Novel Inosine Monophosphate Dehydrogenase Inhibitor VX-944 Induces Apoptosis in Multiple Myeloma Cells Primarily Via Caspase-Independent AIF/Endo G Pathway
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Oncogene (2005) 24, 5888–5896 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc 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-indepen- dent 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).