Azab, Abdel Kareem

Abstract 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. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. 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. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. 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 adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

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 Background: The concept of treatment personalization in cancer often means associating a drug with an actionable mutation. One limitation of this approach is that most tumors have multiple aberrations, which are not actionable. Here we present a predictive simulation based approach which models the molecular architecture and resulting physiology of advanced stage Waldenströms Macroglobulinemia (WM, a B-lymphoplasmacytic lymphoma) as well as the design of novel personalized treatments using existing drug agents for rapid clinical translation with validations. Methods: We used the human WM cell line, RPCI-WM1, as a surrogate model of refractory, advanced patient sub-groups. Publically available as well as proprietary genomic and cytogenetic data was utilized for the creation of an avatar of RPCI-WM1, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were shortlisted. Importantly, this simulation avatar approach not only looks for agents acting on the specific gene mutation, but also predicts the convergence points to be attacked. The personalized simulation avatar technology is a comprehensive functional proteomics representation of WM physiology network. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. A library of over 150 digital functional library of FDA approved drug agents and those in clinical study has been developed and was simulated individually and in combination on the RPCI-WM1 (advanced stage WM patient) avatar. Results: Overall, there were 272 gene aberrations used to create the RPCI-WM1 simulation avatar. Importantly, presence of MYD88L265P mutation, absence of CXCR4 mutations and additional chromosomal aberrations (derivatives, translocations, deletions and amplifications of chromosomes 3, 6, 9, 13, 18 and 19) were taken into account. The RPCI-WM1 patient avatar predicted increased IRAK1/4 engagement due to MYD88 mutation and high copy number (CN) of IL18, to increased NFkB via TAK1, and increased ERK signaling through IRAK1/4 mediated down regulation of DUSP1. The RPCI-WM1 model also indicated high AKT due to indirect convergence of multiple aberrations. Another key characteristic of the genomic aberrations driving proliferative phenotype was low CN of RB1, FOXO3, CDKN2A, CDKN2B, and CEBPa. Modeling predicted sensitivity to the aurora kinase (AURKA) inhibitor, tozasertib and resistance to cell cycle cyclin D-CDK4/6 pathway inhibitors., Although there was no CN variation in AURKA, as per simulation its expression and activity was upregulated due to high CN of ETS1 and PAK1 and increased activation of NFkB, HIF1A and STAT5 in the disease network. Despite high proliferation, LEE011 (representative CDK4/6 inhibitor) was predicted to exert no cytotoxic effect, due to the presence of an RB1 deletion. Through phosphorylation of RB1, the CDK4-CyclinD1 complex aids in release of E2F1 from the RB1 sequestered complex, to drive proliferation. However, predictive modeling suggested that with an RB1 deletion present, this regulation becomes irrelevant and therefore the inhibition of the CDK4/6-CyclinD1 complex would be ineffective. The simulation predictions were experimentally validated. As predicted, AURKA inhibition with tozasertib significantly inhibited viability and proliferation of RPCI-WM1 cells (IC50 ~7nM) whereas inhibition of CDK4/6 with LEE011 had no effect on tumor cell survival. Conclusions: This study demonstrates the utility of a novel technology for rapid translation of a (WM) genetic signature towards a personalized therapeutic strategy. This simulation avatar based approach holistically integrates all genomic aberration information to design personalized therapies architected from FDA approved and/or clinical drug agents with therapeutic potential for WM patients based on unique genetic signatures. Disclosures Vali: CellWorks: Employment. Kumar:CellWorks: Employment. Singh:CellWorks: Employment. Kapoor:Cellworks Research India Limited: Employment. Abbasi:CellWorks: Employment, Equity Ownership.

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.

Abstract Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138+ MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-κB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.