Inhibitor of Apoptosis Proteins As Therapeutic Targets in Multiple Myeloma

Leukemia (2014) 28, 1519–1528 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Inhibitor of apoptosis proteins as therapeutic targets in multiple myeloma V Ramakrishnan1, U Painuly1,2, T Kimlinger1, J Haug1, SV Rajkumar1 and S Kumar1 The inhibitor of apoptosis (IAP) proteins have a critical role in the control of apoptotic machinery, and has been explored as a therapeutic target. Here, we have examined the functional importance of IAPs in multiple myeloma (MM) by using a Smac (second mitochondria-derived activator of caspases)-mimetic LCL161. We observed that LCL161 was able to potently induce apoptosis in some MM cell lines but not in others. Examining the levels of X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2) post LCL161 treatment indicated clear downregulation of both XIAP activity and cIAP1 levels in both the sensitive and less sensitive (resistant) cell lines. cIAP2, however, was not downregulated in the cell line resistant to the drug. Small interfering RNA-mediated silencing of cIAP2 significantly enhanced the effect of LCL161, indicating the importance of downregulation of all IAPs simultaneously for induction of apoptosis in MM cells. LCL161 induced marked up regulation of the Jak2/Stat3 pathway in the resistant MM cell lines. Combining LCL161 with a Jak2-specific inhibitor resulted in synergistic cell death in MM cell lines and patient cells. In addition, combining LCL161 with death- inducing ligands clearly showed that LCL161 sensitized MM cells to both Fas-ligand and TRAIL. Leukemia (2014) 28, 1519–1528; doi:10.1038/leu.2014.2 Keywords: myeloma; apoptosis; IAP; Smac mimetic INTRODUCTION absence of cIAP1 and cIAP2, RIP1 is not ubiquitinated, which signals Abnormalities in the apoptosis (programmed cell death) machinery RIP1 to form a complex with the death receptor complex activating 8,9 are common in various cancers including multiple myeloma (MM) caspase-8 and extrinsic apoptosis. In cells primed to undergo and are an important basis of resistance to existing therapeutic apoptosis, second mitochondria-derived activator of caspases options.1–3 Tumor cells evade apoptosis through numerous (Smac), a mitochondrial proapoptotic protein, is released into the mechanisms that include abnormal activation of signaling cytosol where it binds to IAP proteins relieving their inhibition of 10–12 events leading to increased proliferation and decreased caspases and promoting apoptosis. Tumor cells can evade the apoptosis signals and/or altered balance between anti- and proapoptotic effects of Smac by aberrantly expressing high levels of proapoptotic proteins.1–4 Inhibitors of apoptosis (IAP) proteins, the IAP proteins. Smac mimetics are compounds that bind the IAPs originally identified in baculovirus, are endogenous inhibitors of at Smac binding sites and promote apoptosis. These compounds programmed cell death that are aberrantly expressed in a wide have been shown to sensitize tumor cells to chemotherapies in a variety of tumors.5 Subsequent sequence homology searches led wide variety of malignancies.5 to the identification of eight related proteins in humans, namely, MM cell lines typically express high levels of XIAP,13 and may be cellular inhibitor of apoptosis protein 1 (cIAP1), cellular inhibitor of driven at least in part by cytokines interleukin-6 (IL-6) and insulin- apoptosis protein 2 (cIAP2), X-linked inhibitor of apoptosis protein like growth factor-1, both present at high levels in myeloma (XIAP), survivin, neuronal apoptosis inhibitory protein (NAIP), microenvironment. IL-6 and insulin-like growth factor-1 melanoma-IAP (ML-IAP) and baculovirus inhibitor of apoptosis upregulate XIAP by activating the NF-kB, mitogen-activated repeat containing ubiquitin-conjugating enzyme (BRUCE).5,6 protein kinase and phosphatidylinositol 3-kinase signaling All members of the IAP family are characterized by the presence pathways that are commonly aberrant in MM and other of the baculovirus inhibitor of apoptosis repeat domains.5,6 tumors.14 XIAP downregulation by small interfering RNA (siRNA) Among the IAPs, XIAP, cIAP1 and c1AP2 are the three well- leads to increased drug sensitivity in myeloma cell lines and studied proteins that are expressed in a wide variety of tumors. decreased tumor formation in borderline personality disorder/ Recently, it has been shown that XIAP is the only member of the severe-combined immunodeficiency mice.13 Given this, XIAP IAP family that binds and inhibits the activation of caspases 9 and presents an attractive therapeutic target in myeloma cell lines, 3.7 cIAP1 and cIAP2, on the other hand, are integral members of and XIAP inhibitors need to be investigated for their potential as the tumor necrosis factor (TNF-a) pathway where they associate anti-MM agents as single agents and in combination with existing with receptor-interacting protein (RIP) and TNF receptor- therapies. cIAP1 and cIAP2, on the other hand, have been associated factor (TRAF) and modulate both the canonical and suggested to have a tumor suppressor role, as mutations in noncanonical nuclear factor-kB (NF-kB) pathways.8,9 cIAP1 and these two IAPs have been seen in some MM cell lines and patient cIAP2 ubiquitinate RIP1 through their RING domains. In the cells, which result in activation of the NF-kB pathway.15,16 1Division of Hematology, Mayo Clinic and Foundation, Rochester, MN, USA and 24th Department of Internal Medicine, Hematology University Hospital Hradec Kralove and Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic. Correspondence: Dr S Kumar, Division of Hematology, Mayo Clinic and Foundation, 200 First Street South West, Rochester, MN 55905, USA. E-mail: [email protected] Received 16 December 2013; accepted 20 December 2013; accepted article preview online 9 January 2014; advance online publication, 31 January 2014 IAP inhibition in multiple myeloma V Ramakrishnan et al 1520 However, in another study, it has been shown that IAP expression c in the mitochondria, while apoptotic cells release cytochrome c into increased after chemotherapy in MM patients in association with the cytoplasm. multidrug resistance protein and correlated positively with poor 17 prognosis. Moreover, a Smac-mimetic LBW242 was shown to Western blotting induce cell death in MM cell lines and patient cells, although the Cells were lysed in RIPA buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% ability of the molecule to inhibit the IAPs and the mechanism of Triton X-100, 30 mM sodium pyrophosphate, 5 mM EDTA) containing Halt 18 action of the drug was not investigated in detail. Thus, the precise Phosphatase Cocktail (Thermo Fisher Scientific, Rockford, IL, USA), 1 mM functions of the IAPs in MM are still not very clear. Here, we have phenylmethyl-sulfonyl-fluoride (Thermo Fischer) and protease inhibitor investigated the importance of the IAP proteins by using an orally cocktail (Sigma-Aldrich Corp.). Protein lysate concentrations were bioavailable Smac-mimetic LCL161 in both MM cell lines and measured using BCA assay (Thermo Fisher). Equal amounts of protein patient cells. We observed potent activity of LCL161 in a subset of were loaded on Tris-glycine gels and transferred onto nitrocellulose MM cell lines and patient cells. We have identified cIAP2 and Jak2/ membranes (Bio-Rad, Hercules, CA, USA). All antibodies except the following were purchased from Cell Signaling Technology (Danvers, MA, Stat3 pathway as factors contributing to resistance toward single- USA). cIAP1 antibody was purchased from Epitomics (Burlingame, CA, USA). agent LCL161. Combining LCL161 with Jak2 inhibitors leads to p50, p52, RelB and cRel antibodies were purchased from Santa Cruz synergistic cell death in both MM cell lines and patient cells. Biotechnology (Dallas, TX, USA). Antigen–antibody complexes were detected using enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA). MATERIALS AND METHODS siRNA transfection MM cell lines and patient cells cIAP2 and scrambled siRNAs were purchased from Life Technologies Cell lines MM1S, MM1R, OPM2, RPMI8226 and U266 were kindly provided (Grand Island, NY, USA). One micromolar of either siRNAs was transfected by Dr Jonathan Keats (TGen, Phoenix, AZ, USA). H929 was purchased from using the Amaxa Nucleofector Technology (Kit C-Protocol: X-005) from ATCC (Manassas, VA, USA). DOX40 was kindly provided by Dr William Lonza (Basel, Switzerland) following the manufacturer’s protocol. Following Dalton’s laboratory (Moffitt Cancer Center, Tampa, FL, USA). RM43 was this, LCL161 (2.5 mM) was added or cells were left untreated. After 72 h, kindly provided by Dr Diane Jelinek’s laboratory (Mayo Clinic, Rochester, apoptosis was measured by Annexin V/PI staining and cIAP2 knockdown MN, USA). All cell lines except RM43 were cultured in RPMI 1640 media was confirmed by western blotting. (Mediatech Inc., Manassas, VA, USA) containing 10% fetal bovine serum (Mediatech Inc.), 2 mML-glutamine (Invitrogen, Grand Island, NY, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen). RM43 was ELISA array cultured in the same media but in the presence of 1 ng/ml of IL-6. Freshly Human cytokine enzyme-linked immunosorbent assay (ELISA) plate array obtained bone marrow aspirates from patients were collected with was purchased from Signosis Inc. (Santa Clara, CA, USA). MM1S or MM1R informed consent and were processed to obtain myeloma cells or stromal cells were left untreated or treated with LCL161 (2.5 mM or 1 mM cells as described previously.19–21 All patient cells were cultured in RPMI respectively) for indicated time points. Conditioned media were collected 1640 media that contained 20% fetal bovine serum, 2 mML-glutamine and ELISA array was performed following the vendor’s protocol. (Invitrogen), 100 U/ml penicillin and 100 mg/ml streptomycin. ELISA Drugs Human IP-10, IL-6 and TNF-a quantikine ELISA kits were purchased from LCL161 was synthesized and provided by Novartis (Basel, Switzerland) R&D systems. MM1S and MM1R cells were left untreated or treated with under a Material Transfer Agreement (MTA).

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