Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositolrequiring enzyme 1␣ (IRE1␣) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1␣- IntroductionTreatment for multiple myeloma (MM) has remarkably improved because of novel agents, such as bortezomib, thalidomide, and lenalidomide. [1][2][3] However, MM remains incurable, and nextgeneration novel agents are urgently needed. Because of high levels of endoplasmic reticulum (ER) stress and adaptation by the unfolded protein response (UPR), targeting signaling by the UPR and blocking this key survival pathway represent a new therapeutic strategy. In mammalian cells, protein folding is proportionally fine-tuned to the metabolic state of the cell within its microenvironment. Extracellular insults, such as low nutrients, hypoxia, and multiple drugs, result in the accumulation of misfolded proteins in the ER, thereby causing ER stress and initiating the UPR. 4 The UPR in turn increases the biosynthetic capacity and decreases the biosynthetic burden of the ER, to maintain cellular homeostasis. However, when the stress cannot be compensated by the UPR, cellular apoptosis occurs. 5 The UPR consists of 3 branches of signaling pathways, which initiate from 3 ER transmembrane proteins: inositol-requiring enzyme 1␣ (IRE1␣), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). In the resting state, these proteins are associated with molecular chaperone BiP/GRP78 in the ER. However, when unfolded proteins accumulate in the ER, BiP/GRP78 dissociates from them, thereby inducing UPR signaling. 6 In the UPR, IRE1␣ is activated by oligomerization and autophosphorylation, resulting in activation of its endoribonuclease to cleave and initiate splicing of the X-box binding protein 1 (XBP1) mRNA. A 26-nucleotide intron from XBP1 is removed by activated IRE1␣ endoribonuclease, resulting in a translational frame-shift to modify unspliced XBP1 (XBP1u: inactive) into spliced XBP1 (XBP1s: active). 7 XBP1 is a unique transcription factor that regulates genes responsible for ER-associated degradation (ERAD), such as EDEM, and those responsible for promoting protein folding, such as p58IPK and other ER chaperones. 8 Thus, IRE1␣-XBP1 pathway has a prosurvival role in the UPR. However, under conditions of prolonged and uncompensated stress, the UPR leads to cellular apoptosis, known as the terminal UPR. The proapoptotic transcription factor CHOP, also known as GADD153, is induced via PERK and ATF6 pathways. CHOP causes downregulation of BCL2, thereby leading to caspase-dependent apoptosis. 9 IRE1␣ also has a proapoptotic role: it binds TRAF2 and activates ASK1, which causes JNK activation, thereby leading to caspase-dependent apoptosis. 10 ...

B i o s c i e n c e . N o t f o r d i s t r i b u t i o n .[Cancer Biology & ACKNOWLEDGEMENTSWe are grateful to the following people for providing plasmids or cell lines: Frank B. Furnari, Webster K. Cavenee, William R. Taylor, and Toshio Nikaido. The technical assistance of Susan Su is acknowledged.This work was supported in part by the Kriegel Foundation (A.K.; T.C.C.). NOTE ADDED IN PROOFUsing prostate cancer cell lines, Johnson et al. (Adv Enzyme Regul 2001; 41:221-35) observed differential effects of selective COX-2 inhibitors on apoposis induction. Similar to our data presented here, they found celecoxib to be the most potent compound. Research Paper Differential Effects of Selective COX-2 Inhibitors on Cell Cycle Regulation and Proliferation of Glioblastoma Cell Lines ABSTRACTIt is well established that traditional NSAIDs, which inhibit cyclooxygenase (COX) 1 and COX-2, have the potential to reduce the risk of colorectal cancer. New generation COX inhibitors have been developed that selectively inhibit COX-2, which might cause less side effects while still retaining their therapeutic potential. As patients with brain tumors, such as glioblastoma, exhibit a very poor prognosis, we began to explore whether COX inhibitors could be useful for the treatment of this type of tumor. We found that celecoxib inhibited the proliferation of various glioblastoma cell lines in vitro much more potently than traditional NSAIDs. In addition, although several different selective COX-2 inhibitors potently reduced PGE2 levels in these cells, none of them exerted anti-proliferative effects that were comparable to celecoxib. The addition of external PGE2 to celecoxib-treated cells did not restore proliferation, indicating that growth inhibition by celecoxib was not mediated via the blockage of PGE2 production. In an effort to determine the underlying molecular processes that might mediate celecoxib's potent anti-proliferative effects, we found a loss of the activity of cyclin-dependent kinases, the essential regulators of cell proliferation, which was due to the transcriptional downregulation of cyclin A and cyclin B expression. Taken together, our results show that celecoxib exerts COX-2-independent anti-proliferative effects on glioblastoma cell growth, which are more potent than those of other selective COX-2 inhibitors or traditional NSAIDs, and which are mediated via the transcriptional inhibition of two essential components of the cell cycle machinery, cyclin A and cyclin B.

133 Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures: Tam: MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; MannKind: Membership on an entity's Board of Directors or advisory committees.

The safety of gene therapy using hematopoietic stem cells may be increased by including a suicide gene in the therapeutic vector to eliminate adverse events like insertional oncogenesis while retaining the clinical benefits. We have developed a model of experimental insertional oncogenesis by transducing the murine factor-dependent leukemia cell line Ba/F3 with a bicistronic Moloney murine leukemia virus retroviral vector encoding a murine oncogene (cKit(D814V)) in addition to one of three suicide genes: Herpes simplex virus thymidine kinase (HSV-TK); SR39, an HSV-TK mutant with an increased affinity for the drug substrate Ganciclovir (GCV); or sc39, a splice-corrected version of SR39. Following intravenous challenge with transduced Ba/F3 clones and treatment with GCV, leukemia developed in mice given cells expressing HSV-TK, but not SR39 or sc39. In vitro GCV resistance was observed in heterogeneously transduced Ba/F3 pools at 2.5-14%, and single-nucleotide changes or partial loss of the suicide gene were identified as mechanisms of drug escape. However, GCV treatment resulted in 80-100% survival of mice challenged even with pools of partially resistant Ba/F3 cells expressing SR39 or sc39. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity.

We examined the effects of the 4 major female reproductive hormones, estradiol (E2), progesterone (P4), follicle stimulating hormone (FSH), and luteinizing hormone (LH) on thymidine incorporation in benign and malignant ovarian epithelial tumors cultured in vitro. Treatment of these tumors with E2, FSH and LH resulted in increased thymidine incorporation while treatment with P4 inhibited growth as well as thymidine incorporation. The inhibitory effect of progesterone could not be reproduced by treating the cells with ligands for other steroid hormone receptors known to interact with P4 such as the mineralocorticoid and glucocorticoid receptors. All cells lines expressed at least the PR-A isoform of the progesterone receptor. ORG2058, R5020, RU486, and ZK98299 acted as progesterone receptor agonists with regard to their effect on thymidine incorporation. P4 down-regulated cyclin B1 expression and cdk1 activity and up-regulated the p21 and p27 proteins. Expression of a reporter gene downstream to an AP-1 responsive element in a plasmid construct transfected into ovarian epithelial tumor cells was induced by P4 and inhibited by RU486. We conclude that P4 inhibits cell cycle activity in ovarian epithelial tumors, in part via down-regulation of the cdk1/cyclin B complex. This inhibitory effect may have therapeutic utility against ovarian epithelial tumors.