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  • 1
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    The Functional Role of Microrna 15a/16-1 as Tumor Suppressor Genes in Multiple Myeloma.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 1963-1963
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 1963-1963
    Abstract: Abstract 1963 Poster Board I-986 Background: Multiple Myeloma (MM), a cancer of plasma cells is characterized by frequent chromosomal alterations. Deletion of chromosome 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. When studied in the context of CLL, the miR 15a and 16-1 cluster was associated with a distinct miR signature and clinical outcome. Over-expression of miR16 caused induction of apoptosis and downregulation of the anti apoptotic gene BCL2 in a megakaryocytic leukemia cell line and induced growth arrest in MM cells. Nonetheless, being lost in CLL, MM, MCL and LPL, their functional role has not been studied using a loss-of-function approach in any of these lymphoid malignancies. Here, we describe the generation of an in vivo system for the long term, stable knockdown of miR 15a/ 16-1 expression in myeloma cells to recapitulate the conditions seen in chromosome 13q14 deleted MM. Methods: Using lentiviral vectors to stably express a competitive sponge miR16 inhibitor we set up a system to functionally validate the role of microRNA 15a/16-1 cluster. Pure populations of lentivirally transduced MM cell lines were sorted by flow cytometry using GFP marker. Decreased miRs 15a and 16 expression levels were assessed by Northern blot and R-luciferase reporter system. Cell growth rate was measured using trypan blue counting, and thymidine incorporation. Cell cycle analysis was measured by flow cytometry after staining with PI. Intracellular signal modulation was demonstrated by Western blotting. RNA from MM cell lines expressing the control sponge or sponge16 were hybridized on an Affymetrix U133A 2.0 array chip, and validated using quantitative real time PCR. Xenograft murine models were performed using the stable MM cell lines injected into 6-week old NOD.CB17-PrkdcSCID/J irradiated mice. Results: Selected stable miR knockdown MM cell lines exhibited significantly reduced expression of miRs15a/16-1 as assessd by both by mRNA levels and miR luciferase reporter assays. The knockdown cells showed a significant increase in growth rates (1.5-2 fold) compared to control cells, as measured by viable cell counts and proliferation by thymidine incorporation in vitro. Importantly, miR16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load, invasiveness and host angiogenesis. To further delineate the role of miR15a/16 in MM and to gain additional insight into the possible target genes regulated by this cluster, we performed gene expression-profiling analysis in controls and miR16 deficient MMS1 and RPMI cell lines. Since our sponge system produces downregulation of the miRs, we focused on the upregulated probes. Expression profiling analysis of miR16 deficient cells identified a surprisingly large number of downstream target-genes such as FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. These results were verified both at the mRNA level and the protein level, as well as in other MM cell lines. Moreover, we were able to show that these knockdown cells were partially addicted to some of these pathways using specific drug inhibitors. We further validated designated genes as direct miR16 targets by showing binding sites within the conserved 3' UTR and also within the mRNA coding region, thus indicating that the miRs may have many more possible targets than anticipated by conventional prediction methods. Conclusions: Using this loss-of-function system, which mimics the pleiotropic chronic effects of microRNA loss at the 13q chromosomal deletion, provides a valuable tool to investigate their function as tumor suppressor genes in MM pathogenesis, affecting multiple targets, and may represent a novel potential for therapeutic targeting in MM and other lymphoid malignancies. Disclosures: Munshi: Seattle Genetics, Inc.: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.1963.1963
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
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  • 2
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    Bortezomib Induces Anti–Multiple Myeloma Immune Response Mediated by cGAS/STING Pathway Activation
    American Association for Cancer Research (AACR) ; 2021
    In:  Blood Cancer Discovery Vol. 2, No. 5 ( 2021-09-01), p. 468-483
    Details
    In: Blood Cancer Discovery, American Association for Cancer Research (AACR), Vol. 2, No. 5 ( 2021-09-01), p. 468-483
    Abstract: The proteasome inhibitor bortezomib induces apoptosis in multiple myeloma cells and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine multiple myeloma models, we here show that bortezomib also triggers immunogenic cell death (ICD), characterized by exposure of calreticulin on dying multiple myeloma cells, phagocytosis of tumor cells by dendritic cells, and induction of multiple myeloma–specific immunity. We identify a bortezomib-triggered specific ICD gene signature associated with better outcome in two independent cohorts of patients with multiple myeloma. Importantly, bortezomib stimulates multiple myeloma cell immunogenicity via activation of the cGAS/STING pathway and production of type I IFNs, and STING agonists significantly potentiate bortezomib-induced ICD. Our study therefore delineates mechanisms whereby bortezomib exerts immunotherapeutic activity and provides the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in multiple myeloma. Significance: Our study demonstrates that cGAS/STING-dependent immunostimulatory activity mediates bortezomib anti-myeloma activity in experimental models and associates with clinical response to bortezomib in patients with multiple myeloma. These findings provide the rationale for clinical evaluation of STING agonists to further potentiate anti–multiple myeloma immune response. See related commentary by Zitvogel and Kroemer, p. 405. This article is highlighted in the In This Issue feature, p. 403
    Type of Medium: Online Resource
    ISSN: 2643-3230 , 2643-3249
    URL: Article
    DOI: 10.1158/2643-3230.BCD-21-0047
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2021
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  • 3
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    Targeting the miR-221–222/PUMA/BAK/BAX Pathway Abrogates Dexamethasone Resistance in Multiple Myeloma
    American Association for Cancer Research (AACR) ; 2015
    In:  Cancer Research Vol. 75, No. 20 ( 2015-10-15), p. 4384-4397
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 75, No. 20 ( 2015-10-15), p. 4384-4397
    Abstract: Despite recent therapeutic advances that have doubled the median survival time of patients with multiple myeloma, intratumor genetic heterogeneity contributes to disease progression and emergence of drug resistance. miRNAs are noncoding small RNAs that play important roles in the regulation of gene expression and have been implicated in cancer progression and drug resistance. We investigated the role of the miR-221–222 family in dexamethasone-induced drug resistance in multiple myeloma using the isogenic cell lines MM1R and MM1S, which represent models of resistance and sensitivity, respectively. Analysis of array comparative genome hybridization data revealed gain of chromosome X regions at band p11.3, wherein the miR-221–222 resides, in resistant MM1R cells but not in sensitive MM1S cells. DNA copy number gains in MM1R cells were associated with increased miR-221–222 expression and downregulation of p53-upregulated modulator of apoptosis (PUMA) as a likely proapoptotic target. We confirmed PUMA mRNA as a direct target of miR-221–222 in MM1S and MM1R cells by both gain-of-function and loss-of-function studies. In addition, miR-221–222 treatment rendered MM1S cells resistant to dexamethasone, whereas anti-miR-221–222 partially restored the dexamethasone sensitivity of MM1R cells. These studies have uncovered a role for miR-221–222 in multiple myeloma drug resistance and suggest a potential therapeutic role for inhibitors of miR-221–222 binding to PUMA mRNA as a means of overcoming dexamethasone resistance in patients. The clinical utility of this approach is predicated on the ability of antisense miR-221–222 to increase survival while reducing tumor burden and is strongly supported by the metastatic propensity of MM1R cells in preclinical mouse xenograft models of multiple myeloma. Moreover, our observation of increased levels of miR-221–222 with decreased PUMA expression in multiple myeloma cells from patients at relapse versus untreated controls suggests an even broader role for miR-221–222 in drug resistance and provides a rationale for the targeting of miR-221–222 as a means of improving patient outcomes. Cancer Res; 75(20); 4384–97. ©2015 AACR.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/0008-5472.CAN-15-0457
    RVK:
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    RVK:
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2015
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  • 4
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    Abstract 400: Establishment and characterization of a novel IL6-dependent HLA-A2+ human myeloma cell line LNT1
    American Association for Cancer Research (AACR) ; 2010
    In:  Cancer Research Vol. 70, No. 8_Supplement ( 2010-04-15), p. 400-400
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 70, No. 8_Supplement ( 2010-04-15), p. 400-400
    Abstract: The novel multiple myeloma (MM) cell line LNT1 was established from peripheral blood of a 66-year-old female patient with recurrent plasma cell leukemia of IgG/kappa type. Peripheral blood mononuclear cells from the heparinized peripheral blood separated by Ficoll-Hypaque density gradient centrifugation were cultured in RPMI 1640 medium/20% FBS, 100 U/ml penicillin and 50 μg/ml streptomycin, without additional growth factors. Cells were cultured by replacing ½ of the volume of the culture with fresh medium every 3-4 days for 3 months. In the 4th month, a slow yet sustained growth of cultured cells was noted, and designated LNT1. These cells grow as single cell suspension; IL-6-induced proliferation in a dose-dependent manner with 2 ng/ml of IL-6 inducing maximal DNA synthesis (by [3H]thymidine uptake) and survival (by MTT). In contrast, IGF-1 had no effect. Subsequently, 2 ng/ml of IL-6 was added to cultures and growth now sustained at 7 months. LNT1 was negative for Epstein-Barr virus, as evidenced by PCR using primers specific for EBNA-1. May-Grunwald-Giemsa staining showed that LNT1 cells have a high nucleocytoplasmic ratio with excentrically located nuclei, prominent nucleoli, and a deeply basophilic and vacuolated cytoplasm. Some cells were bi- or multi-nucleated. Surface antigen analysis showed that LNT1 cells express CD138, CD38, HM1.24, CS1, and HLA-A2, but did not express CD19, CD20, CD3, CD16, and CD14. Chromosome analysis at 7 months revealed the karyotype: 43, X, -X, del(1)(p13p22), psu dic(6;1)(q13;p11), del(12)(p11.2), −13, add(19)(q13.4), −22[2] /43, idem, del(2)(q11.2q2?3), der(6)ins(6;?)(q21;?)[7]. DNA fingerprinting was performed by simultaneously amplifying eight short tandem repeat (STR) loci and the amelogenin gene in a multiplex PCR reaction using specific primers to confirm the authentication of cell line. The established LNT1 cell line and the primary tumor showed identical band patterns of these 9 markers and the same immunoglobulin H rearrangement. An unique and identical VH3/IgG fragment was amplified using the appropriate VH family-specific framework region primer in conjunction with CH isotype specific (IgG) primers in DNA samples of both LNT1 cell line and the original tumor. Moreover, identical DNA sequence of VH3/IgG PCR from cell line and primary tumor further confirms clonality. LNT1 cell line also bears a N-RAS G12D mutation, as in the original tumor. Finally, LNT1 cell line is responsive to conventional (Dex, Malphalan) and novel or emerging (bortezomib, lenalidomide, perifosine, AS703026) anti-MM therapies. Therefore, the novel LNT1 cell line will provide a useful model system to study genetics and biology of MM, as well as to evaluate novel therapeutic strategies. Since LNT1 cells highly express HLA-A2, this line also serves a useful model to validate novel tumor-associated antigens for immunotherapy in MM. 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 400.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM10-400
    RVK:
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2010
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  • 5
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    The Transmembrane Receptor Roundabout 1 (ROBO1) Is Necessary for Multiple Myeloma Proliferation and Homing to the Bone Marrow Niche
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 507-507
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 507-507
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-128778
    RVK:
    XA 33000
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
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  • 6
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    The KDM3A-KLF2-IRF4 Axis Maintains Myeloma Cell Survival
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 3633-3633
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 3633-3633
    Abstract: Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase. KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM. KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A. We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of 〉 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells. shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells. KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow. In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. Disclosures Raje: Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.3633.3633
    RVK:
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 7
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    The Clonal Architecture of CXCR4mutations in Waldenstrom's Macroglobulinemia Shows Highly Variable Subclonal Distribution, and Multiple Mutations within Individual Patients Indicative of Targeted Genomic Instability
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 1486-1486
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 1486-1486
    Abstract: Background: Whole genome sequencing (WGS) identified activating CXCR4WHIM somatic mutations in nearly 30% of patients with Waldenstrom's Macroglobulinemia (WM) (Blood 123(11):1637-46). Both nonsense and frameshift CXCR4WHIM mutations occur in WM, with over 30 different types of mutations described within the regulatory carboxyl-terminal domain of CXCR4. CXCR4WHIM mutations almost always occur with activating MYD88 mutations, and impact both disease presentation and treatment outcome (Blood 123(18):2791-6; NEJM 372(15):1430-40.). The clonal architecture of CXCR4WHIM mutations relative to MYD88 mutations and their role in disease evolution remains to be clarified. Methods: We used Sanger sequencing and highly sensitive AS-PCR assays that we developed for the most common CXCR4WHIM mutations (S338X C 〉 A and C 〉 G) to evaluate for CXCR4WHIM mutations. In conjunction with an AS-PCR MYD88L265P assay that we previously developed (Leukemia 28(8):1698-707), we also profiled tumor samples for MYD88L265P and CXCR4S338X mutations in 164 WM, 12 IgM MGUS, 20 MZL, 32 CLL, 14 MM, 7 non-IGM MGUS patients, and 32 healthy donors. Next generation transcriptome sequencing data was also performed for validation in select cases. Results: AS-PCR detected CXCR4S338X mutations in WM and IgM MGUS patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4WHIM mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%), and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and MZL patients, respectively, but not in CLL, MM, non-IGM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4S338X mutations were primarily subclonal, with highly variable clonal distribution (median 35.1%, range 1.2%-97.5%; Figure 1). Sanger sequencing identified 3 patients with multiple CXCR4 mutations, which were shown to be compound heterozygous by TA cloning and sequencing of at least 40 clones. The addition of AS-PCR to the Sanger sequencing results also revealed multiple CXCR4WHIM mutations in many individual patients that included homozygous and compound heterozygous mutations that were validated by next generation sequencing that offered a median of 5,819 (range 5,217-12,235) reads that overlapped the mutated loci. Conclusions: Taken together, these findings show that CXCR4WHIM mutations are more common in WM patients than previously revealed by WGS or Sanger sequencing. Moreover, CXCR4 mutations are primarily subclonal supporting their acquisition after MYD88L265P in WM oncogenesis. The exclusive finding of frameshift and nonsense but not missense variants within the carboxyl-terminal domain of CXCR4 suggests that significant selection pressures exist for activating mutations within the WM clone. Lastly, multiple CXCR4WHIM mutations are common in WM patients indicative of targeted genomic instability within the carboxyl-terminal domain of CXCR4. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.1486.1486
    RVK:
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 8
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    Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells
    American Society of Hematology ; 2016
    In:  Blood Vol. 127, No. 9 ( 2016-03-03), p. 1138-1150
    Details
    In: Blood, American Society of Hematology, Vol. 127, No. 9 ( 2016-03-03), p. 1138-1150
    Abstract: SIRT6 is highly expressed in multiple myeloma cells and blocks expression of ERK-regulated genes. Targeting SIRT6 enzymatic activity sensitizes multiple myeloma cells to DNA-damaging agents.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2015-06-649970
    RVK:
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 9
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    Roundabout 1 (ROBO1)/SLIT2 Is a Novel Signaling Pathway in Multiple Myeloma Promoting Survival and Bone Marrow Niche Interaction
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 485-485
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 485-485
    Abstract: Introduction Molecular mechanisms of multiple myeloma (MM) pathogenesis are unknown, but a role for bone marrow (BM) niche in supporting MM is well established. The transmembrane receptor Roundabout 1 (ROBO1) has been historically annotated as a tumor suppressor gene in solid cancer, and more recently myelodysplastic syndrome. High ROBO1 expression was identified as a poor prognostic factor in newly diagnosed MM; however, its function in MM is unknown. Material and Methods We analyzed ROBO1 and SLIT2 expression in human MM cell lines (HMCL) and a panel of human cancer cell lines via western blot (WB). ROBO1/SLIT2 expression in primary BM samples was assessed via immunohistochemistry and/or WB. We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD). We designed and cloned single guide RNA (sgRNA) into pSpCas9(BB)-2A-GFP vector and developed CRISPR-Cas9-mediated ROBO1 knock out (KO) 293T and OPM2 clones. WT and KO ROBO1 OPM2 cells were used for standard viability, proliferation, and adhesion assays. Full length (FL) and mutant ROBO1 isoforms devoid of extracellular or intracellular domain, with a C-terminus triple FLAG tag were designed and cloned into pCDH lentiviral vectors. WT and ROBO1 KO OPM2 were injected in the BM cavity of femoral bones harvested from donor SCID mice, and implanted subcutaneously into recipient SCID mice to study ROBO1 KO in vivo. Results HMCL, primary MM and BM stromal cells (MM-BMSC) from patients, but not healthy donor BM-resident plasma cells and BMSC, express ROBO1 and SLIT2. Across a panel of myeloid and lymphoid cancer cell lines, high ROBO1 expression was detected only in SU-DHL-4 and BCWM1 cell lines. In newly diagnosed MM patients enlisted in the IMF170 database, high ROBO1 portended worse overall survival compared to low ROBO1 expression (P=0.018). Sh-RNA mediated ROBO1 KD was profoundly and specifically cytotoxic to HMCL, with no cytotoxicity observed in high-ROBO1 expressing SH-SY5Y and HeLa cancer cell lines. Consistent with on target effect, ROBO1 shRNA was not cytotoxic for non-ROBO1 expressing cancer cell lines. To study the functional consequences of ROBO1 loss in MM, we performed CRISPR-Cas9-mediated ROBO1 KO in HMCL. We were unable to establish biallelic ROBO1 KO H929 or U266 clones, suggesting that biallelic loss may be lethal in these HMCL, but created two clones of ROBO1 KO OPM2 cells. In vitro, ROBO1 KO and WT OPM2 cells have comparable viability and proliferation rate, even in the presence of MM-BMSC. However, ROBO1 KO OPM2 cells have profound defects in adhesion to MM-BMSC and BM endothelial cell lines (BMEC). Lentiviral transduction of OPM2 ROBO1 KO cells with FL-ROBO1 addback lentiviral construct completely rescues the BMEC adhesion defect, confirming that loss of ROBO1 is responsible for the defective adhesive phenotype. Interestingly, treatment with heparin abolishes the rescue effect of FL-ROBO1, implying that surface heparan sulfate proteoglycans (HSPG) are necessary for ROBO1-mediated MM-BMEC adhesion. Consistent with a role for ROBO1 in MM-BM niche interaction, ROBO1 KO OPM2 cells show higher failure to engraft in the bone implants compared to ROBO1 WT OPM2 (bone implants without MM engraftment: 5/15 versus 1/15, respectively, P=0.067). PET-CT scan of study mice shows ROBO1 KO tumors to be significantly smaller compared to ROBO1 WT (mean volume 1.2 cm3 vs. 2.9 cm3, P=0.003), suggesting a profound in vivo proliferation/survival defect of ROBO1 KO OPM2. Summary and Conclusions Our data demonstrate that ROBO1 and SLIT2 are highly expressed in HMCL and in the BM of MM patients, but not healthy controls. ROBO1 expression correlates with worse OS in newly diagnosed MM patients; and ROBO1 KD is selectively cytotoxic for HMCL, but not high-ROBO1 expressing SH-SY5Y and HeLa cells. CRISPR-Cas9-mediated ROBO1 KO significantly impairs OPM2 cell adhesion to BMEC and MM-BMSC, which is completely reversed by adding back FL-ROBO1. In vivo, ROBO1 KO OPM2 cells show impaired engraftment to bone implants and significantly decreased growth rate compared to ROBO1 WT OPM2. Altogether, our data demonstrate that ROBO1 is a novel pro-survival protein in MM, mediating MM-BM niche interaction and suggest the therapeutic potential of targeting ROBO1/SLIT2 pathway in MM. Proteomic and RNA-sequencing studies on the downstream signaling effectors of ROBO1/SLIT2 are currently ongoing to elucidate the molecular mechanisms of pathway activation in MM. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Takeda: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Noxxon: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding. Anderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.485.485
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 10
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    Delineation of Canonical and Non-Canonical NF-κB Pathways in Multiple Myeloma: Therapeutic Implications.
    American Society of Hematology ; 2007
    In:  Blood Vol. 110, No. 11 ( 2007-11-16), p. 670-670
    Details
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 670-670
    Abstract: NF-κB pathway plays a crucial role in the pathogenesis in cancer cells including multiple myeloma (MM). The NF-κB complex is dimer in different combinations of Rel family proteins, including p65 (RelA), RelB, c-Rel, p50 (NF-κB1), and p52 (NF-κB2). Recent studies have revealed that NF-κB activity is mediated via two distinct pathways. In the canonical pathway, NF-kB is typically a heterodimer composed of p50 and p65 subunits. In the non-canonical pathway, NF-kB is typically a heterodimer composed of RelB and p100 subunits. We have shown anti-MM activities of IKKβ inhibitors (PS-1145, MLN120B); however, effects of these agents were modest. Our studies therefore suggest that baseline NF-kB activity in MM cells is not totally dependent on the canonical pathway, and that inhibition of only canonical NF-κB pathway may not be sufficient to block total NF-kB activity. In this study, we therefore hypothesized whether non-canonical inhibitors significantly enhanced NF-κB inhibition induced by canonical inhibitors in MM cells. We first examined baseline NF-κB activity using electrophoretic mobility shift assay (EMSA). NF-κB activity varied between cell lines; for example MM.1S, MM.1R and H929 cells have higher level of NF-κB activity than in RPMI8226, INA6 and OPM2 cells. To define the role of canonical and non-canonical pathway, we next examined protein expression of p50, p65 and p52 NF-κB in these cell lines: p65 was highly expressed in all MM cell lines; however, expression of p50 and 52 is variable. Surprisingly, no detectable or weak expression of p50 was observed in U266, RPMI8226, LR5, H929 and OPM2 cell lines, suggesting that baseline NF-kB activity in these cell lines is not maintained only by the canonical pathway. We then attempted to block non-canonical NF-κB pathway in MM cell lines. Specifically since IKKα and IKKβ are client proteins of heat shock protein (Hsp) 90, we examined whether 17AAG could inhibit expression and/or function of IKKα and IKKβ in MM cells. Importantly, both IKKα and IKKβ were significantly downregulated by 17AAG in MM cell lines. To determine whether downregulation of these IKK proteins by 17AAG was due to inhibition of transcription, we next performed real-time quantitative PCR and no significant inhibition of relative expression of IKKβ was observed by 17AAG treatment, suggesting that downregulation of these proteins was a post transcription event. We further examined whether 17AAG enhanced the effect of IKKβ inhibitor MLN120B on NF-κB activity. Although the inhibitory effect by either MLN120B or 17AAG alone on phosphorylation (p) of IκBα triggered by TNFα was marginal, combination treatment of MLN120B with 17AAG almost completely blocked IκBα, suggesting that this combination synergistically inhibit canonical NF-κB activity in MM cells. Importantly, the combination of MLN120B with 17AAG also significantly blocked baseline and TNFα-triggered NF-κB activity, assessed by EMSA, in MM cells. Finally, 17AAG augmented the growth inhibitory effect of MLN120B in the context of bone marrow stromal cells. Taken together, these results showed that baseline and TNFα-triggered NF-κB activities were completely blocked by this combination treatment, and provide the rationale for its clinical evaluation to induce maximum inhibition of NF-κB activity and improve patient outcome in MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V110.11.670.670
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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