Ghobrial, Irene M.

Abstract The objective of this study was to analyze molecular changes that occur at the protein level in WM and IgM-MGUS in order to identify proteins dysregulated at disease progression and design new therapies. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between WM, IgM-MGUS and normal lymphoplasmacytic cells as control. The antibody array is a novel technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto a glass slide spotted with 512 different monoclonal antibodies specific for human proteins. CD 19+ and CD138+ purified lymphoplasmacytic cells were obtained from cryopreserved bone marrow samples of 5 newly diagnosed patients with WM and 5 patients with IgM-MGUS. Control plasma cells were obtained from 3 pooled CD19+ and CD138+ purified normal donor bone marrow cells. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software and changes of protein expression ≥2 fold in 60% of the samples as compared to normal control were identified in both WM and MGUS groups. There were 20 proteins upregulated in WM and 21 proteins in MGUS as compared to normal control cells These included proteins from the small GTPases such as the Ras, Rho and Ran families (Rab 4, MONA, RCC1, and CDC42GAP), Kinases/signaling proteins (ROK alpha, p62 dok, JAK, 14-3-3e), cyclin dependent kinase (CDK2), fatty acid synthase (HDAC3), NFkB pathway (IKKalpha, IKBe), and histone deacetylase. The WM and IgM MGUS differed in only 3 proteins using the 2-fold threshold. The WM samples showed overexpression of the heat shock protein Hsp90, the Ras family protein CDC25C, and the chemotaxis protein p43/EMAPII as compared to the MGUS samples. This is the first proteomic study of WM and MGUS patients. It identifies novel proteins dysregulated in WM that may serve in future clinical trials as targets for therapy such as Hsp90, NFkB, HDAC and fatty acid synthase. In addition, it identifies 3 proteins that identify disease progression from MGUS to WM, which could be useful for monitoring MGUS patients and designing targeted therapies to prevent progression to WM.

Abstract Abstract 2958 PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. Using an antibody-based protein microarray technique, we identified Citron Rho Interacting Kinase (CRIK) as a protein that was highly expressed in primary multiple myeloma (MM) cells compared to normal plasma cell. We therefore sought to investigate the functional role of CRIK in MM cells. Methods: Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM and 4 healthy control using high-throughput proteomic analysis with antibody-based protein microarray (Clontech, CA). MM.1S, OPM2, RPMI8226, and INA6 were used to perform functional validation. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform and qPCR. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, OPM2, and INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by BrdU, MTT, PI and Apo2.7 staining, and flow cytometry analysis, respectively. Results: We first showed that CRIK was overexpressed in 12 patients with MM compared to normal CD138+ cells obtained from healthy controls using high-throughput protein microarray. We further confirmed CRIK expression using immunohistochemistry in the same samples of MM patients. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Knockdown of CRIK in MM cell lines led to an anti-proliferative and pro-apoptotic effect in all MM cell lines tested. Indeed, CRIK-knockdown MM cells were characterized by a reduction of 60–80% in the proliferation rate, supported by reduction of DNA synthesis and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity of caspase-3, caspase-8, caspase-9, and parp cleavage was also demonstrated in CRIK knockdown cells compared to scramble probe transfected cells. We also showed that CRIK knockdown led to cytokinesis in MM cell lines, indicating a possible mechanism of inhibition of proliferation of these cells. Conclusion: In this study, we show that MM cells express a high level of a novel protein CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of MM cells. CRIK could be a critical therapeutic target in MM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract Malignant B-cells characteristically home to the bone marrow and lymph nodes. However, the mechanisms by which cells are recruited into and mobilized from the bone marrow/lymph nodes into the peripheral blood are not well understood. Chemokines such as SDF-1 and it receptor CXCR4 play a central role for lymphocyte trafficking and homing. Downstream activation of the PI3K pathway by chemokines has been implicated in the migration of many cell types. We recently demonstrated that CXCR4 expression correlates significantly with disease progression in CLL. Patients with Rai stage 4 had a higher expression of CXCR4 when compared to patients with Rai stage 0. The expression of CXCR4 was not found on CLL B cells resident within the lymph nodes implying that there may have been downregulation of CXCR4. In this study, we explore the molecular mechanisms involved in homing and migration of B-CLL cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of CLL cells in response to SDF-1. Boyden chamber in vitro migration assays were used with primary CLL peripheral blood cells and the MEK1 CLL cell line. In addition, we expressed fluorescent YFP-CXCR4 in the cells and used confocal microscopy to visualize changes in the subcellular location of the fluorescent CXCR4 before and after SDF-1 stimulation. Inhibitors of the PI3K pathway such as the PI3K inhibitor LY294002 (10uM, one hour pretreatment) and the mTOR inhibitor, rapamycin (200nM overnight) were used. SDF-1 induced a dose dependent migration of primary CLL cells and MEK1 cells, indicating functional CXCR4 receptors. MEK 1 cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping and internalization subcellularly. Pretreatment of the cells with PI3K pathway inhibitors led to inhibition of migration with LY294002 but not with rapamycin. Confocal microscopy demonstrated abrogation of subcellular localization in the presence of LY294002 but not with rapamycin with the addition of SDF-1. These results demonstrate that CLL cells express functional CXCR4 receptora and that the PI3K pathway is essential for SDF-1 dependent CLL migration. In addition, we demonstrate that the CXCR4 receptor is internalized in response to SDF-1 stimulation indicating that CLL cells downregulate the CXCR4 receptor once they home to the bone marrow or lymph nodes where there is abundant SDF-1 in the microenvironment. This leads to the confinement of the cells in the microenvironment preventing further migration of the cells outside of the bone marrow/lymph nodes. Supported in part by CA97274

Abstract Malignant plasma cells characteristically home to the bone marrow (BM). However, the mechanisms by which cells are recruited into and mobilized from the bone marrow into the peripheral blood (PB) are not well understood. In this study, we explore the molecular mechanisms involved in homing and migration of plasma cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of MM cells in response to SDF-1. CXCR4 surface expression was determined by FACS analysis (PE CXCR4, Pharmingen) using samples from bone marrow and peripheral blood of patients diagnosed with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS) and primary systemic amyloidosis (AL). All results were expressed as percent expression in gated CD38+ and CD45+ cells. Boyden chamber in vitro migration assays were performed using Kas6/1MM cells. In addition, live confocal microscopy was used to visualize changes in the subcellular location of YFP-fluorescent CXCR4 before and after SDF-1 stimulation. Cells were pretreated with 10mM LY294002 or 200nM rapamycin to assess the effect of inhibition of PI3K or mTOR on CXCR4 subcellular localization. Immunoblotting was performed to confirm inhibition of the PI3K pathway. Comparisons between groups was performed using Mann-Whitney testing. The median CXCR4 expression is described in table 1. There was a significant difference between expression of BM CXCR4 in MM and MGUS (p=0.02). SDF-1 induced dose dependent migration of Kas 6/1 cells indicating a functional CXCR4 receptor. MM cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping, internalization subcellularly, and production of pseudopodia in response to SDF-1. This process was abrogated with pretreatment with LY294002 and rapamycin. These data demonstrate for the first time that the surface expression of CXCR4 is markedly elevated in the peripheral blood as compared to the bone marrow. Once in the bone marrow, and with the presence of excess SDF-1, the receptor becomes internalized and downregulated. In contrast to MM, CXCR4 expression on plasma cells in the bone marrow of normal and MGUS patients was higher. In addition, we demonstrate that the process of CXCR4 subcellular localization is abrogated by the administration of PI3K inhibitors LY294002 and rapamycin. These data suggest that CXCR4 expression is required for MM cells to circulate, and that downregulation occurs in the BM leading to immobilization of the cells. Future clinical trials using CXCR4 inhibitors, or inhibitors of PI3K and mTOR to prevent the homing and migration of MM cells into the bone marrow may be explored. This was supported in part by CAP50 CA97274. CXCR4 expression N=45 Median CXCR4% expression of total plasma cells Range MM BM (n=23) 27 4.3–90.1 MM PB (n=5) 91 60–95.5 MGUS BM (n=6) 65 41–78.6 AL BM (n=7) 46 17.5–71.6 Normal BM (n=4) 59 6.3–69.5 Kas6/1 cell line 100 -

Abstract Background . Cell-free DNA (cfDNA) sequencing enables serial temporal sampling, which offers the possibility of following the dynamics of biomarkers and clonal evolution in Multiple Myeloma (MM) over time. The use of cfDNA in clinical practice as a molecular biomarker and for monitoring response/resistance is dependent on a comprehensive profile of matched cfDNA and tumor DNA (tDNA) samples. Here we performed Ultra-Low Pass Whole Genome Sequencing (ULP-WGS) followed by whole-exome sequencing (WES) and targeted deep sequencing of matched cfDNA/tDNA samples from MM patients. Methods. We performed next generation sequencing of matched cfDNA/tDNA samples for 63 patients with newly diagnosed or relapsed MM, SMM, or MGUS. Libraries were constructed using the Kappa Hyper kit and sequenced by ultra-low-pass whole-genome sequencing (ULP-WGS, 0.1x coverage) to quantify tumor fraction within cfDNA. WES was performed on 30 matched samples cfDNA/tDNA/germline DNA from 10 patients with more than 5% of tumor fraction. Libraries were hybridized to the Nextera Rapid Capture Exome kit (Illumina) and then sequenced on HiSeq 4000 (Illumina). Targeted deep sequencing was performed on 32 matched cfDNA/tDNA samples from 16 patients using the HaloPlex HS technology (Agilent), allowing for molecular barcoding. Libraries were constructed according to the manufacturer's instructions and sequenced on HiSeq 2500 (Illumina). Sequencing data were analyzed using the Firehose pipelines, including MuTect, ABSOLUTE, ReCapSeg, GISTIC and MutSig. Results. We first used a cost-effective approach to establish the tumor content of cfDNA in a large-scale manner by ULP-WGS. Among 63 tested samples (53 MM, 6 SMM and 4 MGUS patient samples), the tumor fraction within cfDNA ranged from 0 to 81% with a mean of 10%. About 43% of these samples had tumor fraction greater than 5% within cfDNA. To assess whether cfDNA can capture the genetic diversity of MM and inform clinical management, we performed WES of matched cfDNA/tDNA/germline DNA samples for 10 patients (mean target coverage 194x). Copy number alterations (CNAs) assessed by WES (ReCapSeg) were consistent between cfDNA and tumor DNA. Similarly, focal CNAs assessed by GISTIC were consistent between tDNA and cfDNA. We then examined the overlap of somatic single nucleotide variants (SSNVs) between WES of cfDNA and matched tDNA. We found, on average, 100% of the clonal and 96% of the subclonal (range 54-100%) SSNVs that were detected in the tumor were confirmed to be present in cfDNA. Similarly, for mutations detected in the cfDNA, we found, on average, 100% of the clonal and 99% of the subclonal (range 98-100%) SSNVs were confirmed in the tumor. To assess whether targeted deep sequencing of cfDNA could be a good proxy for tumor biopsy we used a targeted deep sequencing approach of known MM driver genes. Libraries were prepared using unique molecular barcodes to avoid duplication rates, for 32 matched cfDNA/tDNA samples from 16 patients with MM. The mean target coverage was 596x. We found similar frequencies of altered MM driver genes in both cfDNA and tDNA, including KRAS, NRAS, and TP53, indicating that cfDNA can be used for precision medicine. Conclusions. Our study demonstrates that both WES and targeted deep sequencing of cfDNA are consistently representative of tumor DNA alterations in terms of CNAs, focal CNAs and SSNVs. This approach could therefore be used to longitudinally follow clonal evolution across the course of the disease and precision medicine in patients with MM. Disclosures Palumbo: Takeda: Employment, Honoraria; Janssen Cilag: Honoraria. Kumar:Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; AbbVie: Research Funding; Onyx: Consultancy, Research Funding. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria. Facon:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy; Janssen: Consultancy, Speakers Bureau; Bristol: Consultancy; Millenium/Takeda: Consultancy; Celgene: Consultancy, Speakers Bureau; Karyopharm: Consultancy. Ghobrial:Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Noxxon: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Amgen: Honoraria.

Abstract Introduction: IgM-secreting myeloma is rare, encompassing about 1% of all the myeloma cases. Given its rarity, the characteristics and survival of these patients have not been extensively studied. Therefore, we carried out a multicenter retrospective study in patients with IgM myeloma. Methods: The study protocol was reviewed and approved by the Institutional Review Board of each participating institution. Clinical data were gathered and included age, sex, hemoglobin, calcium, LDH, estimated glomerular filtration rate (GFR), presence of lytic bone lesions, International Staging System score, cytogenetic abnormalities, final outcome and overall survival (OS) time. OS was defined as the time in months from diagnosis to last follow-up or death. The Chi-square and the rank-sum tests were used to compare categorical and continuous variables, respectively. The Kaplan-Meier method was used to estimate OS and the log-rank waas used to compare groups. The Cox proportional-hazard regression method was used to fit univariate and multivariate survival models, reported as hazard ratio (HR) with 95% confidence intervals (CI). All reported p-values are two-sided, and were considered significant if less than 0.05. Results: A total of 159 patients with IgM myeloma from 20 centers from Europe, USA and Latin America were included in this analysis. Patients were diagnosed between 1996 and 2015. The median age at diagnosis was 65 years (range 37-86 years) with a male predominance (68%). The median serum IgM level was 2510 mg/dl (range 27-12,100 mg/dl) with 25% of patients having a serum IgM within normal limits but an IgM monoclonal spike detectable in serum electrophoresis. Hemoglobin levels <10 g/dl were seen in 49 patients (32%), serum calcium was elevated in 24 (16%), estimated GFR of 60 ml/min or less in 56 (37%), lytic lesions were detected in 86 (58%), and serum LDH was elevated in 28 (24%). ISS scores of 1, 2 and 3 were seen in 53 (36%), 63 (43%) and 31 (21%) of patients, respectively. Although immunohistochemistry and/or flow cytometry data were limited, CD20 expression was seen in 15/26 (58%) and cyclin D1 in 10/15 (67%) patients. The most common cytogenetic abnormalities were t(11;14) in 26/67 (39%), del13q in 25/76 (33%) and del17p in 6/76 (8%) patients. 149 patients (94%) received at least one line of systemic therapy for myeloma of which 13 (9%) received rituximab as part of initial therapy. After a median follow-up of 47 months, 74 patients (47%) have died. The median OS was 62 months (95% CI 51-79 months). Cause of death was known in 38 patients, and the most common was myeloma progression (74%). IN the univariate analysis, prognostic factors associated with a worse OS were age (HR 1.03, 95% CI 1.01-1.06; p=0.01) and ISS score (ISS 2: HR 1.43, 95% CI 0.83-2.48; p=0.2, and ISS 3: HR 3.03, 95% CI 1.54-5.98; p=0.001, using ISS 1 as reference group), while male sex was associated with a better OS (HR 0.56, 95% CI 0.34-0.90; p=0.02). Hemoglobin, calcium, estimated GFR and lytic lesions were not associated with OS. In the multivariate analysis, male sex was associated with a better OS (HR 0.57, 95% CI 0.35-0.95; p=0.03) and ISS with worse OS (ISS 2: HR 1.57, 95% CI 0.89-2.74; p=0.12, and ISS 3: HR 2.76, 95% CI 1.38-5.55; p=0.004, using ISS 1 as reference group). Conclusion: Patients with IgM myeloma apparently present with similar clinical characteristics as patients with non-IgM myeloma. Pathologically, CD20 and cyclin D1 expression is common. The most common cytogenetic abnormalities identified were t(11;14) and del13q. Survival does not appear shorter than non-IgM myeloma patients, and the ISS appears to have similar prognostic value. Disclosures Castillo: Millennium: Research Funding; Janssen: Honoraria; Otsuka: Consultancy; Biogen: Consultancy; Abbvie: Research Funding; Pharmacyclics: Honoraria. Ghobrial:Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria. Hájek:Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; BMS: Honoraria; Takeda: Consultancy. Hungria:International Myeloma Foundation Latin America: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Amgen: Consultancy; Roche: Consultancy; Bristol: Consultancy; Janssen: Consultancy, Speakers Bureau. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau. Reagan:Alexion: Honoraria; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees. Spicka:Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria. Gertz:Celgene: Honoraria; Med Learning Group: Honoraria, Speakers Bureau; Research to Practice: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Research Funding; Novartis: Research Funding; Prothena Therapeutics: Research Funding; Ionis: Research Funding; NCI Frederick: Honoraria; Sandoz Inc: Honoraria; GSK: Honoraria; Annexon Biosciences: Research Funding.

Abstract Background: Oprozomib (OPZ) is an oral epoxyketone proteasome inhibitor that selectively and irreversibly binds to its target. Preliminary findings of OPZ in pts with hematologic malignancies (HM) have been reported previously (Savona, ASH 2012, 203; Kaufman, EHA 2013, P223; Ghobrial, ASH 2013, 3184). Updated safety and efficacy results from the subset of pts with MM enrolled in this ongoing phase 1b/2 study in pts with HM are presented. Methods: This open-label, phase 1b/2 study (NCT01416428) is enrolling adult pts with HM who have relapsed after receiving ≥1 line of therapy. The primary objectives (phase 1b portion) are to determine the maximum tolerated dose (MTD) and the safety and tolerability profile of OPZ. The primary objective (phase 2 portion) is to determine the best overall response rate (ORR; ≥PR). In the phase 1b portion, OPZ is being administered once daily on days 1, 2, 8, and 9 of a 14-day cycle (2/7 schedule) or on days 1–5 of a 14-day cycle (5/14 schedule). The starting dose was 150 mg/day (mg/d); doses were escalated in 30-mg increments up to 330 mg/d. In the phase 2 portion, pts are receiving OPZ (240 mg/d) on the 5/14 schedule (initial phase 2 schedule opened to enrollment). For this report, all enrolled patients with HM are included in the description of the MTD while only the subset of patients with WM is included in the safety and efficacy results. Results: As of July 21, 2014, 106 pts with HM (including 68 pts with MM) were enrolled and treated with the OPZ tablet. Enrollment and baseline demographic information for pts with MM are presented in Table 1. Median treatment duration (phase 1b) was 21.3 weeks (range, 0.3–62.1; 2/7 schedule) and 10.1 weeks (range, 0.3–81.1; 5/14 schedule); preliminary median treatment duration in the ongoing phase 2 portion was 5.4 weeks (range, 0.7–26.7). In all patients with HM, the MTD for the 2/7 schedule was 300 mg/d and 240 mg/d for the 5/14 schedule. Of 3 dose-limiting toxicities (DLTs) on the 2/7 schedule, all 3 DLTs (including hypotension [300 mg/d, n=1], grade 3 diarrhea and grade 4 thrombocytopenia [330 mg/d, n=1 each]) were observed in pts with MM. Of 4 DLTs on the 5/14 schedule, 2 DLTs (grade 3 renal failure [180 mg/d, n=1] and grade 3 tumor lysis syndrome [270 mg/d, n=1]) were observed in pts with MM. The most common adverse events (AEs) in patients with MM are shown in Table 2. Grade 4 AEs included 8 pts (11.8%) with thrombocytopenia, 4 pts (5.9%) with anemia, and 1 pt each (1.5%) with sepsis, leukopenia, decreased platelet count, hyperkalemia, and acute renal failure. Two pts died from upper gastrointestinal bleeding (5/14 schedule, 240 mg/d). One pt (5%) on the 2/7 schedule (phase 1b), 3 pts (15%) on the 5/14 schedule (phase 1b), and 8 pts (30%) on the 5/14 schedule (phase 2) discontinued treatment due to an AE; 6 pts (29%) on the 2/7 schedule (phase 1b), 7 pts (35%) on the 5/14 schedule (phase 1b), and 11 pts (41%) on the 5/14 schedule (phase 2) had their dosage reduced at least once due to an AE. Thirty-four pts in the phase 1b portion were eligible for response. In the phase 1b portion, the ORR in 15 pts on the 2/7 schedule (all were carfilzomib [CFZ]-naïve) was 33.3%; the clinical benefit rate (CBR) was 46.7% (3 very good partial response [VGPR], 2 partial response [PR], and 2 minimal response [MR]). Among 19 pts on the 5/14 schedule (phase 1b portion; all were CFZ-naïve), the ORR was 36.8%; the CBR was 42.1% (1 complete response, 2 VGPR, 4 PR, and 1 MR). In the 5/14 schedule (phase 1b), the ORR among bortezomib [BTZ]-refractory pts (n=7) was 14.3%. On the phase 2 portion (5/14 schedule), the ORR among CFZ-refractory pts (n=11) and CFZ-sensitive pts (n=12) was 27.3% (3 PR) and 33.3% (2 VGPR and 2 PR), respectively; the ORR among BTZ-refractory pts (n=12) was 25.0%. Conclusions: The MTD of OPZ was 300 mg/d in the 2/7 schedule and 240 mg/d in the 5/14 schedule; these MTDs were determined from all patients with HM. The most common grade 3 AEs were diarrhea, nausea, and vomiting; grade 4 AEs were infrequent. Additional measures will be taken to improve gastrointestinal tolerability. Single-agent OPZ continues to have promising antitumor activity. Enrollment of pts with MM is continuing on the phase 2 study in both treatment schedules. Extended-release OPZ tablets will be introduced and assessed for safety, activity, and pharmacokinetics. Updated results will be presented at the meeting. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Vij: Celgene: Honoraria, Research Funding; Onyx: Honoraria, Research Funding; Sanofi: Honoraria; Jannsen: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Array: Honoraria. Off Label Use: Carfilzomib as treatment in multiple myeloma and solid tumors. Savona:Incyte: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Siegel:Celgene: Honoraria, Speakers Bureau; Onyx: Honoraria, Speakers Bureau; Millennium: Honoraria, Speakers Bureau. Kaufman:Millennium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria; Merck: Research Funding. Ghobrial:BMS: Advisory Board Other; Celgene: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Onyx: Advisory Board, Advisory Board Other. Paner:Celgene Corporation: Honoraria. Jagannath:Millenium: Honoraria; Celgene: Honoraria; Onyx: Honoraria; Merck: Honoraria; Ortho: Membership on an entity's Board of Directors or advisory committees; im: Membership on an entity's Board of Directors or advisory committees; Medicom Worldwide: Membership on an entity's Board of Directors or advisory committees; Optum Health Worldwide: Membership on an entity's Board of Directors or advisory committees; PER group: Membership on an entity's Board of Directors or advisory committees. Jakubowiak:BMS: Advisory Board Other, Consultancy, Honoraria; Celgene: Advisory Board, Speaking, Advisory Board, Speaking Other, Consultancy, Honoraria; Janssen: Advisory Board, Speaking, Advisory Board, Speaking Other, Consultancy, Honoraria; Millennium: Advisory Board, Advisory Board Other, Consultancy, Honoraria; Novartis: Advisory Board Other, Honoraria; Onyx: Advisory Board, Speaking Other, Consultancy, Honoraria; Skyline Dx: Advisory Board Other, Honoraria. Mikhael:Onyx: Research Funding; Celgene: Research Funding; Sanofi: Research Funding; Novartis: Research Funding. Kapoor:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding. Neuman:Onyx: Employment. Lee:Onyx Pharmaceuticals, an Amgen subsidiary: Employment.

Abstract Background. Endothelial progenitor cells (EPCs) are circulating precursors with the capacity to differentiate into endothelial lineage cells through a process known as “vasculogenesis” thus contributing to vessel formation. We studied the role of endothelial progenitor cells in multiple myeloma (MM) pathogenesis. Methods. EPC levels were evaluated in peripheral blood (PB) of patients with smoldering MM (SMM), and active myeloma (MM) and in PB of healthy controls, by using flow-cytometry (CD34+VEGFR2+ cells), and by performing endothelial colony forming assays. EPC levels were studied in PB from transgenic Vk*MYC mice harboring either early MM (smoldering-like stage); or late (active MM-like stage); as well as in mice injected intravenously with either murine MM 5TGM1-turboRFP+ cells or human MM1S-GFP+/luc+ cells. Healthy syngeneic mice were used as controls. GFP-bone marrow (BM) transplantation and sub-cutaneous femur implantation were performed in recipient mice to study EPC BM mobilization (GFP+CD34+VEGFR2 cells) and EPC BM recruitment to the implanted femurs during 5TGM1 MM model progression. Transgenic ID1+/-ID3-/- mice (with a defect in EPC mobilization and differentiation ability) were injected with transplantable Vk*MYC cells and followed for survival. Wild type littermates were used as controls. Similar experiments were performed using transgenic ID1+/-ID3-/- mice transplanted with BM of wild type littermates. Therapeutic activity of DC101 anti-murine VEGFR2 Ab was evaluated in the MM1S human orthotopic xenograft model, using both early and late treatment approaches. Results. EPC levels were significantly increased in PB of MM patients, including SMM, compared to healthy controls (P<0.05). PB mononuclear cells (PBMCs) from SMM and aMM patients showed an increase ability to form both EC-CFUs (P<0.01) and ECFCs compared to those from healthy controls (P<0.05). EPC levels were significantly increased in PB of transgenic Vk*MYC mice at both early and advanced stage of MM development compared to healthy syngenic mice. In orthotopic MM models we found that levels of circulating EPCs increase early after i.v. injection of either human MM1S-GFP+/luc+ cells (1.5 and 2 times higher after 1 week and 2 weeks, respectively) or murine 5TGM1-turboRFP+ MM cells (2.5 and 3 times higher after 2 and 3 weeks, respectively) compared to naïve mice. By coupling GFP-BM transplantation and subcutaneous femur implantation, we generated a new model that allows the study of BM derived-cell trafficking from one site to another of the BM. The levels of BM-derived GFP+ EPC increased early after i.v. injection of 5TGM1-turboRFP+ cells (2 times increase after 2 weeks, P<0.01); also, the percentage of both total GFP+ cells (7.92% versus 2.635%, P<0.05) and GFP+ EPCs (0.22% versus 0.068%, P<0.05) increased in the BM of implanted femurs of tumor cell injected mice compared to controls. Survival of ID1+/-ID3-/- mice was significantly increased compared to that of wild type littermates after injection of MM Vk*MYC cells (62 versus 27 days median survival time, P=0.0005); this survival advantage was lost after wild type BM was transplanted in ID1+/-ID3-/- mice (34 versus 35 days median survival time). In human MM1S orthotopic xenograft model, treatment with anti-murine VEGFR2 DC101 Ab was able to inhibit MM progression when treatment was started early during smoldering like stages, but not when it was started after significant tumor burden (50.5, 41.5 and 40 days median survival time in DC101 early, late and untargeted IgG treated mice respectively) Conclusion. These results show that EPC mediated vasculogenesis is an important pathogenic process that occurs during the early/smoldering MM stage. Knockout mouse experiments confirm that EPCs are essential for MM tumor progression. These results indicate that anti-angiogenic agents could be effective as an early therapeutic intervention in SMM patients. Disclosures Ghobrial: Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Background: Oprozomib (OPZ) is an oral epoxyketone proteasome inhibitor that has shown promising antitumor activity in patients (pts) with hematologic malignancies (HM), including WM (Kaufman, EHA 2013, P223; Ghobrial, ASH 2013, 3184). Updated safety and efficacy results from the subset of pts with WM enrolled in this ongoing phase 1b/2 study in pts with HM are presented. Methods: This open-label, multicenter, phase 1b/2 study (NCT01416428) is enrolling adult pts with HM who have relapsed after receiving ≥1 line of therapy. The primary objectives of the phase 1b portion of the study are to determine the maximum tolerated dose (MTD) and the safety and tolerability profile of OPZ. The primary objective of the phase 2 portion of the study is to determine the best overall response rate (ORR; ≥minimal response [MR]). In the phase 1b portion, single-agent OPZ is being administered once daily on days 1, 2, 8, and 9 of a 14-day cycle (2/7 schedule) or on days 1–5 of a 14-day cycle (5/14 schedule). The starting dose was 150 mg/day (mg/d); doses were escalated in 30-mg increments. In the phase 2 study, pts have received 240 mg/d on the 5/14 schedule, which was the initial phase 2 schedule opened to enrollment. For this report, all enrolled patients with HM are included in the description of the MTD while only the subset of patients with WM is included in the safety and efficacy results. Results: As of July 21, 2014, 106 pts with HM (including 36 pts with WM) were enrolled and treated with the OPZ tablet. Enrollment and baseline demographic information for pts with WM are presented in Table 1. For WM patients in the phase 1b portion, median treatment duration was 17.1 weeks (range, 3.1–51.4; 2/7 schedule) and 51.7 weeks (range, 3.9–74.9; 5/14 schedule); preliminary median treatment duration in the ongoing phase 2 portion was 8.1 weeks (range, 0.7–22.0). In all pts with HM, the MTD for the 2/7 schedule was 300 mg/d and 240 mg/d for the 5/14 schedule. None of 3 dose-limiting toxicities (DLTs) on the 2/7 schedule occurred in patients with WM. Two of the 4 DLTs observed on the 5/14 schedule occurred in pts with WM (grade 4 tumor lysis syndrome [240 mg/d, n=1] and grade 3 vomiting [270 mg/d, n=1]). The most common adverse events (AEs) in pts with WM are shown in Table 2. Grade 4 AEs were thrombocytopenia (n=1; phase 1b; 2/7 schedule) and influenza and tumor lysis syndrome (n=1 each; phase 1b; 5/14 schedule). No on-study deaths occurred in pts with WM. In the phase 1b portion, two pts (25%) in the 2/7 schedule and 1 pt (9%) in the 5/14 schedule discontinued treatment due to an AE; 6 pts (35%) discontinued treatment due to an AE in the phase 2 portion. In the phase 1b portion, 3 pts (38%) (2/7 schedule) and 6 pts (55%) (5/14 schedule) had their dosage reduced at least once due to an AE; 10 pts (59%) in the phase 2 portion had their dosage reduced at least once due to an AE. In the phase 1b portion, all 19 pts were eligible for response and were carfilzomib (CFZ)-naïve. The ORR in 8 pts enrolled on the 2/7 schedule was 38% (3 pts had a partial response [PR]). Among 11 pts enrolled on the 5/14 schedule, the ORR was 73% (1 complete response, 1 very good partial response, 5 PRs, and 1 MR); the ORR for BTZ-refractory pts (n=4) was 75%. All 17 pts enrolled on the phase 2 portion were response-eligible. The ORR in the phase 2 portion was 59% (there were 5 PRs and 5 MRs). The ORR among CFZ-naïve pts (n=16) in the phase 2 portion was 56; the ORR among BTZ-refractory pts (n=3) was 67%. Conclusions: The MTD of OPZ was 300 mg/d in the 2/7 schedule and 240 mg/d in the 5/14 schedule; these MTDs were determined from all pts with HM. In pts with WM who received single-agent OPZ, the most common grade 3 AEs were neutropenia and diarrhea; grade 4 AEs were infrequent. Additional measures will be taken to improve gastrointestinal tolerability. Single-agent OPZ continues to have promising antitumor activity in pts with WM. Enrollment of patients on the 2/7 schedule is continuing; the target enrollment for the phase 2 portion of the study is 66 patients. Extended-release OPZ tablets will be introduced and assessed for safety, activity, and pharmacokinetics. Updated results will be presented at the meeting. Disclosures Siegel: Celgene: Honoraria, Speakers Bureau; Onyx: Honoraria, Speakers Bureau; Millennium: Honoraria, Speakers Bureau. Off Label Use: Monoclonal antibody/toxin fusion protein. For treatment of multiple myeloma. Kaufman:Millennium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria; Merck: Research Funding. Raje:Celgene: Consultancy; Millennium: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Onyx: Consultancy; Acetylon: Research Funding; Ely Lilly: Research Funding. Mikhael:Onyx: Research Funding; Celgene: Research Funding; Sanofi: Research Funding; Novartis: Research Funding. Kapoor:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding. Treon:Onyx: Consultancy, Research Funding. Neuman:Onyx: Employment. Lee:Onyx Pharmaceuticals, an Amgen subsidiary: Employment. Ghobrial:BMS: Advisory Board Other; Celgene: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Onyx: Advisory Board, Advisory Board Other.

Abstract Abstract 1825 Poster Board I-851 Purpose Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. We have previously demonstrated that Citron Rho Interacting Kinase (CRIK) is overexpressed in primary multiple myeloma (MM) cells, as compared to the normal plasma cell counterpart, using an antibody-based protein microarray technique. We therefore sought to investigate the functional role of CRIK in MM cells. Methods We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM.1S, RPMI8226, and INA6 MM cell lines were used in this study. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by thymidine uptake, MTT, PI and Annexin/PI staining and flow cytometric analysis, respectively. Results Overexpression of CRIK has been confirmed in primary CD138+ tumor cells isolated from bone marrow of 12 patients with MM, as compared to normal plasma cells obtained from healthy donors. We found that CRIK-knockdown exerted an anti-proliferative and pro-apoptotic effect only in IL-6-dependent MM cell line INA6; in contrast, no effect on proliferation and survival was observed in MM1.S and RPMI8226. Indeed, INA6 CRIK-knockdown cells were characterized by a reduction in the proliferation rate, associated with decreased S-phase and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity was also demonstrated in CRIK knockdown cells compared to scramble probe transfected or non-transfected cells. We also showed that CRIK knockdown led to cytokinesis in INA6, indicating a possible mechanism for inhibition of proliferation of these cells. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Conclusion In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of IL-6 dependent MM cells. Moreover, CRIK protein expression correlated with poor survival in patients with MM. Disclosures Anderson: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.