee the complexities of personalized medicine. Journal of Molecular Signaling, 7, 12. Thomas, A., El Rouby, S., Reed, J.C., Krajewski, S., Silber, R., Potmesil, M. & Newcomb, E.W. (1996) Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance. Oncogene, 12, 1055–1062. Tse, C., Shoemaker, A.R., Adickes, J., Anderson, M.G., Chen, J., Jin, S., Johnson, E.F., Marsh, K.C., Mitten, M.J., Nimmer, P., Roberts, L.,

Tahir, S.K., Xiao, Y., Yang, X., Zhang, H., Fesik, S., Rosenberg, S.H. & Elmore, S.W. (2008) ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Research, 68, 3421– 3428. Wickremasinghe, R.G. (2008) Why is CLL refractory to bortezomib? Blood, 112, 3540– 3541. Wierda, W.G., Chiorazzi, N., Dearden, C., Brown, J.R., Montserrat, E., Shpall, E., Stilgenbauer, S., Muneer, S. & Grever, M. (2010) Chronic lymphocytic leukemia: new concepts for future therapy. Clinical Lymphoma, Myeloma and Leukemia, 10, 369–378.

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 78–88