ALBERT

Description: Introduction Multiple myeloma (MM) is characterized by dissemination and accumulation of plasma cells in the bone marrow (BM), which promotes tumor cell growth and therapy resistance. ROBO1 is a conserved transmembrane receptor of the Ig superfamily with no intrinsic catalytic activity, and its role in MM pathogenesis is unknown. Material and Methods We first analyzed ROBO1 expression via western blot and/or immunohistochemistry (IHC). Gene expression profiling in a cohort of 170 newly diagnosed MM patients (IFM170) was used to compare ROBO1 expression across primary MM and BM stroma cells (BMSC), and normal BM plasma cells (PC). We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD) and CRISPR-Cas9 for ROBO1 knock out (KO). For protein structure-function and rescue studies, ROBO1 KO MM cells were transduced with a lentiviral vector expressing either full-length (FL) or truncated ROBO1 mutants devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived truncating mutations, with a C-terminus triple FLAG tag. FLAG immunoprecipitation (IP) followed by mass spectrometry or western blotting and immunofluorescence (IF) were used to identify ROBO1 interacting partners and ROBO1 cellular localization. We used a hydrogel encapsulation system to study proliferation in a 3D system. To study extramedullary and intramedullary MM growth in vivo, WT and ROBO1 KO OPM2 were injected either subcutaneously (plasmacytoma model) or intra-medullary in femoral bones of donor mice which were then implanted subcutaneously in recipient SCID mice (µ-SCID model). PET-CT was used to assess tumor volume. Mouse tumors were retrieved for IHC and RNA extraction followed by RNA sequencing. To study dissemination and homing, KO and FL addback OPM2 cells were injected intravenously in SCID mice. Femurs and plasmacytoma were retrieved at endpoint for IHC. Results ROBO1 is highly expressed in human MM cell lines and primary MM cells with highest expression in cells carrying the high risk t(4;14) cytogenetic and low/absent expression in normal PC. Of human cancer cell lines, ROBO1 expression was limited to late B cell lineage; and ROBO1 KD was selectively cytotoxic against MM, but not other hematologic cancers. ROBO1 KO significantly decreases proliferation in a 3D culture system and tumor growth in extramedullary (mean tumor volume KO versus WT plasmacytoma: 457 versus 1323 mm3, p value= 0.02) and intramedullary (mean tumor volume KO versus WT: 823 versus 2684 mm3, p value= 0.001) murine models of human MM. ROBO1 KO MM cells show decreased adhesion to BM endothelial and BMSC, which is fully rescued by FL ROBO1 addback. To address whether ROBO1 loss alters dissemination/homing of MM cells in vivo, we injected mice intravenously with ROBO KO or FL addback OPM2 cells. While ROBO1 KO resulted in a modest, non-statistically significant prolongation in mouse OS (90 versus 75 days, respectively, p value 0.2), the pattern of disease was strikingly different. As expected, ROBO1 FL mice developed hindlimb paralysis with extensive BM infiltration with MM. Importantly, ROBO1 KO mice demonstrated reduced BM infiltration and developed solitary plasmacytoma. We next showed that ROBO1 C-terminus domain is necessary and sufficient to rescue ROBO1 KO proliferative defect while expression of ROBO1 truncations, including patient-derived frameshift mutations, acted as dominant negative. IP showed avid interaction of ROBO1 with ABL1. Interestingly, we showed that the cytosolic domain of ROBO1 undergoes cleavage and translocates to the nucleus, where its function is now being studied. Conclusions We show that ROBO1 is necessary for MM homing to the BM niche and for MM growth within and outside the BM space. ROBO1 cytosolic domain undergoes proteolytic cleavage and translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO defective proliferation. Based on our data, we propose a dual model for ROBO1 in MM: the full transmembrane receptor is involved in regulating adhesion, dissemination and homing of MM cells within the BM niche; the cleaved intracellular C-terminus domain participates in transcriptional regulation, promoting MM proliferation. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Disclosures Roccaro: Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding. Ghobrial:Takeda: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Description: Abstract 616 Introduction Although marked heterogeneity in phenotypes of MM is known, classification of MM based on molecular expressions remains to be determined. During analysis of microarray data utilizing purified MM cells obtained from bortezomib-sensitive and resistant cases, we identified a 100-fold higher expression of CD125 in bortezomib-resistant MM cells. Because CD125, also known as interleukin-5 receptor alpha chain, is expressed on mature B cells undergoing class switch recombination, the existence of CD125 in MM cells may suggest that MM cells are at a less mature differentiation stage than plasma cells. We further tried to classify MM patients by analyzing correlations of CD125-expressions with other surface antigen expressions and endoplasmic reticulum stress (ER stress) response: the latter plays a crucial role in resistance to bortezomib. Correlation of CD125 expression with sensitivity to bortezomib therapy was also evaluated. Materials and Methods Bone marrow samples were obtained from MM patients under written informed consentaccording to Helsinki Declaration and processed for purification using CD138 immunomagnetic beads. Expressions of CD125 and spliced-XBP-1 (spliced-XBP-1 presence is a hallmark of activated ER stress response), were evaluated using real time PCR. Flowcytometry analysis was performed using gating CD38 bright population at SRL laboratory Inc. (Tokushima, Japan). Response to bortezomib was assessed according to the international myeloma working group criteria. Results CD125 expression was found in MM cells at various levels. When it was compared to other surface antigen expressions, MM cells with expression of CD125 tend to express both CD20 and CD45 (p

Description: Key Points LAPTM5 c403t and HCLS1g496a are potentially novel contributors for the genetic predisposition to familial WM. LAPTM5 c403t and HCLS1g496a represent possible candidates for screening in familial WM.

Description: Platelets play important roles in both physiological and pathological conditions. For instance, platelets may facilitate cancer metastasis by protecting circulating tumor cells (CTCs) from shear stress and immunological assault during their intravascular phase and by supporting CTCs extravasation. Moreover, soluble factors released from activated platelets enhance proliferation and migration of endothelial cells, thereby promoting tumor angiogenesis. Correlations between increased platelet counts and shorter survival time have been described for many solid tumors. However, the role of platelets/megakaryocytes (MKs) in regulating tumor progression and dissemination in Multiple myeloma (MM) has not been previously examined. We measured the platelet aggregation-inducing abilities of MM cell lines and found MM cell lines (MM.1S, OPM-2, KMS-11, U266, and H929) induced platelet aggregation. This was not observed using leukemia cells (K562) and primary healthy donor-derived peripheral blood mononuclear cells. We next investigated whether platelets interact with MM cells within the bone marrow (BM) niche, in vivo. In order to identify platelets/MKs, femurs were harvested from MM.1S GFP+-harboring mice and stained with a DyLight649-conjugated anti-GPIb-beta antibody. The femurs were rendered transparent by the CUBIC method as previously described (Cell 159, 911-24 (2014)) and examined using confocal microscopy; and found that platelets/MKs co-localized within MM.1S GFP+-infiltrated BM niches. Further confirmation of the co-localization of MM cells and MKs was performed using immunohistochemistry (CD138+ and GPIb-alpha+). We next investigated the effect of platelets on MM cell proliferation: MM cells, where co-cultured with platelets and found that platelets enhanced the proliferation rate of MM cells in a platelet number-dependent manner, as shown by using BrdU (p

Description: Background. p53 is a well defined tumor suppressor involved in the modulation of cell proliferation, cell cycle progression and programmed cell death. BLIMP-1 plays a crucial role in modulating B-cell differentiation towards Ig-secreting plasma cells, and it acts as a tumor suppressor, as documented in both diffuse large B-cell lymphoma and Burkitt lymphoma. Whether B-cell specific loss of both p53 and BLIMP-1 may favor a B-cell lymphoma phenotype remains unanswered. We therefore aimed to generate in vivo dual p53/BLIMP-1-floxed conditional inactivation in B-cells, and to define the functional relevance of both p53 and BLIMP-1 n B-cell lymphomagenesis in vivo Methods.Cre recombinase under the control of CD19 promoter (C57BL/6 CD19Cre/Cre) mice were crossed with either C57BL/6 BLIMPflox/flox or C57BL/6 p53flox/flox mice to achieve deletion of BLIMP or p53, respectively, in B cells. Secondly, CD19Cre/Cre BLIMPflox/flox mice were crossed with CD19Cre/Cre p53flox/flox to achieve concomitant deletion of both BLIMP and p53 in B cells (CD19Cre/Cre BLIMPflox/flox p53flox/flox), referred as CD19/Bl-/p53- mice. Transgenic experimental mice (CD19/Bl-/p53-) where characterized for B cell infiltration using immunohistochemistry, flow cytometry; clonotypic immunoglobulin heavy-chain rearrangement was assessed by Southern Blotting. Whole exome sequencing was performed using DNA isolated from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and from matched tail-derived tissues, used as germline (Illumina HiSeq 2500 platform; Agilent SureSelectXT). MTT assay was used to BTK-inhibitor-dependent cytotoxicity using CD19/Bl-/p53-derived B220 cells. Results.We generated dual p53/BLIMP-1-floxed conditional inactivation in B-cells, using mice expressing Cre recombinase under the control of CD19 promoter. 100% of the CD19/Bl-/p53- mice presented with diffuse lymphadenomegalies, and splenomegaly, hepatomegaly (90.3% and 77.4%, respectively). Other clinical manifestations included presence of ascites and hind lymb paralysis (12.9% and 19.3%, respectively). The CD19/Bl-/p53- showed worse survival compared to Bl-/p53- mice non-expressing the CD19/Cre recombinase, CD19/p53-, or CD19/Bl- (363, 469.5, 460.5, and 770 days, respectively). H.E. staining of CD19/Bl-/p53--derived lymph nodes, defined a nodal architecture with a monomorphic population of large sized atypical lymphoid cells with finely clumped and dispersed chromatin, and multiple basophilic medium sized, paracentrally situated nucleoli. A "starry sky" pattern was also observed. Overall, these features are compatible with a high-grade lymphomas. IHC analysis confirmed a marked positivity for B220 staining (TdT, Bcl6, CD138 and CD4, CD8 negative). Tumors were confirmed to be B220+/IgM+, with either Igk- or Ig-lambda-restriction as demonstrated by flow cytometry; and either mono- or bi-clonal, as demonstrated by Southern blotting, thus further confirming the clonal transformation induced by dual BLIMP/p53 deletion in B cells. Whole exome sequencing was performed from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and identified 143 SNVs. Among them, non-synonymous somatic mutations were mapped on genes involved in the regulation of focal adhesion, PDGF signaling, p53-downstream pathway, and lipoprotein metabolism. B220+ cells selected from CD19/Bl-/p53--derived lymph nodes were implanted subcutaneously into recipient SCID/Bg mice (n: 10), and presented with 100% engraftment, with a monomorphic lymphoid infiltration of B220+ and IgM+ cells. B220 positive cells were selected from the s.q. tumor and intravenous injected into recipient SCID/Bg (n: 10) and BL/6 mice (n: 10). Engraftment was demonstrated in all the mice, where hepatomegaly, splenomegaly and hind lymb paralysis were observed. Infiltration of B220+ cells was documented within bone marrow, liver and spleen. We next investigated the anti-tumor activity of BTK-inhibitor, and found that B220+ cells selected from lymph nodes harvested from CD19/Bl-/p53-mice were sensitive to ibrutinib treatment. Conclusion. These studies demonstrate that the specific dual inactivation of p53 and BLIMP in B-cells promotes oncogenic transformation, resulting in aggressive B-cell lymphoma development. Disclosures Ghobrial: Celgene: Other: Advisory Board; BMS: Other: Advisory Board; Amgen: Other: Advisory Board; Takeda: Other: Advisory Board; Janssen: Other: Advisory Board. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

Description: Abstract 2844 Poster Board II-820 Introduction: Skeletal complications including bone fracture, bone pain and hypercalcemia are major clinical events in patients of multiple myeloma (MM). Osteoclastgenesis is known to be induced by free receptor activator of nuclear factor kappa β ligand (RANKL) and inhibited by dimerization of RANKL and osteoprotegerin (OPG). OPG is also known as a soluble inhibitor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); therefore, a possible role of TRAIL as an osteoclast inducer is suggested, although the association of TRAIL with bone lesions in MM is a matter of debate. We thus investigated the expression of TRAIL mRNA in purified MM cells and analyzed its association with skeletal-related events. Patients and Methods: MM cells were purified from bone marrow samples from 40 MM patients by CD138-immunomagnetic beads (Miltenyi Biotech, Paris, France). TRAIL mRNA expression in purified MM cells was analyzed by real time PCR(ABI PRISM 7700 Sequence Detector, Applied Biosystems). Simultaneous analysis of serum TRAIL concentrations, analyzed by ELISA (Diaclone, Cedex, France), and TRAIL mRNA-expression levels was also performed in 23 cases. Each of these patients was given a score called skeletal-related event score (SRE score) according to the skeletal complications (pathological fracture, bone-associated plasmacytoma, 〉12mg/dL hypercalcemia, and receiving an pathological fracture-related operation or radiation therapy). Results: Significant association (p=0.0006) was seen between TRAIL mRNA expression levels and the SRE score (Fig.1). Serum calcium levels also had significant association to TRAIL mRNA expression levels (p=0.0050). On the other hand, no association of TRAIL mRNA with hemoglobin (p=0.3970) and platelets (p=0.9401) was seen. Serum TRAIL concentrations in MM cases, which were equivalent to those in healthy individuals, did not correlate to TRAIL mRNA expression levels in purified MM cells (p=0.4094). Conclusions: The data suggest that MM patients with high TRAIL expression in MM cells tend to have more skeletal complications, which may be mediated by increased osteoclastgenesis. Since serum TRAIL concentrations did not correlate with TRAIL mRNA levels in MM cells, increased TRAIL expression in bone marrow microenvironment could be important. Despite of previous reports suggesting TRAIL-induced apoptosis of hematopoietic cells, the observed high TRAIL expression did not correlate with anemia or thrombocytopenia in our cases. Although mechanisms regulating TRAIL expression in MM cells and protection from TRAIL-induced apoptosis remain to be determined, our findings may introduce a new strategy targeting TRAIL to reduce skeletal events in MM. Disclosures: No relevant conflicts of interest to declare.

Description: 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

Description: Background The extracellular matrix (ECM) is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM), interactions between MM cells and the bone marrow (BM) microenvironment, of which the ECM forms a major component, are critical to the pathogenesis of the disease and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Until recently ECM proteins have proven difficult to characterize due to their biochemical properties and large size. Recent advances in proteomics have led to the characterization of the ECM and ECM-associated proteins (“matrisome”) in normal human tissues and tumors using a systematic and comprehensive approach. Methods Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x106) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after 2 weeks in order to mimic early tumor development (luminescence = 1x105 p/sec/cm2/sr) or 5 weeks (1x108 p/sec/cm2/sr) to model more advanced MM. Human bone marrow aspirates; Whole bone marrow was obtained from newly diagnosed MM patients (n=9) and healthy human donors (ND) (n=4) following written informed consent. ECM proteins were enriched from bone marrow samples obtained from MM patients, NDs and mice according to previously published methods.Tandem Mass Spectrometry (LC-MS/MS): Peptides were run using reversed-phase microcapillary liquid chromatography – tandem mass spectrometry (LC-MS/MS) on a high resolution hybrid Orbitrap Elite mass spectrometer. MS/MS data were searched against the UniProt Human database using MASCOT to identify proteins. Spectral counts were used as a semi-quantitative measure of abundance. ECM proteins were defined according to the in-silico definition of the matrisome. Validation of expression of ECM mRNA in MM cell lines (MM1s, RPMI-8226 and U266) and in CD138+ cells and bone marrow stromal cells (BMSC’s) from MM patients in comparison to NDs was performed using qRT-PCR. Results Primary myeloma sample ECM; Using a spectral count of 2 as a cutoff of peptide abundance we identified a total of 536 unique proteins in ND bone marrow of which 35 are defined as matrisome proteins. 982 unique proteins were enriched from whole bone marrow samples of newly diagnosed MM patients of which 26 are defined as matrisome proteins, 7 unique proteins were identified as ECM or ECM-associated in newly diagnosed patients which were not detected in the ND samples including PRG3, FGG, LEG10, TLN1 and PLEC. Critical ECM components such as laminins, matrix metalloproteinases and collagens were also found to be significantly altered in newly diagnosed MM with evidence of destruction of ECM components in active disease. Tumor Xenograft ECM; In mice with an earlier phase of human MM1s cell tumor burden we detected a total of 329 unique proteins of which 48 were defined as matrisome proteins, 23 of these proteins were unique to the earlier phase of MM in these mice. Mice with more advanced tumor development had unique ECM proteins which were not detected in the earlier disease stage including collagens, laminins and matrix metalloproteinases, indicating that these ECM components may be critical for re-modelling the ECM in MM. Interestingly, in our xenograft model of MM we were able to detect both human and mouse ECM components indicating that the tumor ECM is secreted from both the murine stroma and the human MM cells and allowing delineation of the source of individual ECM components. This indicates that as MM progresses certain ECM components, including FBN1, which were initially derived from stroma are later derived from MM cells. Differential expression of ECM components, including FBN1 between normal and malignant plasma cells was confirmed using qRT-PCR. Conclusions We have performed proteomic profiling of the unique tumor ECM in MM using mass spectrometry with a view to determining the specific components that may be altered with disease progression. Through this approach plasma-cell-derived ECM can be identified with a view to developing therapeutic strategies in this disease. Disclosures Glavey: BMS: Consultancy, Research Funding. Palumbo:Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Description: Introduction.Exosomes are small vesicles (50-100 nm) of endocytic origin, which are released in the extra-cellular milieu by several cell types. Exosomes play a role in tumor progression since they have been shown to carry and transfer microRNAs (miRNAs) to recipient cells. In this study, we sought to characterize circulating exosomes in Multiple Myeloma (MM) patients and to assess the prognostic value of circulating exosomal miRNAs in a cohort of 112 newly diagnosed MM patients. Methods. Exosomes were isolated from peripheral blood (serum samples) using an ultracentrifugation protocol and Exoquick® solution. They were characterized using electron microscopy with immunogold labeling for the detection of CD63 and CD81, as well as for size using Nanosight® analysis. MiRNAs were isolated using miRNeasy micro kit (Qiagen®) and profiled using nCounter miRNA expression assay (Nanostring® Technologies) in 5 healthy donors (HD) and 10 MM patients. We next studied a cohort of 112 newly diagnosed MM patients uniformly treated with Bortezomib-Dexamethasone (4 cycles) followed by high dose Melphalan and autotransplant on the IFM 2005-01 Phase III Trial. After exosome isolation and miRNA extraction, we performed a low density qRT-PCR miRNA array using Taqman Array Micro Fluidic Cards for a panel of 23 specific miRNAs. Data were normalized by robust median global normalization and conditional inference tree was used to select the cut-point for miRNA levels. Kaplan-Meier estimation, Log-Rank test and Cox regression were used to assess progression-free survival (PFS). Results. Circulating exosomes were first characterized from 5 HD and 10 MM patients. CD63 and CD81 expression was demonstrated by immunogold labeling and electron microscopy. The size (approx. 100nm) and concentration of peripheral blood exosomes did not differ between HD and MM patients using Nanosight® analysis. We next performed a miRNA array (Nanostring) and identified differentially expressed miRNAs in circulating exosomes from MM patients compared to HD. Notably, the miR-17/92, -106b/25, -106a/363 clusters were more highly expressed and the cluster miR-15a/16 and let-7 family members were down regulated in circulating exosomes from MM patients as compared to HD. We then designed a panel of 23 specific miRNAs from circulating exosomes in MM and performed a Taqman Low Density Array on a cohort of 112 patients who were newly diagnosed and therapy-naive in the IFM 2005-01 Phase III Trial or treated as such. The mean patient’s age was 61 years, 36% of patients had a 13q deletion, 7% a t(4;14) and 4% a 17p deletion. ISS stage I, II and III distribution was 37%, 39% and 21% respectively. The median follow up was 5.4 years. Among tumor suppressor miRNAs, we found that patients with lower let-7e levels before treatment had significantly shorter PFS compared to patients with higher levels of let-7e (P

Description: Introduction: Citron rho-interacting serine/threonine kinase (CIT) is a serine/threonine kinase which is a key component of the midbody and is essential for cytokinesis. CIT localizes to the central spindle and midbody and functions to promote efficient cytokinesis. CIT knockdown may disrupt cytokinesis and therefore cell growth. CIT has been reported to be upregulated and important for growth of several cancers. However, the significance of CIT has not been investigated in the field of multiple myeloma (MM). We therefore dissected the role of CIT in MM growth in vitro and in vivo. Materials and methods: CIT gene expression in MM cells was compared to normal plasma cells using public-available gene expression profile (GEP) data set (GSE6477). Kaplan-Meier curve for MM patient survival between high and low CIT expressing patients were examined by using the GEP data set (GSE4581). Protein expression of CIT in MM cells was confirmed by proteomic analysis and immunohistochemistry. Knockdown of CIT was performed in MM cell lines MM1s and OPM2 using lentiviral shRNAs. CIT knockdown was confirmed by reduced CIT mRNA in comparison to a scrambled control. Differences in cell proliferation and cell cycle between CIT knockdown cells and scramble control were analyzed by using thymidine uptake and PI staining, respectively. Cytokinesis failure was analyzed by immunofluorescence using alpha-tubulin antibody and DAPI. shCIT OPM2 (n=7) and the scrambled control cells (n=8) were injected subcutaneously into SCID-Bg mice (5x106 cells/mouse) and were followed for tumor development and survival. Results: CIT expression was significantly higher in MM patients’ plasma cells compared to healthy donors in GEP (p=0.02), proteomic analysis and immunohistochemistry. Also CIT expression was higher in relapsed patients compared to newly diagnosed patients by GEP. MM patients with high CIT expression had significantly worse overall survival compared to low CIT expressing patients (p=0.04). CIT knockdown MM cell lines showed reduced cell proliferation and G2 cell cycle arrest by thymidine uptake and PI staining compared to the scrambled control. Significantly, large amount of multinucleated cells, which indicates cytokinesis failure, were observed in the CIT knockdown cells compared to scrambled control. Reduced tumor growth (p