Purified Tumor Cells Research Articles

An Investigational Novel Orally Bioavailable Proteasome Inhibitor MLN9708/MLN2238 Triggers Cytotoxicity In Multiple Myeloma Cells Via p21- and Caspase-8-Dependent Signaling Pathway

AbstractAbstract 2992 Background and Rationale:Therapeutic targeting of Ubiquitin-Proteasome Signaling (UPS) is exemplified by the FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM). As with other agents, however, dose-limiting toxicities and the development of resistance limit its long-term utility. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a selective, orally bioavailable proteasome inhibitor currently in phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. Similar to bortezomib, MLN2238 is a boronic acid analog; however, it is distinct from bortezomib since preclinical data demonstrates it has a shorter proteasome dissociation half-life, as well as improved pharmacokinetics, pharmacodynamics, and antitumor activity in xenograft models versus bortezomib. In the present study, we examined the anti-tumor activity of MLN2238 in MM cells using in vitro model systems. Methods and Model:We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, OPM2, H929, LP1, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability, proliferation, and apoptosis assays were performed using Trypan blue, MTT, CellTiter-Glo, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Statistical significance of data was determined using a Student t test. Results:We first confirmed the functional specificity of MLN2238 using different experimental strategies: 1) Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates showed that MLN2238, like bortezomib, primarily inhibits chymotrypsin-like proteasome activity (EC50 = 9 ± 2.3 nM versus bortezomib: 5.5 ± 1.5 nM); 2) Treatment of MM.1S MM cells with MLN2238 induces a time- and dose-dependent accumulation of ubiquitinated proteins; and 3) Washout experiments showed that MLN2238 is a reversible proteasome inhibitor (P < 0.001; n=3). We next examined the effects of MLN2238 in MM cells. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, OPM2, H929, LP1, KMS-12BM, and INA-6) and primary patient cells for 48h significantly decreased their viability (IC50 range 12.5 – 25 nM) (P < 0.001; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for MLN2238. MLN2238-triggered apoptosis was confirmed in MM.1S and H929 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, MLN2238 induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that MLN2238-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of p53, Noxa, PUMA, Bid, E2F, and pAKT; 3) downregulation of MM cell growth and survival proteins: NF-kB, Bcl2, phospho-Rb protein, and cyclin-A; 4) inhibition of migration of MM cells and angiogenesis; and 5) increased expression of CDK inhibitor p21. Importantly, blockade of p21 using both siRNA strategy and p21- knockout HCT116 cells showed significant abrogation of MLN2238-induced cell death (P value < 0.001; n=3). We next examined the requirement for caspase-8 versus caspase-9 during MLN2238-induced apoptosis. Incubation of MM.1S cells with pan-caspase inhibitor (Z-VAD-FMK) markedly abrogates MLN2238-induced apoptosis. Inhibition of caspase-8 (IETD-FMK) led to a significant decrease in MLN2238-triggered cell death, whereas inhibition of caspase-9 (LEHD-FMK) only moderately blocked MLN2238-triggered decreased viability in MM.1S cells (P < 0.001, n=3). These data suggest that MLN2238-induced apoptosis in MM cells is predominantly mediated by caspase-8 extrinsic apoptotic pathway. Finally, combination of MLN2238 with lenalidomide or dexamethasone triggered synergistic anti-MM activity. Conclusions:Our preclinical study supports further clinical evaluation of MLN9708, either alone or in combination, as a potential MM therapy. Disclosures:Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Anti-Myeloma Activity of Enzymatically Activated Melphalan Prodrug J1

AbstractAbstract 1838 Background and Rationale:The alkylating drug melphalan is routinely used in clinical protocols for the treatment of multiple myeloma (MM). Importantly, clinical trials in MM have effectively utilized combination of melphalan with proteasome inhibitor bortezomib and prednisolone (VMP regimen) to reduce toxicity, overcome drug resistance and enhance cytotoxicity. These findings highlight the utility of conventional alkylating agent, and importantly, provide impetus to develop conventional agents based prodrugs with a potent cytotoxic activity. In this context, pharmacological screening of alkylating oligopeptides led to the identification of a novel melphalan-containing prodrug J1 (L-melphalanyl-p-L-fluoro phenylalanine ethyl ester) - a new molecular entity with a more distinct activity profile than melphalan (Gullbo J, et al., Anticancer Drugs 2003,14:617–24; Gullbo J, et al., Invest New Drugs 2004, 22:411–20; Wickstrom M, et al., Mol Cancer Ther 2007, 6:2409–17). J1 is rapidly incorporated into the tumor cells cytoplasm, followed by intracellular hydrolysis in part mediated by aminopeptidase N (APN), resulting in a 10-fold greater release of free intracellular melphalan than exposure to melphalan at the same molar concentration (Gullbo J, et al., J Drug Target 2003,11:355–63; Wickstrom et al., Biochem Pharmacol 2010, 79(9):1281-90). In vitro studies showed a greater cytotoxic potency of J1 versus melphalan against different human solid cancers; however, its effect in MM is undefined. In the present study, we examined the anti-tumor activity of J1 in MM cells using both in vitro and in vivo model systems. Methods and Models:We utilized MM.1S, MM.1R, RPMI-8226, melphalan-resistant derivative of RPMI-8226 (LR-5), KMS12BM, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability-, proliferation-, and apoptosis assays were performed using Trypan blue, MTT, thymidine incorporation, and Annexin V/Propidium iodide staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymologic assays. Statistical significance of data was determined using Student t test. Results:As aminopeptidase N (APN) has been shown to play a key role in conversion of J1 into melphalan in solid tumors, we first examined both expression and enzymatic activity of APN in MM cells. Immunoblot analysis showed a high expression of APN in various MM cell lines. Similarly, colorimetric analysis of APN enzymatic activity using the APN substrate L-alanine-4-nitro-anilide demonstrated elevated APN activity in MM cells. Moreover, pre-treatment of MM cells with APN inhibitor Bestatin showed a moderate, but significant blockade of J1-induced cytotoxicity in MM cells (P < 0.05; n=3). We next examined the effects of J1 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 0.5 – 1.0 uM; P < 0.001; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index forJ1. Of note, the IC50 range of melphalan for MM cell lines is 10–40 uM. J1-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by marked increase in Annexin V+ and PI-cell population (P < 0.001, n=3). Importantly, J1induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that J1-triggered apoptosis in MM cells is associated with 1) activation of caspase-7, caspase-8, caspase-9, caspase-3, and PARP; 2) induction of phospho-c-Jun and phospho-JNK, p53, and p21; 3) release of mitochondrial apoptogenic protein cytochrome-c; 4) inhibition of VEGF-induced migration of MM cells and angiogenesis; and 5) induction of DNA damage response, evidenced by increase in phospho-histone H2AX. Pre-treatment of MM cells with pan-caspase inhibitor Z-VAD-fmk attenuated J1-triggered MM cell apoptosis (P value < 0.001; n=3). Finally, treatment of tumor-bearing mice with J1 (3 mg/kg, twice a week for 2 weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Conclusions:Our study provides the rationale for clinical protocols evaluating J1, either alone or in combination, to improve patient outcome in MM. Disclosures:Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Spira:Oncopeptide AB: Employment, Equity Ownership. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept

The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence-activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis. Mononuclear cells were collected from 10 L of blood by leukapheresis, to which carboxyfluorescein succinimidyl ester prelabeled CaOV-3 tumor cells were spiked at a ratio of 26 to 10⁶ leukocytes. Elutriation separated the spiked leukapheresates primarily by cell size into distinct fractions, and leukocytes and tumor cells, characterized as carboxyfluorescein succinimidyl ester positive, EpCAM positive and CD45 negative events, were quantified by flow cytometry. Tumor cells were isolated from the last fraction using FACS or anti-EpCAM coupled immunomagnetic beads, and their recovery and purity determined by fluorescent microscopy and real-time PCR. Leukapheresis collected 13.5 x 10⁹ mononuclear cells with 87% efficiency. In total, 53 to 78% of spiked tumor cells were pre-enriched in the last elutriation fraction among 1.6 x 10⁹ monocytes. Flow cytometry predicted a circulating tumor cell purity of ~90% giving an enrichment of 100,000-fold following leukapheresis, elutriation, and FACS, where CaOV-3 cells were identified as EpCAM positive and CD45 negative events. FACS confirmed this purity. Alternatively, immunomagnetic bead adsorption recovered 10% of tumor cells with a median purity of 3.5%. This proof of concept study demonstrated that elutriation and FACS following leukapheresis are able to enrich and isolate tumor cells from a large blood volume for molecular characterization.

Statistical evaluation of a molecular classification tool for discriminating cancer recurrence versus new primary cancer formation.

e21059 Background: Second cancer formation is an increasingly important problem because of longer survival of cancer patients and improved imaging and sampling technologies. Traditional microscopic criteria (morphologic similarity, immunostain patterns) are not reliable or may not be applicable (eg, fine needle aspirates that disrupt morphology; multiple cancer sites in same organ) when determining the relatedness of 2 cancers. We created a classification rule to discriminate metastasis from new primary based on comparing the mutational profiles of both cancers. Methods: Archival specimen pairs from 2 cohorts consisted of 63 pts with breast, colorectal and lung cancer and known metastasis and 21 pts with known independent second primary cancers in which one tumor involved breast, colon or lung. Classification into either cohort was independent of molecular results. Extracted DNA from microdissected targets of pure tumor cells taken from routine histology slides were evaluated for 17 oncogene or tumor suppressor gene mutations. We created a distance metric to evaluate the degree of difference in each mutational marker for each pair of tumors in each patient. Low distance implies similar mutations (metastasis); high distance, dissimilar mutations (new primary). Using a training set representing subsets of each cohort, we evaluated reference levels and classification rules. Results: We found that a difference in 4 markers indicated a new primary. This rule achieved 100% accuracy in evaluating the training set. Further evaluation with a test set will be completed shortly. Conclusions: Microscopic analysis has many limitations for determining the relatedness of 2 separate cancers. This classification rule for interpreting the results of comparative mutational profiles may provide a robust way to definitively determine if 2 cancers represent 1 primary with metastasis or 2 separate primaries. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration RedPath Integrated Pathology RedPath Integrated Pathology RedPath Integrated Pathology

Breast cancer metastasis gene signature: The search for the holy grail.

1075 Background: Lymph node status remains one of the most useful prognostic indicators in breast cancer, however, current methods to assess nodal status disrupt the lymphatic system, lead to secondary complications, and 10-33% of women diagnosed with negative lymph nodes will develop metastases. Identification of molecular signatures discriminating primary tumors from patients with the propensity to metastasize from those without would allow for stratification of patients requiring surgical assessment of lymph node status and identification of node negative patients at high-risk of developing metastatic disease. Methods: Frozen breast specimens were collected from women with negative (n=39) and positive (n=35) lymph node status. Patients with positive BRCA1/BRCA2 status, neoadjuvant therapy or negative lymph node status who later developed distant metastasis were not included in this study. RNA was isolated from pure tumor cell populations after laser microdissection and gene expression data generated using HG U133A 2.0 arrays (Affymetrix). Data was randomly split into a training set (29 negative and 25 positive) and a validation set (10 negative and 10 positive) and a classifier was built using a forward-building Bayesian naïve classifier. Results: Neither age at diagnosis nor pathological characteristics including ER, PR, or HER2 status differed significantly between the two groups. A median prediction probability >95% was achieved with the Bayesian classifier using 17 markers (mean probability 94.31%, median 95.05%, min 74.62%, and max 99.91%) in the training set. The 17 marker panel was, however, only able to correctly classify 9/20 samples in validation set, resulting in approximately random classification. Conclusions: The inability to derive molecular profiles of metastasis may reflect tumor heterogeneity (e.g., different methods of metastasis for each tumor subtype), the presence of only a few cells within the primary tumor that have metastatic potential, or an inherited host susceptibility to metastasis, rather than molecular properties of the tumor itself. Together, these data suggest that a molecular signature that can discriminate between primary breast tumors with and without lymph node metastases does not exist. No significant financial relationships to disclose.

Abstract 3357: Isolation of tumor cell subpopulations using semi-automated tissue dissociation and magnetic cell separation

Abstract The analysis of tumor resident cell and stem cell populations requires proper methods for tissue dissociation and cell purification. Over the past few years, there has been a particular focus on the study of tumor infiltrating lymphocytes (TIL) and cancer stem cells (CSC). TIL can be isolated and characterized using conventional lymphocyte markers whereas CSC-specific cell surface markers are currently established (1). Two important markers, namely CD24 and CD44, have been shown to define CSC in various neoplasms such as breast cancer (2) or colon cancer (3). We have established methods for the enzymatic and mechanical dissociation of solid tumors and optimized them according to the specific needs of a given tissue or cell type. Solid tumors are built up of a mixture of cell types, which are interconnected to each other and surrounded by an extracellular matrix composed of a variety of proteins and polysaccharides. The major goal was to disrupt the extracellular matrix and cell adhesion components without harming the integrity of the cell membrane. This was achieved by a combination of varying enzyme mixes, mechanical forces, incubation periods, and temperatures. The automation of all mechanical steps (gentleMACS™ dissociator) led to reproducible and moreover user independent results with reduced overall processing times. Viable tumor infiltrating lymphocytes (TIL) as well as tumor cells were obtained from melanoma metastases in high numbers. The isolated TIL expanded significantly faster than those from manually dissociated tumors and showed functionality in vitro and in vivo. As for cancer stem cells, we present novel tools for the immunomagnetic isolation of CD44+ and CD44+/CD24- cells. Using magnetic cell sorting (MACS®), a subpopulation of human teratocarcinoma cells could be isolated to a purity of 92% based on their expression of CD44. Furthermore, a combined depletion and positive selection approach allowed for the enrichment of a CD44+/CD24- human CML subpopulation from a starting frequency of 34% to final purity of 95%. The separation of pure tumor cell populations will be necessary for their further characterization and also targeted drug screening approaches. The methods presented here are broadly applicable as they can be easily adapted to other cancer tissues and cell types and should help to optimize and standardize the future basic and clinical cancer research. 1. Trumpp, A. and Wiestler, O. D. (2008); Nature Clin. Prac. Oncology.; Vol. 5 No. 6 2. Al-Hajj, M. et al. (2003); Proc. Natl. Acad. Sci. USA; Vol. 100 No. 7 3. Du, L. et al. (2008); Clin. Cancer Res.; 6751 14 (21) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3357.

The impact of RNA standardization and heterogeneous gene expression on the results of cDNA array of human breast carcinoma

cDNA microarray is an established technique. However, difficulties such as handling tissue samples under RNase-free conditions, the heterogeneous tumor composition, i.e. non-malignant versus malignant cells and different pathologic types of malignant cells, and lack of appropriate reference may limit the potentially benefit of this method in clinical use. In this study, we examined how standardization of gene expression to total mg RNA or mg tissue and tumor heterogeneity affect the final results. We found that the gene expression of human breast tumors was approximately 9 times higher in malignant tissue as compared to the non-malignant tissue when expressed per total mg RNA, but approximately 40 times higher when expressed per mg tissue. Genes that were expected to act as housekeeping genes (PUC18, RPL and beta-actin) varied between different parts of the tumor and also between non-malignant and malignant tissues, excluding them as reference genes. We also found that the gene expression differed in various parts of the breast tumor, probably due to a mixture of different types of cells, i.e. non-malignant and malignant cells. To find out if the variations in the gene expression were due to cell heterogeneity we used microdissection to collect malignant cells separately. We found that the gene expression was markedly different in the isolated malignant cells as compared to the gene expression of the bulk tumor tissue. Thus, to be able to evaluate results from cDNA array gene expression experiments it is, to our opinion, necessary to work with pure tumor cell populations, until solid information is available on the impact of stromal component. Housekeeping genes should be handling with care and mg tissue may be preferred instead of microg RNA for standardization.

Computational Dissection of Tumor Expression Profiles.

Abstract Gene expression profiling has yielded important insights about tumor biology that may improve treatment decisions in patients. However, it is difficult to collect a specimen of pure tumor cells, and thus microarray measurements usually reflect the contribution of tumor cells as well as stromal and other normal cells. We applied unsupervised matrix factorization methods to gene expression data to derive several sets of co-expressed genes, or signatures, that together comprise a set of independent descriptors of breast tumors. Some of these signatures correspond to specific cell types (adipocytes, lymphocytes, fibroblasts), while others reflect well-known tumor-intrinsic expression programs (ER, ERBB2, proliferation). We confirmed the specificity of the signatures using expression data from purified normal cells and tumor cell lines, microdissected tumors, and bulk tumors with corresponding histological cellularity estimates. In several data sets, cell-type signatures were highly variable and anticorrelated with tumor-intrinsic signatures, confirming that variation in normal cell content is a potential source of measurement bias.Overall, these results provide an intuitive framework for the interpetation of tumor expression profiles and may lead to an improved understanding of the physiological mechanisms involved in tumor development. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1166.

Abstract IA-4: Studying tumor evolution in mouse models of cancer

Abstract Like other solid tumors, lung adenocarcinoma progresses through multiple stages including tumor initiation, expansion in the lung, and metastasis of the primary tumor to distant organs. Dissection of this process requires quantitative and tractable models that recapitulate the human disease at the genetic and histological level. We have employed autochthonous mouse models of lung adenocarcinoma that recapitulate tumor initiation, progression and metastasis in the human disease both genetically, and pathologically. We have established oncogenic Kras as the initiating event in lung adenoma and amplification of the oncogenic signaling is associated with progression to adenocarcinoma. The p53 tumor suppressor limits tumor progression in this model. Restoration of the Trp53 locus has pinpointed the adenoma-adenocarcinoma transition as the stage in which p53 function is critical for tumor suppression. Expression of oncogenic Kras in the absence of p53 leads to the development of distant metastases after long latency. This suggests that unknown cooperating genetic events are required for metastatic spread and we are utilizing multiple tools to identify these changes. Use of lentiviral vectors that drive stable GFP expression in tumors allows for identification and purification of tumor cells. Additionally, identification of the lentiviral integration site in each tumor allows the clonal relationship between metastases and the primary tumors to be unequivocally established. Gene expression and DNA copy number analyses of metastasis-derived cell lines and primary lung tumors have uncovered a metastasis associated gene expression signature and recurrent genomic alterations. Integration of these datasets, other genome-wide analyses and human tumor data are providing insights into the molecular mechanism and biology of lung cancer progression and metastasis. Citation Information: Cancer Res 2009;69(23 Suppl):IA-4.

NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Abstract 3842Poster Board III-778 Background and RationaleVascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and MethodsWe utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. ResultsTreatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. ConclusionOur preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures:Chauhan:Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

PR-924, a Selective Inhibitor of the Immunoproteasome Subunit LMP-7 Blocks Multiple Myeloma Cell Growth Both in Vitro and In Vivo.

Abstract 612 Background:Therapeutic targeting of Ubiquitin-Proteasome pathway is exemplified by the recent FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of multiple myeloma (MM). As with other agents, dose-limiting toxicities and the development of drug resistance limit its long-term utility. The β5, β1 and β2 catalytic subunits within the 26S proteasome mediate chymotrypsin-like, caspase-like, and trypsin-like activities, respectively. Importantly, these catalytic subunits have corresponding immunoproteasome components LMP-7, LMP-2 and multicatalytic endopeptidase complex subunit-1 (MECL-1), which regulate immune cell function and cytokine production; however, the role of the immunoproteasome in MM cells is still unclear. Recent studies have therefore focused on the discovery and development of small molecule inhibitors of the immunoproteasomes, which will both delineate the function of immunproteasomes and allow for specific therapeutic targeting of the UPS in order to reduce off-target activities and associated toxicities. Here, we examined PR-924, an LMP-7-selective peptide-ketoepoxide proteasome inhibitor related to carfilzomib. PR-924, like carfilzomib, contains a ketopoxide pharmacophore that covalently modifies proteasomal N-terminal threonine active sites. We examined the effects of PR-924 in MM cell lines and primary patient cells in vitro. To determine the in vivo efficacy of PR-924, we utilized two xenograft models of human MM in SCID mice, a subcutaneous tumor plasmacytoma model and the SCID-hu model, which best reflects the human MM-BM microenvironment in vivo. Methods and Model:We utilized MM.1S, MM.1R, RPMI-8226, U266, DOX40, KMS12, LR-5, OPM1, OPM2 and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability and apoptosis assays were performed using Trypan blue, MTT and Annexin V staining. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, caspase-7, PARP, Bcl-2, BID, or GAPDH. For tumor xenograft studies, CB-17 SCID male mice (n = 10; 5 mice/each group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum-free RPMI-1640 medium. When tumors were measurable (∼150 mm3) 2-3 weeks after MM cell injection, mice were injected IV with either PR-924 (6 mg/kg BW) or vehicle twice weekly. Mice were sacrificed when their tumors reached >2 cm3. In the SCID-hu model, 2 × 106 INA-6 cells were injected directly into human bone chips implanted subcutaneously in SCID mice (n=10: 5 mice/EA group), and MM cell growth was assessed by serial measurements of circulating levels of soluble human interleukin-6 receptor (shulIL6R) in mouse serum. Statistical significance of differences observed in PR-924 vs. vehicle treated mice was determined using a Student t test. Results:PR-924 significantly inhibits growth of all the MM cell lines in a time- and dose-dependent manner (IC50 range: 3-5 μM; P <0.005 for all cell lineIt alsos. reduced the viability of primary patient cells (P < 0.05; n=5), without significant effects on normal peripheral mononuclear cells. The PR-924-triggered decrease in MM cell viability is due to apoptosis, as evidenced by Annexin V/PI staining. Moreover, PR-924-induced apoptosis in MM.1S and MM.1R MM cells is associated with activation of caspase-3, caspase-8, caspase-9, caspase-7, BID and PARP. In vivo PR-924 triggered significant tumor growth inhibition in tumor plasmacytoma xenografts (2.3 fold decrease in tumor volume in mice receiving PR-924 versus mice injected with vehicle alone; P value = 0.01). Similarly, a significant reduction in the shuIL6R levels (3.4 fold decrease; P value = 0.02) was observed in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated, as evidenced by the lack of weight loss even after three weeks of treatment. Importantly, treatment of tumor bearing mice with PR-924, but not vehicle alone, significantly prolonged survival (P < 0.005). Conclusion:Our preclinical findings establish immunoproteasome LMP-7 as a novel therapeutic target in MM. Disclosures:Aujay:Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership.

Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Abstract 1845Poster Board I-871Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures:Trudel:Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

NRF2 at the Interface of Oxidative and Endoplasmic Reticulum Stress Pathways Is a Critical Integrator of Bortezomib Response in Mantle Cell Lymphoma in Vitro and In Vivo.

Abstract 727The proteasome inhibitor bortezomib (BZM) is effective as single-agent in relapsed and refractory mantle cell lymphoma (MCL) but more than half of patients remain insensitive to BZM. Suggested mechanisms of action include activation of Noxa, p53, oxidative, and endoplasmic reticulum (ER) stress. To define mechanisms relevant for BZM-induced cytotoxicity we pursued two approaches: first, we characterized gene expression changes in 10 MCL cell lines exposed for 24h to 10nM BZM, a concentration that kills >50% of sensitive, but <20% of resistant cells. Secondly, we analyzed gene expression changes in tumor cells of patients with leukemic MCL undergoing BZM treatment in a clinical trial. RNA was extracted from MCL cell lines at early (1h and 3h), intermediate (6h) and late (24h) time points. Virtually no changes in gene expression were detectable at 1 and 3h of drug exposure and only about 100 genes changed by 6h. After 24h of treatment 524 genes were significantly changed in sensitive and 271 genes in resistant cell lines respectively. The delayed onset of gene expression changes is consistent with the reversibility of BZM toxicity for up to 8 hours. Using Ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) we identified two dominant responses induced by BZM: 1) an oxidative stress response mediated by NRF2 and related transcription factors, and 2) an ER stress/ubiquitin proteasome response (FDR by GSEA <0.1). Both responses were primarily apparent in sensitive cell lines. A set of 20 experimentally validated NRF2 target genes was used as a core NRF2 signature: this signature was increased 15-fold on average in sensitive cell lines but only 2-fold in resistant cell lines (P=0.006). Similarly, an XBP1 and ATF6 signature, reflecting activation of the ER stress response, was stronger induced in sensitive than in resistant cell lines (average 1.9-fold vs 1.3-fold; P=0.003). Activation of these stress pathways upon BZM treatment was confirmed by demonstrating accumulation of nuclear NRF2 in sensitive Jeko1 but not in resistant Mino cells. Also markers of ER stress such as phosphorylation of ER resident nuclease Ire1 that splices the transcription factor XBP1 and activation of ATF3, ATF4, and CHOP downstream of PERK were readily detected in Jeko1 but not in Mino cells. Finally, Noxa, the BH3-only protein primarily responsible for BZM-induced apoptosis, was only induced in sensitive Jeko1 cells. We next analyzed the effect of BZM on purified tumor cells from five patients with leukemic MCL treated with BZM (1.5mg/m2, day 1, 4, 8 and 11). Two patients showed a >50% reduction in circulating tumor cells after 2 injections of drug (day 8) and >75% reduction after 4 injections (day 2, sensitive), while in three patients there was no change or an intermediate response (resistant). Western blotting demonstrated Noxa up-regulation in circulating tumor cells of sensitive but not resistant samples. This is consistent with the demonstrated importance of Noxa for induction of apoptosis in response to BZM in cell line studies. Next we performed gene expression profiling immediately before, at 6h, and 24h after the first and 24h after the second dose of BZM. Using IPA and GSEA up-regulation of the ubiquitin/proteasome pathway and the NRF2-mediated oxidative stress response was again prominent, but an ER stress response was less apparent. XBP1 splicing was not detected in tumor cells from sensitive samples indicating that an ER stress response was not fully activated by BZM in vivo. Consistent with in vitro data the NRF2 signature was induced 2.3-fold on average in sensitive but not in resistant samples (P<0.05). Intriguingly, baseline expression of the NRF2 signature genes was significantly higher in resistant than in sensitive cells (P=0.0007). In summary, we identify NRF2 as critical integrator of different stress pathways in response to BZM in MCL. Thus, rapid induction of NRF2 target genes might be a useful biomarker of BZM-induced cellular stress and predict clinical response. Our data suggest a more complex function of NRF2 than previously appreciated. NRF2-regulated genes serve primarily homeostatic roles and enable cells to deal with oxidative and xenobiotic insults; a function that may come to play in BZM resistant cells with higher baseline expression of NRF2 target genes. On the other hand, our data suggest a possible pro-apoptotic role of acute induction of high levels of NRF2 that is currently under investigation. Disclosures:No relevant conflicts of interest to declare.

Clonal Origins of 'late' Relapses in ETV6-RUNX1 Acute Lymphoblastic Leukemia..

Abstract 1583Poster Board I-609Approximately a quarter of B cell precursor childhood acute lymphoblastic leukemia (ALL) is characterized by an ETV6-RUNX1 (TEL-AML1) fusion gene and has an overall good prognosis. The majority of these children will be treated on the standard risk arm of the United Kingdom ALL treatment protocols. Relapse usually occurs after cessation of treatment but remarkably can present many years later. The incidence of ETV6-RUNX1 at relapse has been reported to be less than or similar to de novo ALL. Molecular studies on neonatal bloodspots and on twins with concordant ALL have demonstrated the prenatal origin of major subtypes of childhood ALL, including most ETV6-RUNX1 fusion gene positive cases. In addition these investigations have suggested the existence of a preleukaemic stem cell requiring additional mutations or ‘hits' in order to develop frank leukemia. To understand the genetic basis and clonal origin of late relapses we have compared the profiles of genome-wide copy number alterations (CNA) at relapse versus presentation in samples matched with remission DNA from 24 patients. The selected samples had tumor cell purity >75% before DNA extraction. DNA copy number alteration data was generated using the Affymetrix 500K SNP arrays. LOH analysis was performed using CNAG 3.0 and dCHIP 2008. Overall we identified 168 CNA at presentation and 252 at relapse (excluding deletions at IgH and TCR loci), equating to 6.96 and 10.3 CNA at presentation and relapse respectively. Although the number of CNA increased at relapse, no single gene or pathway was uniquely targeted in relapse. The most frequent alterations involved loss of 12p3.2 (ETV6), 9p21.3 (CDKN2A/B), 6q16.2-3 and gain of 21q22.1-22.12. A novel observation was gain of part or whole of chromosome 16 (2 patients at presentation, 5 at relapse) and deletion of the oncogene Plasmocytoma Variant Translocation 1 (PVT1) in 3 patients. Pathway analysis demonstrated frequent involvement at presentation and relapse of genes implicated in both B cell development (44 versus 46%) and cell cycle control (46 versus 71%). In order to study the clonal origin of relapse, we devised a classification describing the change in CNA between presentation and relapse in each individual patient. The clonal relationship between the presentation and relapse clone was established by the persistence of both the ETV6-RUNX1 fusion and at least 1 Ig and/or TCR rearrangement. We used a classification focussed on ‘driver' CNA, defined as CNA that target genes functionally involved in leukemogenesis or CNA that are recurrently targeted as described in the literature. The four categories of relapse were type 1 (the dominant clone at presentation presented unchanged at relapse), type 2 (the relapse clone was derived from the major subclone at presentation with additional CNA), type 3 (the relapse clone was derived from a minor clone at presentation with gains and losses of CNA) and type 4 (the relapse clone is derived from an ancestral or preleukemic clone at initial presentation with all CNA gained). Twenty-one of the 24 patients were classifiable in this way (Figure 1). Although comparative relapse / presentation CNA profiles cannot identify precise clonal origins of relapse, the data indicate that irrespective of time to relapse (<2 to 9.9 years), the relapse clone appeared to be derived from either a major or minor clone at diagnosis with none (0/6) of the very late relapses (>5 years) derived from pre-leukemic cells lacking CNA. This data indicate diverse clonal origins of relapse and extended periods of dormancy, possibly via quiescence, for stem cells in ETV6-RUNX1+ ALL.Relapse typeRemission duration (years)< 22 - 5> 51••2••••••••••3•••••••4••Figure 1. Each patient is represented by a black dot. Each patient is classified on the basis of the relapse type and remission duration. DisclosuresNo relevant conflicts of interest to declare.

Comparative Genomic Hybridization, Gene Expression Profiling and Whole Genome Sequencing Analysis of Disease Progression in Myeloma Reveals Vigorous Clonal Evolution in Patients with High Baseline Genetic Risk.

Abstract 2810Poster Board II-786We wished to explore the genetic events associated with disease progression and development of drug resistance in multiple myeloma (MM). To do so 11 patients were studied in whom at least two (range 2-3) temporally distinct samples of tumor DNA and RNA were available. The baseline genetic initiating event was defined for all patients (3 were genetic high risk; one with t(14;16) two with t(4;14)) as well as the gene expression profile (GEP) defined risk score using the Little Rock 70 or 17 gene panel (only one, the t(14;16) was GEP defined high risk). High resolution array CGH and gene expression were then performed on each sample. Of the 8 patients with a “low risk” tumor initiating event and low risk GEP score, 6 patients had no, or only one, copy number abnormality (CNA) change between the two temporally distinct MM samples. In stark contrast the 3 genetic high risk at baseline had between 17 and 40 distinct CNA changes at the time of progression. For all 11 patients 89 CNA were acquired with progression whereas 19 previously abnormal regions disappeared suggesting clones with these abnormalities were extinguished by the therapy received. In total we detected 0-40 CNA changes between the various timepoints, median 1, mean 10.7. The acquisition of new CNA was much more common than the loss of CNA. We then focused more specifically on the t(4;14) patient with the highest number of CNA changes. This patient has a well documented clinical course of having a sustained two year VGPR to Len/dex and then progressing while still taking Len/dex. Comparison of the pre and post-Len/dex samples identified 40 CNA changes(the most of any pair studied to date). Only six CNA were shared between the two samples, which included deletions of chr4, 9, 12, 13, and X plus a t(4;14) translocation. These likely represent the initiating “driver” tumor events. The new CNA we identified originated from both remodeled genomic changes and the emergence of unique changes, indicating a new tumor clone had emerged while the previously dominant clone had regressed (e.g. a deletion of a large segment of chromosome 8 at diagnosis was no longer observed in the relapse sample). The newly acquired CNA encompassed 3968 genes (13.7% of the genes in the genome), however, only 1235 of these genes (4%) were expressed in this patient at diagnosis (1188 in the typical myeloma patient). Since 1235 genes is still a large number we hypothesized that whole genome sequencing (WGS) would help elucidate the mechanism of lenalidomide resistance. We isolated DNA from germline tissue and CD138 purified tumor cells including: diagnostic, first relapse and second relapse samples. Utilizing SOLiD (Applied Biosystems, Foster City, CA) sequencing technology, we have completed fragment library WGS on both the germline and the final tumor samples. Quality control measures report the average number of sequence reads per start point to be less than 1.2, indicating the library is primarily composed of unique molecules. In addition, approximately 40% of the sequence reads map uniquely to the genome. Together these quality measures indicate our sample libraries are complex and provide good representation of the genome. Data on the whole genome sequence of myeloma at diagnosis and at the time of progression will be presented. Disclosures:Bergsagel:Celgene: Consultancy.

Identification of molecular targets associated with transformed diffuse large B cell lymphoma using highly purified tumor cells

Follicular lymphoma (FL) frequently transforms into the more aggressive diffuse large B cell lymphoma (DLBCL-tr), but no protein biomarkers have been identified for predictive or early diagnosis. Gene expression analyses have identified genes changing on transformation but have failed to be reproducible in different studies, reflecting the heterogeneity within the tumor tissue and between tumor samples. Gene expression analyses on Affymetrix Human Genome U133 Plus 2.0 arrays were performed, using flow cytometry sorted tumor cells derived from FL and transformed DLBCL. To identify molecular targets associated with the transformation, subsequent immunohistochemistry (IHC) analyses of the corresponding proteins were performed. Using highly purified cells, this study identified 163 genes, which were significantly deregulated during the transformation in a majority of cases. Among the upregulated transcripts, 13 genes were selected for validation using IHC, based on the availability of commercial antibodies, and galectin-3 and NEK2 proteins specifically identify DLBCL-tr, when compared with FL. We demonstrate that by purifying tumor cells through cell sorting, thereby reducing the heterogeneity due to infiltrating cells, it was possible to identify distinct differences between tumor entities rather than variations due to cellular composition. Galectin-3 and NEK2 both identified a subgroup of DLBCL-tr, and the function of these protein markers also suggests a biological role in the transformation process.

Targeting aldehyde dehydrogenase: a potential approach for cell labeling

To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. The average radiochemical yields for the synthesis of [(125)I]FMIC and [(125)I]DEIBA were 70+/-5% and 47+/-14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

MicroRNA Expression, Chromosomal Alterations, and Immunoglobulin Variable Heavy Chain Hypermutations in Mantle Cell Lymphomas

The contribution of microRNAs (miR) to the pathogenesis of mantle cell lymphoma (MCL) is not well known. We investigated the expression of 86 mature miRs mapped to frequently altered genomic regions in MCL in CD5(+)/CD5(-) normal B cells, reactive lymph nodes, and purified tumor cells of 17 leukemic MCL, 12 nodal MCL, and 8 MCL cell lines. Genomic alterations of the tumors were studied by single nucleotide polymorphism arrays and comparative genomic hybridization. Leukemic and nodal tumors showed a high number of differentially expressed miRs compared with purified normal B cells, but only some of them were commonly deregulated in both tumor types. An unsupervised analysis of miR expression profile in purified leukemic MCL cells revealed two clusters of tumors characterized by different mutational status of the immunoglobulin genes, proliferation signature, and number of genomic alterations. The expression of most miRs was not related to copy number changes in their respective chromosomal loci. Only the levels of miRs included in the miR-17-92 cluster were significantly related to genetic alterations at 13q31. Moreover, overexpression of miR-17-5p/miR-20a from this cluster was associated with high MYC mRNA levels in tumors with a more aggressive behavior. In conclusion, the miR expression pattern of MCL is deregulated in comparison with normal lymphoid cells and distinguishes two subgroups of tumors with different biological features.

Functional pathway mapping of human glioblastoma multiforme and brain metastases for patient tailored therapy

2076 Background: Genome scanning analysis of human glioblastoma multiforme (GBM) has suggested that this form of cancer is a protein pathway disease. Since genomic analysis cannot directly predict protein activation, analysis of protein pathway activation is required. With the current focus on targeted translational therapeutic modalities, a functional understanding of the GBM signaling repertoire is critical, and yet largely unknown. Methods: Twelve tumors were included in this study: 10 GBMs (9 primary, 1 recurrent) and two brain metastases (1 breast and 1 lung). Pure tumor cell populations were obtained from fixed frozen tissue sections using Laser Capture Microdissection. Protein pathway mapping was performed using Reverse Phase Protein Microarrays (RPMA) whereby the activation of 85 key signaling proteins was quantitatively measured at once. Unsupervised and supervised analysis was used to explore pathway activation. Results: Unsupervised hierarchical clustering of all tumors in the study set revealed largely patient-specific signaling portraits yet also identified distinct pathway subsets. The two metastatic tumors clustered separately and distinctly from the GBMs. The GBM specimens clustered according to pathway activity. Statistical analysis demonstrated significant correlations between certain phosphorylated endpoints detected and overall survival. Phosphorylation of cofilin (S3) was associated with shorter survival time, while Stat1 (Y701) and Shc (Y317) phosphorylation were both positively correlated with longer overall survival. Conclusions: This study represents the most comprehensive proteomic analysis of human GBM pathway mapping to date. Since certain pathway biomarkers are themselves being targeted by current investigational therapies, the ability to map pathway activation and identify critical pathway biomarkers can lead to targeted therapeutics tailored to each patient's tumor. The ability to segregate short from long-term survivors according to protein pathway activation is promising. [Table: see text]

Combination of a Novel Proteasome Inhibitor NPI-0052 and Lenalidomide Trigger in Vivo Synergistic Cytotoxicity in Multiple Myeloma

Background: Our previous study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action, and importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. These preclinical data provided the basis for the ongoing phase-I clinical trial of NPI-0052 in relapsed/refractory MM patients. Recently, a Phase-1/2 clinical trial of bortezomib with Lenalidomide and low dose dexamethasone demonstrated safety and remarkable efficacy in newly diagnosed MM patients. Given that the combination of bortezomib with Lenalidomide has proven a successful treatment strategy, coupled with our findings that NPI-0052 is a potent proteasome inhibitor, we determined whether combining NPI-0052 with Lenalidomide triggered synergistic/additive anti-MM activity.Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies including Lenalidomide or bortezomib. Informed consent was obtained from all patients in accordance with the Helsinki protocol. Cell viability and apoptosis assays were performed using MTT and Annexin V staining. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, BIM, p-JNK or tubulin. In vitro and in vivo proteasome activity assays were performed using fluorogenic peptide substrates. All animal studies were approved by the DFCI Institutional Animal Care and Use Committee. CB-17 SCID male mice (n = 30; 5 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (~150 mm3) three weeks after MM cell injection, mice were treated with oral doses of vehicle alone, NPI-0052 (0.15 mg/kg), Lenalidomide (2.5 mg/kg), Lenalidomide (5.0 mg/kg), NPI-0052 (0.15 mg/kg) plus Lenalidomide (2.5 mg/kg) or NPI-0052 (0.15 mg/kg) plus Lenalidomide (5.0 mg/kg) on a twice weekly schedule for NPI-0052 and four consecutive days weekly for Lenalidomide for four weeks. Statistical significance of differences observed in NPI-0052, Lenalidomide or NPI-0052 plus Lenalidomide-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism.Results: Combining NPI-0052 and Lenalidomide induces synergistic/additive anti-MM activity in vitro using MM cell lines (P<0.005, n=3, CI < 1) or patient CD138-positive MM cells (5 patients, P< 0.004). NPI- 0052 plus Lenalidomide-induced synergistic apoptosis is associated with:•activation of caspase-8, caspase-9, caspase-3, and PARP;•induction of c-Jun-NH2-terminal kinase;activation of BH-3 protein BIM;inhibition of migration of MM cells and angiogenesis;suppression of chymotrypsin-like, caspase-like and trypsin-like proteolytic activities in an additive manner; andinhibition of NF-kappa B signaling.Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus Lenalidomide-induced apoptosis (61 ± 7.1% decrease in cell death; P < 0.003, n=2). Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus Lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low dose combination NPI-0052 (0.15 mg/kg) and Lenalidomide (2.5 or 5.0 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.03), and prolongs survival (4–5 months in mice receiving combined drugs, P = 0.001). Immununohistochemistry analysis of MM tumors excised from NPI-0052 plus Lenalidomide-treated mice showed growth inhibition (Ki-67), apoptosis (TUNEL assay, caspae-3 activation), a decrease in associated angiogenesis (Factor VIII and VEGF receptor), and additive inhibition of proteasome activity. Taken together, our study provides the preclinical rationale for clinical protocols evaluating Lenalidomide together with NPI-0052 to improve patient outcome in MM.