Novel Inosine Monophosphate Dehydrogenase Inhibitor VX-944 Induces Apoptosis in Multiple Myeloma Cells Primarily Via Caspase-Independent AIF/Endo G Pathway

Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway

Kenji Ishitsuka1, Teru Hideshima1, Makoto Hamasaki1, Noopur Raje1, Shaji Kumar1, Klaus Podar1, Steven Le Gouill1, Norihiko Shiraishi1, Hiroshi Yasui1, Aldo M Roccaro1, Yu-Zu Tai1, Dharminder Chauhan1, Robert Fram2, Kazuo Tamura3, Jugnu Jain2 and Kenneth C Anderson*,1

1Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA; 2Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, USA; 31st Department of Internal Medicine, Fukuoka University, 7-45-1 Nanakuma Jonan Fukuoka 814-0180, Japan

Inosine monophosphate dehydrogenase (IMPDH) is a Introduction rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharma- Multiple myeloma (MM) remains incurable despite ceuticals, Cambridge, MA, USA) is a small-molecule, conventional therapies including high-dose chemother- selective, noncompetitive inhibitor directed against human apy with stem cell support. Importantly, novel agents IMPDH. In this report, we show that VX-944 inhibits like thalidomide, the immunomodulatory drug Revli- in vitro growth of human multiple myeloma (MM) cell mid, and the proteasome inhibitor bortezomib can lines via induction of apoptosis. Interleukin-6, insulin-like achieve responses in patients with relapsed and refrac- growth factor-1, or co-culture with bone marrow stromal tory MM, and are now under evaluation as treatment cells (BMSCs) do not protect against VX-944-induced for patients earlier in their disease course (Richardson MM cell growth inhibition. VX-944 induced apoptosis et al., 2002, 2003, 2004). Nonetheless, there remains an in MM cell lines with only modest activation of caspases urgent need for the development of new therapeutic 3, 8, and 9. Furthermore, the pan-caspase inhibitor strategies for MM treatment. z-VAD-fmk did not inhibit VX-944-induced apoptosis Inosine monophosphate dehydrogenase (IMPDH) is and cell death. During VX-944-induced apoptosis, expres- a rate-limiting enzyme required for the de novo synthesis sions of Bax and Bak were enhanced, and both apoptosis- of guanine nucleotides from IMP (Zimmermann et al., inducing factor (AIF) and endonuclease G (Endo G) were 1998). There are two IMPDH isoforms in mammalian released from the mitochondria to cytosol, suggesting that cells: type I is constitutively expressed in most cell types, VX-944 triggers apoptosis in MM cells primarily via a and catalytically similar type II is activated in prolifer- caspase-independent, Bax/AIF/Endo G pathway. Impor- ating cells (Nagai et al., 1991; Senda and Natsumeda, tantly, VX-944 augments the cytotoxicity of doxorubicin 1994). Gene expression of IMPDH type II is elevated in and melphalan even in the presence of BMSCs. Taken patient MM cells compared with normal control plasma together, our data demonstrate a primarily non-caspase- cells (Takebe et al., 2004). Conversely, inhibition of dependent apoptotic pathway triggered by VX-944, IMPDH induces depletion of guanine nucleotide pools, thereby providing a rationale to enhance MM cell followed by decreased DNAand RNAsynthesis cytotoxicity by combining this agent with conventional (Laliberte et al., 1998). Recently, IMPDH has been agents which trigger caspase activation. shown to bind nucleic acids in vitro and in vivo, Oncogene (2005) 24, 5888–5896. doi:10.1038/sj.onc.1208739; suggesting that it may have a direct role in replication published online 6 June 2005 and transcription (McLean et al., 2004). IMPDH inhibitors induce cell-cycle arrest and decrease T- and Keywords: multiple myeloma; IMPDH inhibitor; B-cell responses effectively, both in vitro and in vivo. VX-944; apoptosis; AIF/Endo G pathway Consequently, they have been evaluated primarily as immunosuppressive rather than anticancer therapies (Eugui and Almquist, 1990; Eugui et al., 1991; Turka et al., 1991; Jain et al., 2001). Speciﬁcally, the IMPDH inhibitors benzamide riboside (BR) and mycophenolic acid (MPA) were found to induce growth inhibition, but not cytotoxicity, in the panel of 60 cancer cell lines *Correspondence: KC Anderson; E-mail: [email protected] derived from hematological and solid tumors at the Received 28 December 2004; revised 22 March 2005; accepted 22 March National Cancer Institute (http://dtp.nci.nih.gov). More 2005; published online 6 June 2005 recently, however, BR and MPAhave been shown to VX-944 induces apoptosis in MM cells K Ishitsuka et al 5889 induce apoptosis in selected cancer cells (Hunakova et al., 2000; Szekeres et al., 2002; Yalowitz et al., 2002; Messina et al., 2004). Although activation of caspases and inhibition of PARP have been reported as possible mediators of apoptosis triggered by MPAand BR (Yalowitz and Jayaram, 2002; Gu et al., 2003), their mechanisms of action are not fully characterized. MPA and tiazofurin, another IMPDH inhibitor, have been evaluated in cancer animal models (Sweeney et al., 1972; Ahluwalia et al., 1984), as well as in clinical trials, for the treatment of leukemia and CML, respectively (Knudtzon and Nissen, 1972; Wright et al., 1996). Both compounds showed some objective responses, but gastrointestinal intolerance (MPA) or neurotoxicity (tiazofurin) has limited further investigation. Hence, evaluation of more selective and well-tolerated IMPDH inhibitors is needed to determine the therapeutic potential of this approach in the treatment of malig- nancy. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor of both human IMPDH isoforms (Ki 6–10 nM). In this study, we evaluated the cytotoxicity of VX-944 against MM cells in vitro. We demonstrate that VX-944 inhibits growth of MM cell lines by induction of apoptosis, primarily via a caspase-independent pathway.

Results

VX-944 inhibits growth of MM cell lines The effect of VX-944 on growth of MM cell lines was determined using the MTT assay. VX-944 significantly inhibited the growth of RPMI8226, MM.1S, and U266 Figure 1 Effects of VX-944 on proliferation of MM cell lines and cells in a dose-dependent fashion, with 50% inhibition viability of normal PBMNCs, assessed by MTT assay after 48 h culture. VX-944 inhibited proliferation of MM cell lines in a dose- (IC ) values at 48 h of 450, 450, and 600 nM, 50 dependent manner (a); conversely, addition of guanosine signifi- respectively. VX-944 also inhibited growth of drug- cantly reversed the inhibitory effects of VX-944 in RPMI8226 cells resistant cell lines, including doxorubicin (Dox)-resistant (b). VX-944 did not reduce the viability of PBMNCs derived from RPMI8226-Dox40, melphalan (Mel) resistant three healthy subjects (c) RPMI8226-LR5, and Dex (dexamethasone) resistant MM.1R cells, with IC50 values similar to the parental drug-sensitive cell lines (Figure 1a). The degree of growth inhibition at 72 h was observed to be similar to lymphocytes as compared to cancer cells. Increased that at 48 h. The effects of VX-944 on DNAsynthesis expression of type II isoform has been closely linked were also determined by measuring [3H]thymidine with an increase in total IMPDH activity in cancer cells incorporation during the last 8 h of 48-h cultures. IC50 (Nagai et al., 1991). values determined by this method were lower than those determined in the MTT assay: 350 nM for RPMI8226, VX-944 induces caspase-independent apoptosis 100 nM for MM.1S and 75 nM for U266 cells (data not in MM cells shown). Growth inhibition induced by VX-944 is significantly reversed by the addition of guanosine, In order to further characterize the cytotoxic effects of which is converted to guanine and thereby circumvents VX-944 on MM cell lines, apoptosis induced by VX-944 the effect of inhibition of IMPDH on guanine nucleotide was analysed. ANNEXIN V/PI staining showed 13.4 levels (Zimmermann et al., 1998) (Figure 1b). Impor- and 16.8% of ANNEXIN V þ /PIÀcells, as well as 40.4 tantly, VX-944 did not reduce the viability of peripheral and 55.5% of ANNEXIN V þ /PI þ cells, after 72 h- blood mononuclear cells (PBMNCs) derived from exposure to 800 nM VX-944 in RPMI8226 and MM.1S healthy individuals at 48 h at concentrations up to cells, respectively (Figure 2a). VX-944-induced apopto- 6400 nM (Figure 1c). This is consistent with the sis was further confirmed by TUNEL assay cultured for expression of type II IMPDH, which is lower in normal 96 h with 800 nM of VX-944 (Figure 2b).

Oncogene VX-944 induces apoptosis in MM cells K Ishitsuka et al 5890

Figure 2 Effects of VX-944 on induction of apoptosis. Induction of apoptosis induced by VX-944 (800 nM) was shown by ANNEXIN V-PI staining at 48 and 72 h (a) and TUNEL assay at 96 h (b)

We next examined the apoptotic pathway induced by Caspases 6 and 7, mediators of caspase 3-independent VX-944. Cleavage of caspase-8, -9, and -3 triggered apoptosis (Miyashita et al., 1998; Kagawa et al., 2001; by VX-944 in MM.1S and RPMI8226 cells was modest, Pirnia et al., 2002), were not activated by VX-944 (data and there was no cleavage of DFF45 (Figure 3a). not shown). To further conﬁrm caspase-independent

Oncogene VX-944 induces apoptosis in MM cells K Ishitsuka et al 5891

Figure 3 VX-944 induced modest cleavage of caspase 3, 8 and 9 in MM.1S cells and RPMI8226 cells, demonstrated by Western blotting (a; control; b, cells treated with 800 nM of VX-944 for 48 h; c, cells treated with z-VAD-fmk 25 mM for 2 h, followed by 800 nM of VX-944 for 48 h; d, control; e, cells treated with 4 mM of As2O3 for 12 h; f, cells treated with z-VAD-fmk 25 mM for 2 h, followed by 4 mM of As2O3 for 12 h). Cytotoxicity (b) and apoptosis (c) induced by VX-944 were not blocked by pretreatment with z-VAD-fmk. Cells were treated with 800 nM of VX-944 for 48 h, with or without z-VAD-fmk (25 mM) pretreatment. Cytotoxicity was determined by counting viable cells by trypan-blue exclusion dye. Representative data from triplicate experiments are shown (b; MM.1S and RPMI8226). The percentage apoptotic cells after treatment with 800 nM of VX-944 for 48 h, with or without pretreatment of z-VAD- fmk (25 mM) for 2 h, was determined by ﬂow-cytometric analysis for APO2.7 staining. Block of As2O3-induced apoptosis conﬁrmed the inhibiting activity of z-VAD-fmk (c)

apoptosis, the effects of z-VAD-fmk on cytotoxicity without z-VAD-fmk (25 mM) pretreatment for 2 h. and apoptosis induced by VX-944 were next determined. Neither growth inhibition nor cytotoxicity of VX-944, Z-VAD-fmk is a broad-speciﬁcity caspase inhibitor as determined by trypan blue exclusion, was reduced with second-order inactivation rates; 7100, 16 000, by pretreatment with z-VAD-fmk (Figure 3b). Cyto- 18 000, 180 000, 280 000 MÀ1 sÀ1 for caspases 6, 3, 7, 9, toxicity of lower concentrations of VX-944 (200 and and 8, respectively (Garcia-Calvo et al., 1998). MM.1S 400 nM) was similarly unaffected by pretreatment with cells (3 Â 105/ml) and RPMI8226 cells (1.5 Â 105/ml) z-VAD-fmk (data not shown). Moreover, pretreatment were exposed to 800 nM VX-944 for 48 h, with or with z-VAD-fmk did not block induction of apoptosis

Oncogene VX-944 induces apoptosis in MM cells K Ishitsuka et al 5892 by VX-944 at 48 h in both cell lines (Figure 3c). MM.1S cells treated with arsenic trioxide (As2O3; 4 mM) for 48 h, with or without z-VAD-fmk pretreatment, were a control for caspase-dependent apoptosis and the inhibitory activity of z-VAD-fmk (Hayashi et al., 2002; McCafferty-Grad et al., 2003). Modulation of pro- and anti-apoptotic proteins by VX-944 was next evaluated. MM.1S and RPMI8226 cells were treated with 800 nM VX-944 for 12, 24, and 48 h; cell lysates were prepared as described previously. Of bcl-2 family member proteins, Bax and Bak were markedly upregulated in VX-944-treated cells, without signiﬁcant changes in Bcl-2, Mcl-1, XIAP, and Bad (Figure 4a). Furthermore, translocation of mitochondrial proapoptotic proteins, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) to cytosolic fractions was observed (Figure 4b).

Effect of VX-944 on MM cells cultured with exogenous IL-6, IGF-1, and bone marrow stromal cells (BMSCs) We next examined the effect of VX-944 on MM cells in the presence of exogenous IL-6 and IGF-1, as well as on Figure 4 VX-944 enhances expression of Bax and Bak proteins, determined by Western blotting of whole cell lysates MM cell growth in the BM microenvironment. Neither (a). Mitochondrial proteins AIF and Endo G were released IL-6 nor IGF-1 protected against VX-944-induced to cytosolic fraction after VX-944 (800 nM) treatment in MM.1S growth inhibition (Figure 5a and b). Binding of cells (b)

Figure 5 VX-944 overcomes protective effects of IL-6 (a), IGF-1 (b) and adherence to patient BMSCs in MM.1S cells (c, d). DNA synthesis was determined by measuring [3H]thymidine incorporation during the last 8 h of 48 h cultures (*Po0.05)

Oncogene VX-944 induces apoptosis in MM cells K Ishitsuka et al 5893 MM.1S cells to BMSCs triggers DNAsynthesis, which Discussion was also abrogated by VX-944 (Figure 5c and d). Importantly, VX-944 at similar concentrations did not In this study, we ﬁrst showed that the novel IMPDH affect the viability of BMSCs, as determined by MTT inhibitor VX-944 directly inhibits growth of MM cell assay (data not shown). To further delineate the effect of lines. Growth inhibition of MM cell lines, including VX-944 on growth signaling in MM.1S cells, MM.1S Dox-, Mel-, and Dex-resistant lines, was observed at an cells were stimulated with IL-6 (20 ng/ml) or IGF-1 IC50 of o600 nM by MTT assay. Importantly, our (25 ng/ml) for 10 min following pretreatment with studies further showed that up to 6.4 mM VX-944 did not 800 nM of VX-944 for 3, 6, and 9 h. No signiﬁcant inhibit the viability of normal PBMNCs, consistent with inhibition of STAT3, p42/44 MAPK or Akt signaling a Phase I pharmacokinetic study of VX-944 in which was noted in VX-944-treated MM.1S cells (data not high-plasma concentrations were achieved without toxic shown). effects. These data therefore demonstrate that VX-944 effectively inhibits growth of MM cell lines, including Combination with VX-944 and Dox or Mel augments drug-resistant cells, at clinically achievable concentra- inhibition of growth of MM.1S cells tions. Neither co-culture with BMSCs, which mimics the BM microenvironment of MM patients, nor exogenous Sequential treatment of MM.1S cells with VX-944 IL-6 and IGF-1, which are growth and survival factors followed by Dox or Mel enhanced the cytotoxicity of for MM cells, protected MM cells against VX-944- each drug (Figure 6a and b). Additive effects were also induced cell death (Klein et al., 1995; Chauhan et al., observed in MM.1S cells cultured with BMSCs derived 1997; Jelinek et al., 1997; Ge and Rudikoff, 2000). from MM patient (Figure 6c and d). Although Gu et al. (2003) have reported that MPA

Figure 6 Sequential treatment with VX-944 followed by Dox (a, c) or Mel (b, d) demonstrates additive cytotoxicity in MM.1S cells. MM.1S cells were treated with VX-944 for 24 h, followed by addition of combination agent. Cell growth was determined at 72 h by MTT assay (a, b). MM.1S cells cultured with BMSCs were treated with VX-944 for 24 h, followed by the addition of combined agent. DNAsynthesis is determined by incorporation of [ 3H]thymidine during the last 8 h of 48 h culture (c, d)(*Po0.05)

Oncogene VX-944 induces apoptosis in MM cells K Ishitsuka et al 5894 causes downregulation of STAT3 and 5, p42/44 MAPK, In conclusion, we have shown that the novel IMPDH and Akt pathway in a IL-3 dependent murine hemato- inhibitor VX-944 appears to induce apoptosis mainly poietic cell line (Gu et al., 2003), our study did not reveal via a caspase-independent, Bax/AIF/Endo G pathway inhibition of STAT3, p42/44 MAPK, or Akt signaling in in MM cells. VX-944 induces cell death in drug-resistant VX-944-treated MM.1S cells, suggesting that growth MM cells and overcomes the growth-promoting activity inhibition induced by VX-944 is independent of these of IL-6, IGF-1 and BMSCs. These results suggest that pathways. VX-944, used either alone or in combination with other Caspases are believed to be the key executors of drugs, represents a promising novel targeted approach apoptosis. However, recent studies suggest that caspase to enhance MM cell cytotoxicity and improve patient activation is not the sole pathway for inducing apoptosis outcome in MM. or necrosis by death stimuli (Jaattela and Tschopp, 2003; Abraham and Shaham, 2004; Lockshin and Zakeri, 2004). For example, caspase-independent cell Materials and methods death in MM cells has been reported in As2O3-treated RPMI8226 cells and patient MM cells (McCafferty- Cells Grad et al., 2003). Since most drugs used to treat MM Dex-sensitive MM.1S and Dex-resistant MM.1R human MM induce cell death via activation of caspases, agents such cell lines were kindly provided by Dr Steven Rosen (North- as VX-944 that can kill MM cells via caspase- western University, Chicago, IL, USA). RPMI 8226-Dox40 independent pathways may be particularly useful for (Dox-resistant) and RPMI 8226-LR5 (Mel-resistant) human combination with conventional and novel drugs to MM cell lines were kindly provided by Dr William Dalton overcome drug resistances. (Moffit Cancer Center, Tampa, FL, USA). RPMI-8226 and During VX-944-induced apoptosis, activation of U266 cells were obtained from the American Type Culture caspases 3, 8 and 9 was only modest; caspases 6 and 7, Collection (Rockville, MD, USA). Fresh PBMNCs obtained known mediators of caspase 3-independent apoptotic from three healthy subjects after informed consent were signaling (Miyashita et al., 1998; Kagawa et al., 2001; separated from heparinized peripheral blood by Ficoll- 1 Pirnia et al., 2002), were not activated. Cleavage of Hipaque density sedimentation. Cells were cultured at 37 C in RPMI 1640 containing 10% fetal bovine serum (FBS; DFF45, which inactivates the inhibitory function of Sigma, St Louis, MO, USA), 2 mML-glutamine, 100 U/ml DFF40 and related nuclear DNAfragmentation (Liu penicillin, and 100 mg/ml streptomycin (Gibco, Grand Island, et al., 1998), was not observed. In addition, pretreat- NY, USA). ment with z-VAD did not significantly inhibit VX-944 MNCs in BM specimens, obtained from patients with MM triggered cytotoxicity or apoptosis. Therefore, VX-944 after informed consent, were separated by Ficoll-Hipaque appears to induce apoptosis primarily via a caspase- density sedimentation and used to establish long-term BMSC independent mechanism. cultures, as described (Uchiyama et al., 1993; Hideshima et al., We next determined whether VX-944 induced Bax 2001). and Bak protein expression, as well as translocation of AIF and Endo G to the cytoplasm. The Bax/AIF/Endo Reagents G pathway is a major caspase-independent apoptotic VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) was cascade (Daugas et al., 2000; Ahn et al., 2004; Cande dissolved in dimethyl sulfoxide (DMSO) at 10 mM stock et al., 2004; Cregan et al., 2004). In this pathway, solution and stocked at À201C. Guanosine and Dox (Sigma, enhanced Bax and Bak expression induces mitochon- St Louis, MO, USA) were dissolved in sterile water at 1 and drial membrane permeabilization, thereby releasing AIF 3.45 mM, respectively. Mel (Sigma, St Louis, MO, USA) was and Endo G from the mitochondria to the cytosol and dissolved in DMSO at 20 mM. Recombinant human IL-6 and nucleus, with subsequent chromatin condensation and IGF-1 (R&D Systems, Minneapolis, MN, USA) were recon- cell death. Conversely, reports that apoptosis induced stituted with sterile PBS containing 0.1% FBS and 10 mM via Bax/AIF/Endo G pathway can be caspase-depen- acetic acid containing 0.1% FBS, respectively. Pan-caspase dent show that the release of AIF and Endo G from inhibitor z-VAD-fmk (Bachem, Bubendorf, Switzerland) was dissolved in methanol. mitochondria is blocked by z-VAD (Arnoult et al., 2003; Cande et al., 2004). In our study, the release of these proapoptotic proteins is likely caspase-independent, Cellular proliferation and DNA synthesis assay since caspase activation was modest. Colorimetric assays were performed to evaluate drug activity. As most conventional and novel drugs induce MM cell lines and MM patient BMSCs were cultured in 96- apoptosis in MM cells via activation of caspases, agents well culture plates with VX-944 in 100 ml of media for 48 h, killing MM cells via caspase-independent pathways may pulsed with 10 ml of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5- overcome drug resistances. Therefore, we next examined diphenyl tetrasodium bromide (MTT; Sigma, St Louis, MO, the effect of combining VX-944 and other drugs in vitro. USA) to each well for 4 h, followed by 100 ml isopropanol The additive effect of VX-944 and Dox or Mel shown in containing 0.04 N HCl. Absorbance readings at a wavelength of 570 nm and reference wave length of 630 nm were taken on a our study indicates that VX-944 can be combined with spectrophotometer (Molecular Devices Corp., Sunnyvale, CA, these drugs to enhance their efficacy. Sequential treat- USA). ment with VX-944, followed by Dox or Mel, holds great DNAsynthesis was measured by [ 3H]thymidine uptake, as promise, since these combinations inhibited the growth described previously (Hideshima et al., 2000). Cells in 96-well of MM cells even in the presence of BMSCs. culture plates were pulsed with 0.5 mCi/well of [3H]thymidine

Oncogene VX-944 induces apoptosis in MM cells K Ishitsuka et al 5895 (Perkin-Elmer, Boston, MA, USA) during the last 8 h of AIF, phospho-Akt, and Akt (Cell Signaling, Beverly, MA, culture, harvested onto glass filters with an automatic cell USA); phospho-p42/44 MAPK, phospho-STAT3, bcl-2, and harvester (Cambridge Technology, Cambridge, MA, USA), Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and and counted using the LKB Betaplate scintillation counter Endo G (Axxora, San Diego, CA, USA) antibodies. Immuno- (Wallac, Gaithersburg, MD, USA). All experiments were blotting with anti-alpha-tubulin Ab (Sigma, St Louis, MO, performed in triplicate. USA) and anti-voltage-dependent anion channel (VDAC) Ab (Calbiochem, San Diego, CA, USA) confirmed equivalent Detection of cytotoxicity and apoptosis protein loading. Cytotoxicity was determined by trypan blue exclusion assay (Sigma, St Louis, MO, USA). Viability was expressed as Effect of IL-6, IGF-1, and BMSCs on VX-944-induced growth percent viable cells. Apoptosis in VX-944-treated cells was inhibition assesed by annexin V and PI staining, TUNEL assay, and MM.1S cells were cultured for 48 h with VX-944, in the APO 2.7 staining. For annexin V and PI staining, cells were presence or absence of IL-6 or IGF-1. To evaluate growth suspended in binding buffer (10 mmol/l HEPES, pH 7.4, stimulation in MM cells adherent to BMSCs, MM.1S cells 140 mmol/l NaCl, 2.5 mmol/l CaCl2), followed by incubation were cultured in BMSC-coated 96-well plates for 48 h, in the with annexin V-FITC (MBL, Nagoya, Japan) and PI (MBL, presence or absence of VX-944. DNAsynthesis in these Nagoya, Japan) for 5 min at room temperature. To detect cultures was measured by [3H]thymidine uptake. DNAstrand breaks, the TUNEL assay was performed using a commercial kit (MBL, Nagoya, Japan). In brief, cells were fixed and permeabilized by 4% paraformaldehyde and 70% Combination with other drugs ethanol, followed by incubation with a mixture of FITC-dUTP MM.1S cells were cultured with VX-944 for 24 h, followed by and TdT for 1 h at 371C. For detection of mitochondrial addition of either Dox or Mel. Cellular growth was determined membrane protein 7A6 expressed in apoptotic cells, cells were by MTT assay after 72 h treatment. Cells were also incubated incubated with APO 2.7 reagent (Immunotech, Marseille, with VX-944 in BMSC-coated 96-well plates, followed by France) for 20 min. Expression of Annexin V and PI, TUNEL, addition of either of Dox or Mel at 24 h. DNAsynthesis was and APO 2.7 was determined using an EPICS XL flow measured by the addition of [3H]thymidine uptake for the last cytometer. 8 h of the 48 h cultures.

Western blotting Acknowledgements MM cells were harvested, washed twice with ice-cold PBS, and We acknowledge K Lin, R Hoover, Y Yao, M Harding, J lysed in lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, Thomson, M Partridge and C Sorensen at Vertex for critical 1% NP-40, 5 mM EDTA, 5 mM NaF, 2 mM Na3V4,1mM reading of the manuscript or advice during the course of these PMSF, 5 mg/ml leupeptine, and 5 mg/ml aprotinin after VX-944 studies. This study is supported by National Institutes of treatment. Subcellular proteins were extracted from 2 Â 107 Health Grants Specialized Programs of Research Excellence viable cells using mitochondria isolation kit (Pierce, Rockford, (SPORE) IP50 CA10070-01, PO-1 78378, and RO-1 CA IL, USA). Whole-cell lysates or fractionated proteins were 50947; the Doris Duke Distinguished Clinical Research subjected to SDS–PAGE, transferred to nitrocellulose mem- Scientist Award (KCA); the Multiple Myeloma Research brane, and immunoblotted with anti-caspase-3, caspase-6, Foundation (TH); and the Cure for Myeloma Research Fund caspase-7, caspase-8, caspase-9, DFF45/35, Bax, Bak, XIAP, (KCA).

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