Haug, Jessica

Introduction: While the molecular target of immunomodulators such as pomalidomide (POM) and lenalidomide (LEN) has been identified, the mechanisms underlying therapeutic resistance remain incompletely understood. The uniformly emerging resistance to therapy over time in the absence of identifiable cereblon pathway mutations in the majority of patients raises questions about alternative mechanisms including aberrant gene expression. Methods: We performed gene expression profiling using an Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray on CD138+ bone marrow cells from patients with relapsed / refractory (RRMM) and newly diagnosed (NDMM) multiple myeloma prior to initiating treatment. Patients were treated on two phase II clinical trial protocols (MC0789: POM ± dexamethasone in RRMM; MC0884: LEN ± dexamethasone in NDMM) between 2007 and 2012. We categorized patients based on their IMWG response as non-responders (SD) and responders (VGPR+). We selected 15 responders and 15 non-responders from MC0789 (n = 30) and compared overall survival, gene expression patterns, and involved cellular pathways between the two groups. We selected 5 responders and 5 non-responders from MC0884 (n = 10) for targeted validation of differentially expressed candidate genes. After data quality control and normalization of gene expression values, differential gene expression was estimated using limma. Statistical significance was adjusted for multiple testing in the discovery set using a false discovery rate-based approach for genome-wide experiments (q-value). We used Gene Ontology and PANTHER pathway analysis for functional annotation of differentially expressed genes. Overall survival estimates were calculated using the Kaplan-Meier method. Computation and visualization were performed in R. Results: Median age at treatment initiation on MC0789 was 65 years (40 - 82), 65% of the patients were male. Pomalidomide resistance was associated with an increase in mortality (median overall survival 1.6 versus 6.4 years, p = 0.009, Kaplan-Meier plot). There were 1076 differentially regulated genes between responders and non-responders (521 up- and 555 down-regulated, q < 0.050 for all genes, volcano plot). Expression of CRBN was 1.5-fold down-regulated in non-responders (q = 0.005). Supervised hierarchical clustering of the top 500 differentially expressed genes demonstrated distinct patterns in pomalidomide-resistant disease (heatmap). Gene ontology analysis revealed protein synthesis as one of the most enriched biological processes (bar graph). Pathway analysis showed a 6-fold enrichment (FDR = 0.007) of the ubiquitin proteasome pathway in pomalidomide-resistant disease. Differentially expressed genes involved key protein degradation pathways, epigenetic modifiers, and transcription factors. Targeted validation in MC0884 revealed 13 common genes with at least 1.5-fold differential expression (5 up- and 8 down-regulated), 12 of which have previously been implicated in the regulation of apoptosis, tumor glucose metabolism, Rho and Wnt signaling, miRNA-driven resistance to chemotherapy, and ubiquitin-dependent protein degradation (Table and Sankey diagram). The most up-regulated gene in non-responders was MYRIP, a gene coding for a vesicle trafficking protein associated with platinum resistance and suppression of pro-apoptotic BCL-2 family members in solid malignancies. The most down-regulated gene was FRZB, a gene coding for a negative regulator of Wnt signaling, previously implicated in the progression of monoclonal gammopathy of undetermined significance to multiple myeloma. Conclusions: Overall survival of patients with pomalidomide-resistant RRMM remains poor. Pomalidomide resistance was associated with differential gene expression in several potentially targetable cellular pathways beyond the known drug target cereblon. Targeted validation of candidate genes revealed common cellular pathways in immunomodulator-resistant disease. Elucidating the exact molecular mechanisms underlying immunomodulator resistance is of considerable interest for biomarker development and the identification of novel therapeutic targets and warrants further exploration. Figure Disclosures Lacy: Celgene: Research Funding. Dispenzieri:Celgene: Research Funding; Alnylam: Research Funding; Intellia: Consultancy; Janssen: Consultancy; Pfizer: Research Funding; Akcea: Consultancy; Takeda: Research Funding. Kumar:Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Introduction: Recent advancements in the treatment of multiple myeloma (MM) have centered on engaging the immune system to target multiple myeloma cells. Although these therapies are being combined with immunomodulatory imide drugs (IMiDs) and corticosteroids, the individual contributions of these drugs on the immune system of MM patients has not been examined in the upfront setting. In this study, we examined the peripheral blood immunophenotypes of newly diagnosed multiple myeloma (NDMM) patients receiving the IMiD lenalidomide with or without the corticosteroid dexamethasone. Methods: To characterize immunophenotypes, we utilized flow cytometry to profile white blood cell populations from 35 patients enrolled in a clinical trial testing the efficacy of lenalidomide with and without dexamethasone in NDMM (NCT00772915). In this trial, all patients were initiated on single-agent lenalidomide. Dexamethasone was initiated in patients that did not meet desirable responses or for disease progression. At each cycle, peripheral blood was stained with a 17-marker antibody panel against several immune lineages and functional surface markers. We grouped patients into two groups: 1) lenalidomide alone or 2) lenalidomide with dexamethasone according to their treatment regimen at each cycle timepoint. Results: First we confirmed anti-myeloma cell activity for both groups by measuring a steady decline in circulating plasma cells in both groups. Examining peripheral blood immunophenotypes showed an expected decrease in T cells and a smaller decrease in B cells in both groups of patients. Closer inspection of B cell populations revealed a switch towards a more immature B cell phenotype in both treatment groups. This was measured as a switch from CD19-CD20+ cells to CD19+CD20- B cells. Inspection of T cell subsets revealed that patients receiving single-agent lenalidomide had a sustained decrease in the levels of CD4+ T cells and increase in the levels of CD8+ T cells. This was seen in both naïve and regulatory T cells evidenced by a decrease in the CD4/CD8 ratio among CD28+ T cells as well as CD25+ T cells. Importantly, this alteration did not lead to sustained alterations in the overall level of CD25+ or CD28+ T cells, and the addition of dexamethasone reverses this trend. In addition to the effects seen on T and B cell numbers, we detected expansions of NK cell populations in patients receiving lenalidomide alone. This expansion is detected as an overall increase in CD56+ mononuclear cells with the majority of cells being CD56+CD3- cells. Conclusions: Our data show that lenalidomide and dexamethasone therapy have shared but distinct effects on peripheral blood immunophenotypes in NDMM. Both drugs alter B cells numbers and populations leading to an expansion of CD19+CD20- B cells. However, lenalidomide alone decreases the CD4/CD8 T cell ratio; and, lenalidomide more strongly expands NK cell populations. The addition of dexamethasone reverses this trend and leads to a restoration of the CD4/CD8 ratio. This suggests that lenalidomide without dexamethasone might be counterproductive in immunotherapies intended to recruit CD4+ T cells. Conversely, lenalidomide alone could increase the efficacy of immunotherapies dependent on NK cell recruitment such as antibody-dependent cellular cytotoxicity (ADCC). This information may benefit future investigations of immune responses in MM patients and improve the adoption of immunotherapies to MM patients. Figure Disclosures Kumar: Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

Introduction: High-risk multiple myeloma can be defined by the presence of specific cytogenetic abnormalities (structural) or by characteristic changes in bone marrow and peripheral blood biomarkers (functional). While both entities are characterized by therapeutic resistance, frequent disease relapses, and adverse survival outcomes, the underlying molecular mechanisms remain incompletely understood. Methods: We performed gene expression profiling (GEP) using an Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray on CD138+ bone marrow cells from 137 patients diagnosed with multiple myeloma between 2004 and 2012. All patients underwent Fluorescence In-situ Hybridization (FISH) evaluation, plasma cell labeling, International Staging System (ISS) risk stratification, and GEP prior to initiating treatment with novel agents. The presence of del(17p), t(4;14), t(14;16), and t(14;20) on FISH, a plasma cell labeling index (PCLI) > 2%, and ISS stage III were considered high-risk abnormalities: FISH-HR (n = 15, structural high-risk, at least one high-risk FISH lesion), PCLI-HR (n = 20; functional high-risk, PCLI > 2%), and ISS-HR (n = 12; functional high-risk, ISS stage III). For each HR group we sampled standard risk (SR) controls in a 4:1 ratio. After data quality control and normalization, differential gene expression was estimated using limma. Statistical significance was adjusted for multiple comparisons using a false discovery rate-based approach for genome-wide experiments (q-value). We employed PANTHER pathway analysis for the differentially expressed genes in each HR group. We implemented a simple gene expression score (GES) by calculating the sum of quartiles of the normalized gene expression values for genes differentially expressed in more than one HR group (GES = ΣUP(quartile - 1) + ΣDN(4 - quartile)) and externally validated its prognostic significance (UAMS TT2 / TT3, GSE24080). Survival outcomes were analyzed using the methods described by Kaplan, Meier, and Cox. Computation and visualization were performed in R. Results: Median age at diagnosis was 63 years (32 - 87), 53% of the patients were male. High-risk disease was associated with inferior overall survival, regardless of the used definition (left Kaplan-Meier plots): FISH-HR (HR 4.3, 95% CI 1.9 - 9.8, p < 0.001), PCLI-HR (HR 2.7, 95% CI 1.4 - 5.3, p = 0.004), and ISS-HR (HR 2.8, 95% CI 1.2 - 6.5, p = 0.015). There were 59 (FISH-HR), 424 (ISS-HR), and 507 (PCLI-HR) differentially expressed genes (q < 0.050 for all genes, volcano plots). PCLI-HR and FISH-HR demonstrated a predominance of transcriptional up-regulation while ISS-HR had a balanced gene expression profile with a similar number of genes being up- and down-regulated. The involved cellular pathways were different across the HR groups except for anti-apoptotic signaling (bar graphs). All HR groups had distinct gene expression profiles with no complete overlap between all HR groups. There were 71 genes with overlap between two HR groups (69 up-regulated, 2 down-regulated, Venn diagrams). The median GES was 97 (18 - 206, higher numbers indicating higher expression of up-regulated and lower numbers of down-regulated high-risk genes) in 559 patients treated on UAMS TT2 / TT3 (GSE24080). Tertiles of the GES were associated with event-free survival (HR 1.4, 95% CI 1.2 - 1.6, p < 0.001) and remained independently prognostic after adjusting for age, sex, and ISS stage (HR 1.3, 95% CI 1.1 - 1.5, p < 0.001). Conclusions: High-risk multiple myeloma remains associated with inferior overall survival, regardless of the used definition (structural or functional). The subtypes of high-risk disease have distinct gene expression profiles and involve different cellular pathways, providing important clues to the underlying biology. A 71 gene signature derived from the different high-risk subtypes was of prognostic significance in a clinical trial population after adjusting for known prognostic factors. Figure Disclosures Lacy: Celgene: Research Funding. Dispenzieri:Akcea: Consultancy; Intellia: Consultancy; Janssen: Consultancy; Pfizer: Research Funding; Takeda: Research Funding; Celgene: Research Funding; Alnylam: Research Funding. Stewart:Takeda: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Ono: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy. Bergsagel:Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

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 Background: Epigallocatechin-3-gallate (EGCG) is a polyphenol extracted from green tea that has been studied for cancer prevention and treatment. Green tea and its extracts have been an integral part of traditional medicines from Asia and are thought to have considerable medicinal value. EGCG has been studied in many tumor types and in vitro studies have been encouraging. It is currently in Phase I/II clinical trial in the treatment of chronic lymphocytic leukemia. Here we examined the activity of EGCG in the setting of myeloma and its potential mechanisms of action. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of EGCG was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Fresh myeloma cells were obtained from bone marrow aspirates and subjected to ACK lysis to remove red cells. These cells were cultured in RPMI1640 with or with out EGCG and apoptotic rates were determined by Apo 2.7 expression on gated plasma cells. Flow cytometry was used for determining the phosphorylation state of various signaling proteins. Anti-angiogenic activity of EGCG was measured using a human in vitro angiogenesis kit (Angiokit). Results: EGCG treatment of myeloma cell lines resulted in dose- and time-dependent cytotoxicity to the cells. EGCG was cytotoxic to several myeloma cell lines, including IL-6 dependent cell lines KAS-6/1 and ANBL-6 as well as drug resistant cell lines MM1.R, Dox40 and LR5. The effect was maintained when the cells were grown in co-culture with marrow stromal cells, indicating the ability to overcome microenvironment mediated resistance. EGCG induced apoptosis of myeloma cell lines, apoptotic cells being measured as Annexin (+)/PI (−) cells by flow cytometry. It induced a dose-dependent apoptosis of freshly isolated myeloma cells with increased effect seen on the CD45+ myeloma cells compared to CD45- cells, reflecting increased activity on the proliferative fraction. Down regulation of Akt phosphorylation was seen following treatment of myeloma cells with EGCG. Using the Angiokit, we were able to demonstrate inhibition of tubule formation by EGCG at concentrations lower than those cytotoxic to myeloma cells. It was also able to overcome the angiogenic activity of VEGF. Conclusion: We demonstrate potent anti-myeloma activity of EGCG in vitro. In vivo studies in animal models of myeloma are ongoing. We demonstrate, as in other cell lines, inhibition of Akt pathways as a potential mechanism of action of EGCG. Ongoing studies are exploring other mechanisms. Most importantly, we demonstrate anti-angiogenic activity of this compound in the setting of myeloma, a finding in concordance with the inhibition of VEGF activity seen with other tumor systems.

Abstract Background: The clonal plasma cells in multiple myeloma have relatively low rates of apoptosis. We have previously shown that the apoptotic rates of the plasma cells have prognostic value in these patients. However, several methods are used both for targeting the myeloma cells for study as well as for detection of apoptosis in these cells. It is not clear which method is optimal for studying apoptotic rates of plasma cells from primary patient samples. We examined two different ways of studying the plasma cells, namely CD138 magnetic bead based positive selection of myeloma cells (sorted) and gating on the plasma cells in whole bone marrow (unsorted) using CD38/CD45 expression patterns. Additionally, we compared two different methods of identifying apoptotic plasma cells. Methods: Paired samples processed by two different methods were studied. The bone marrow aspirate samples were initially processed using ACK lysis to remove the red cells. The samples were then studied using flow cytometry (unsorted sample) or subjected to a second step of magnetic bead based separation of myeloma cells based on CD138 expression (sorted sample). Each of the samples was studied using Annexin/PI and Apo 2.7 expression to identify the plasma cells undergoing apoptosis. Plasma cells were identified in the unsorted samples by virtue of their CD38/CD45 expression. Samples were studied at collection and after 48 hours of culture. Results: At collection, the median percentages of cells considered apoptotic by annexin/PI (Annexin pos, PI neg) were higher than that detected by the Apo 2.7 method (Apo 2.7 + cells), for both sorted (6.5% vs. 1.5%) and unsorted cells (26% vs. 10%). However, after 48 hours of culture the percentage of apoptotic cells detected using the Apo 2.7 method were higher than that by Annexin/PI for both sorted (22.5% vs. 14.5%) and unsorted cells (43.5% vs. 10%). When the 48 hr study was compared with the initial study, percent apoptotic cells detected by Apo 2.7 had increased at 48 hrs for both sorted and unsorted cells. However, the percentage of apoptotic cells by annexin/PI had decreased in the unsorted cells at 48 hrs compared to initial study. The sorted samples had an overall increase in apoptosis at 48 hours as measured by annexin/PI. We then looked at the percentage of apoptotic cells detected by either method on initial study and compared the sorted cells and unsorted cells. Using either method, the percentage of apoptotic cells was significantly less (P=0.01; paired t-test) among the sorted cells compared to the unsorted cells. Conclusion: Previous studies have shown that CD138 expression is lost from PCs as they enter apoptosis. Here we show in paired samples that the percentage of apoptotic myeloma cells significantly decreases following CD138 sorting. CD138 sorting should not be used when studying apoptosis of myeloma cells in patient samples. Use of unsorted cells also allows for examination of the effects of interventions on other cell populations as well. When serial studies of apoptosis are done with unsorted samples, we recommend using Apo 2.7 rather than Ann/PI, since the percentage of apoptotic cells by Ann/PI expression actually appear to decrease with time in culture, thus making it difficult to assess effects of intervention such as in drug treatment studies. We hypothesize that this is due to an artificially high level of Annexin positivity at the initial study that might be brought on by non-specific damage of the membranes secondary to cell processing.

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.