Ramakrishnan, Vijay

Abstract Abstract 4034 Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4078 Background: Cytokine stimulated signaling pathways contribute to multiple myeloma (MM) disease progression and in acquired resistance to current treatment options making MM an incurable malignancy. It is very well documented that HGF is a cytokine that is secreted by bone marrow stromal cells which has an autocrine and paracrine role in the disease progression in a myeloma setting. HGF binds to its receptor tyrosine kinase on MM cells, MET and this binding at the extracellular domain results in activation of MET which interacts with several of its target proteins resulting in increased survival, increased proliferation, cell cycle progression, motility, migration and invasion. In normal bone marrows, co-expression of MET and its ligand HGF is a rarity whereas co-expression is a common feature of MM. Elevated levels of HGF have been observed in serum and bone marrows of MM patients with a negative correlation to disease progression. In addition, increased HGF levels cause abnormal and reduced bone formation in patients. HGF gene levels in MM samples have been observed to be significantly up regulated in myeloma cells when compared to normal cells. Recently, studies have identified that HGF facilitates the MM cells to adhere to fibronectin, a bone marrow matrix protein, thereby positively impacting MM cell invasion and proliferation. Overall, HGF and its receptor mediated pathway influences tumor progression in myeloma by targeting several different aspects of the disease biology and hence is a very attractive and potentially very important target for improving treatment regimens in a myeloma setting. Methods: MK2461 was synthesized by Merck Inc. (Whitehouse Station, NJ, USA). Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various durations. Results: MK2461 treatment led to dose and time dependent cytotoxicity in a few myeloma cell lines (OPM2, DOX40, RPMI8226 and LR5) but not in others (MM1S, MM1R, H929 and U266). The IC50 values for the sensitive lines varied from 1μ M (OPM2) to 10μ M (DOX40, RPMI8226 and LR5). However, MK2461 significantly inhibited the proliferation of MM cells at sub IC50 concentrations in all cell lines tested except MM1S. This inhibition of proliferation was observed when cells were co-cultured with stromal cells or cytokines, namely VEGF, IL6 or HGF. Culturing MM cells with increasing doses of HGF was still unable to protect them from drug induced inhibition of proliferation. MK2461 was able to induce time dependent increase in apoptosis (as measured by annexin/PI), decrease in proliferation (as measured by BrdU assay) and induction of cell cycle arrest in the drug sensitive cell lines. This effect was not observed in MM1S cells. Exploring the mechanism of action of the drug indicated that MK2461 treatment led to down regulation of pc-Met, pGab1, pAkt and pErk in both the drug sensitive (OPM2) and drug resistant (MM1S) cell lines. However, proteins down stream of Akt in the PI3K/Akt pathway, namely pGSK3β, p70S6K, Bcl2, cyclin E and cyclin D3 were down regulated only in OPM2 cells. On the contrary, we observed up-regulation of these proteins in the drug resistant cell line offering a possible explanation for the drug resistant phenotype. We have also examined combinations of MK2461 with inhibitors of PI3K/Akt pathway. Conclusion: These studies demonstrate significant in-vitro activity of MK2461 in MM. Our results suggest the presence of two populations one very sensitive to MK2461 and one insensitive. Differential effects on the signaling pathways provide important clues to the mechanisms of action of c-met inhibitors in myeloma. The results form the basis for clinical evaluation of MK2461 in MM. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Abstract Abstract 2998 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. Inhibitors of apoptosis (IAP) proteins represent a conserved group of proteins that are important regulators of apoptosis. X-linked IAP (XIAP) is the best studied IAP and inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP and levels are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in the myeloma microenvironment. IL6 and IGF1 up-regulate XIAP by activating the NF-κB, MAPK and PI3K signaling pathways that are commonly aberrant in MM and other tumors. XIAP mRNA contains an internal ribosomal entry site (IRES) sequence in the 5` untranslated region. IRES sequences enable direct ribosome recruitment and aid in translation that is independent of cap-mediated translation. Thus, molecules like mTOR inhibitors might not lead to XIAP downregulation. This was observed when rapamycin, an mTOR inhibitor, was used on MM cell lines. XIAP protein levels were not reduced due to the IRES sequence which leads to translation that is independent of the 5` cap and 4EBP1. Thus XIAP inhibitors might be able to overcome resistance associated with rapamycin treatment. Methods: LC161 was synthesized by Novartis Inc. (Basel, Switzerland). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: LC161 treatment resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 72 hours, with minimal increase seen up to 96 hours of incubation. At 72 hours of incubation, the median inhibitory concentration varied considerably between various cell lines with an IC50 range of 2.5–25μ M. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Dose-dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Primary myeloma cells from patients were treated with increasing doses of the drug and dose dependent increase in apoptosis was observed. Immunoblotting studies demonstrated dose dependent significant down regulation of Xiap, cIAP1, cIAP2 and surviving and up-regulation of activated caspases 3, 8 and 9 and PARP. Furthermore, LC161 resulted in down regulation of pAkt, canonical and non-canonical NF-κB, pJNK, p-p38MAPK, c-Myc, Bcl-xL and Mcl1 and up-regulation of pErk and Bcl-2. We are currently examining basal levels of expression of the IAP proteins (Xiap, cIAP1 and cIAP2), pAkt and pErk in various MM cell lines to identify marker proteins that might predict response to this class of drug. In addition, our initial studies of LC161 in combination with the proteasome inhibitor bortezomib demonstrated synergy in killing MM cells in vitro. Additional combinations including inhibitors of the Akt/mTOR pathway and MEK/Erk pathway are currently been done. Conclusion: These studies demonstrate significant in vitro activity of LC161 in MM. Our results suggest the presence of two populations one very sensitive to IAP inhibition and one relatively less sensitive. Our current studies will help identify marker proteins that might predict response to LC161 treatment. We are currently testing LC261 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of LC161 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Abstract Abstract 2848 Poster Board II-824 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. IL-6 is a critical cytokine involved in myeloma cell proliferation and survival and exerts its activity primarily through the JAK/STAT pathway. In addition to IL6, other cytokines are also believed to cross talk with the JAK/STAT pathway, making it a crucial interface for survival signals. It has been implicated in myeloma cell interaction with the microenvironment and resistance to apoptotic stimuli from different drugs, and represents a potential therapeutic target. We examined the pre-clinical activity of a novel JAK2 tyrosine kinase inhibitor TG101209. Methods: TG101209 (N-tert-butyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-ylamino)-benzenesulfonamide) was synthesized by TargeGen Inc. (San Diego, CA, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and using Apo2.7 in primary patient cells. CD45 expression was estimated using flow cytometry and cells were gated by their CD45 expression to assess differential effects of the drug. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: TG101209 resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 48 hours, with minimal increase seen up to 96 hours of incubation. At 48 hours of incubation, the median inhibitory concentration was between 2 and 4uM with similar IC50 seen for myeloma cell lines sensitive or resistant to conventional therapies. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Increasing doses of IL6 was not able to rescue the cells from the drug. Dose dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Addition of the pan-caspase inhibitor zvad-fmk did not prevent drug-induced apoptosis confirming non-caspase mediated mechanisms of cell death as well. Primary myeloma cells from several patients were treated with increasing doses of the drug and IC50 similar to cell lines were seen in 8/10 patient samples tested. Interestingly, evaluation of U266 cell lines, which have a mix of CD45+ and negative cells as well as primary patient cells demonstrated more profound cytotoxicity and anti-proliferative activity of the drug on the CD45+ population relative to the CD45- cells. Immunoblotting studies demonstrated significant down regulation of IL-6 induced pSTAT3 with minor effects on the pERK and pAkt. The effect on pSAT3 was sustained compared to that on pERK and pAkt. This was accompanied by significant down regulation of Bcl-xL. Studies in a mouse model of myeloma are planned. Conclusion: These studies demonstrate significant in-vitro activity of JAK2 inhibition in multiple myeloma. In particular, the preferential targeting of CD45 cells, considered to reflect the proliferative compartment in myeloma holds out the promise for more sustained impact on the disease from a therapeutic standpoint. This is likely explained by the increased sensitivity of the CD45 cells to cytokines as a result of higher expression of different cytokine receptors as has been previously shown. This leads to increased activity of and dependence of the cells on the JAK-STAT pathway and likely explains the increased effect of the pathway inhibition. These studies form the framework for clinical evaluation of the drug in the setting of myeloma. Disclosures: Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding.

Abstract Abstract 2943 Background: Inhibitor of apoptosis (IAP) proteins represents a conserved group of proteins that are important regulators of cell survival and apoptosis. X-linked IAP (XIAP) is the best studied IAP that inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP. The levels of XIAP are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in myeloma microenvironment. The other two main IAP proteins, namely cIAP1 and cIAP2 are not direct inhibitors of caspases. Instead, they modulate the levels of various signaling pathways by ubiquitinating proteins within the pathways. The NFKB pathway could be activated or inhibited by cIAP1 and 2. In MM, deletions of cIAP1 and cIAP2 have been shown to activate non-canonical NFKB pathway, which indicates a possible tumor suppressor role of these proteins. We wanted to investigate the role of the three IAPs by using a small molecule inhibitor. Our studies clearly indicate the importance of inhibiting all the three IAPs for the induction of apoptosis in MM cells. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. siRNA to cIAP2 was purchased from Invitrogen and was electroporated into MM1S cells. Results: We first examined baseline levels of cIAP1, cIAP2 and XIAP in several MM cell lines and a few patient cells. We observed that the IAPs were constitutively expressed in MM cells. We then wanted to examine the functional significance of these IAP proteins in MM cells. For this, we used an IAP inhibitor LCL161. We observed that LCL161 was able to induce cytotoxicity and inhibit proliferation of MM cells, albeit with differences observed between cell lines. We then examined the factors contributing to resistance in the less sensitive cell lines. For this we chose H929, a sensitive cell line and MM1S, a less sensitive cell line to LCL161. Upon treatment with LCL161, cIAP1 and XIAP were down regulated accompanied by increase in levels of activated caspases 9, 8 and 3 in both H929 and MM1S cells. Using LCL161 in combination with a caspase 9 or a caspase 8 or a pan caspase inhibitor showed clearly that the extrinsic pathway is more involved in the LCL161 induced cell death process. LCL161, however, was unable to inhibit cIAP2 in the less sensitive cell line MM1S whereas cIAP2 was not found to be expressed in H929 cells. It has been shown that cIAP1 is required for ubiquitination and degradation of cIAP2. Therefore, cIAP1 down regulation by LCL161 could actually be contributing to the lack of down regulation of cIAP2 and the observed resistance to LCL161. In order to test this, we used a siRNA to cIAP2 and transfected it into MM1S cells by electroporation. We observed that the siRNA reduced cIAP2 levels and in combination with LCL161 led to marked increase in cells undergoing apoptosis. We also examined signaling pathways after treatment with LCL161 and observed upregulation of both canonical and non-canonical NFKB pathways and Jak/Stat pathway in MM1S cells and not in H929 cells. Combining LCL161 with a Jak2 specific inhibitor SD-1029 synergized in inducing cell death in MM1S and other cell lines less sensitive to LCL161. We are currently testing this combination in MM patient cells. Conclusion: These studies demonstrate the importance of inhibiting cIAP1, cIAP2 and XIAP together in MM cells. Furthermore, by this study we were able to identify resistance mechanisms that are upregulated due to inhibiting the IAP proteins and the importance of using agents that inhibit the IAPs along with inhibitors of these pathways in inducing apoptosis in MM cells. The findings from these studies form the basis of evaluation of IAP inhibitors in combination with a Jak/Stat pathway inhibitor in patients with MM. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

Abstract Abstract 1848 Background: Aurora kinases play an important role in cell division by controlling chromatid segregation. Aberrant functions of aurora kinases result in genetic instability, a condition often seen in cancers. While Aurora A is important in the alignment of the sister chromatids, Aurora B, a spindle fiber associated kinase, heavily influences the equal division of sister chromatids and along with the Aurora C kinase also assists in a regulated cell division. Inhibition of either of the Aurora family kinases affects chromosomal alignment and segregation during the course of cell division resulting in polyploidy, cell growth arrest and ultimately cell death. VEGF has been implicated in the increased angiogenesis in MM patients. Increase in VEGF levels leads to upregulation in the Ras/Mek/Erk pathway and increased angiogenesis, cell proliferation and decreased apoptosis. Inhibiting this pathway has shown to induce apoptosis in MM cells. We therefore investigated the role of a small molecule inhibitor A-1014907 that inhibits both aurora kinases and VEGF stimulated Ras/Mek/Erk pathway. Methods: A-1014907 was synthesized and provided by Abbott Laboratories Ltd. Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: A-1014907 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested with IC50 values between 50–100nM. Similar extents of inhibition of proliferation was also observed when MM cells were co-cultured with bone marrow stromal cells or HUVEC cells or tumor promoting cytokines IL6, IGF and VEGF. The increase in cytotoxicity was due to apoptotic cell death observed in both MM cell lines and patient cells. Cell cycle assays demonstrated that A-1014907 was able to induce cell cycle arrest at the G2/M stage of cell cycle followed by polyploidy indicative of Aurora B inhibition. We also observed an increase over time the proportion of cells in sub G0/G1 stage indicative of cell death. Western blots were performed to understand the mechanism of action of A-1014907. We observed that A-1014907 was able to significantly down regulate Aurora B activity as measured by pHistone H3 (Ser 10) down regulation. This was accompanied by up regulation of p21 and down regulation of CDK4 and cyclin E both indicative of G2/M arrest. We also observed the ability of A-1014907 to inhibit the Ras/Mek/Erk pathway by measuring levels of pErk post treatment with the drug. A-1014907 potently inhibited pErk levels and this down regulation was also observed when MM cells were co-cultured with either VEGF or HUVEC cells. Furthermore, A-1014907 at sub IC50 doses induced synergistic cell death of MM cell lines when combined with sub IC50 doses of dexamethasone Conclusion and current studies: A-1014907 clearly inhibits the Ras/Mek/Erk pathway and aurora B activity and induces apoptosis in MM cell lines and patient cells. We are currently using siRNA to aurora A in combination with A-1014907. We are also examining the mechanism of action of dexamethasone in combination with A-1014907. All this will help to design clinical trials with A-1014907 either alone or in combination with other anti-MM agents in MM patients. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

Abstract Abstract 2948 Background: Deletions and mutations in the tumor suppressor protein p53 are an uncommon observation in new multiple myeloma (MM) patients and are observed more commonly in patients with advanced disease. p53 deletion has been observed to correlate with poor overall and progression free survival in MM patients. Wild type p53 modulates the expression levels of a broad array of proteins involved in cell cycle progression, apoptosis ultimately leading to cell cycle arrest and apoptosis. p53 is negatively regulated by MDM2. MDM2 binds to and ubiquitinates p53 marking it for proteasomal degradation. In addition, MDM2 is a direct downstream regulator of p53. Targeting the p53-MDM2 interaction by developing agents that bind to the p53 binding motif of MDM2 and reactivating p53 has therefore been an active area of research. Here, we present results from our pre-clinical studies using AT219, a small molecule inhibitor that binds to MDM2 preventing its interaction with p53. Methods: AT219 was obtained from Ascenta Therapeutics. Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Akt1/2 kinase inhibitor (Akti) was purchased from Sigma. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Mitocapture and cytochrome-c assays were also performed to confirm the induction of apoptosis in MM cell lines. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various time points. Results: AT219 induced potent cytotoxicity in MM cell lines MM1S, MM1R and H929, all three expressing wild type p53 with IC50 values of 2.5–5μM. Similar effects were observed when the above mentioned cell lines were treated with AT219 and the inhibitory effect of proliferation of these cells were examined. When MM1S or H929 cells were cultured with bone marrow stromal cells (BMSCs) derived from MM patients or with one of the three tumor promoting cytokines implicated in MM (IL6, IGF or VEGF) and treated with AT219, the drug was able to inhibit the proliferation of both cell lines to similar extents as observed when cultured independently without BMSCs or the cytokines. The increase in cytotoxicity was found to be due to cells undergoing apoptosis as observed when MM1S or H929 cells were cultured with AT219 and % apoptotic cells were measured as measured by annexin/PI, mitocapture and cytochrome c assays. AT219 was also observed to induce more potent apoptosis in primary cells obtained from new MM patients with wild type p53 than in cells obtained from relapsed MM patients with wild type p53. AT219 clearly upregulated p53 as observed by performing immunoblots after treatment with the drug in MM1S and H929 cells. In addition, MDM2 and p21 were also found to be significantly upregulated and Bax was slightly upregulated post drug treatment. Bcl2, Mcl1 and Xiap levels were down regulated. In MM1S cells AT219 treatment resulted in a slight down regulation of pAkt (Ser 473). However, in H929 cells we observed a transient upregulation of pAkt following AT219 treatment. This prompted us to test AT219 in combination with Akti on MM cell lines. Our results on both MM1S and H929 cells using AT219 in combination with Akti demonstrated synergy. We are currently testing this combination in primary cells drawn from MM patients with both wild type p53 and those with p53 deletions and mutations. Conclusions: Our studies validate the anti-MM activity of AT219 in MM patients with wild type p53. In addition to using AT219 in combination with Akti, we are testing AT219 in combination with existing anti- MM chemotherapeutic agents. Interesting results from our studies will form the basis for clinical evaluation of AT219 as a single agent or in combination with an Akt inhibitor or other agents in MM patients. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

Abstract Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and a Results: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX. Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. Disclosures No relevant conflicts of interest to declare.