Long Non-Coding RNAs Annotation and Their Involvement in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4420-4420 ◽  
Author(s):  
Arantxa Carrasco ◽  
Teresa Ezponda ◽  
Cem Meydan ◽  
Marta Kulis ◽  
Raquel Ordoñez ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cell ◽  
Cell Lines ◽  
Promoter Region ◽  
Research Funding ◽  
Epigenetic Mechanisms ◽  
Non Coding Rnas

Abstract Increasing amount of evidence indicates that the deregulation of non-coding elements is a common feature of cancer and therefore, its investigation may uncover new molecular oncogenic mechanisms. In multiple myeloma (MM), the altered expression of a small number of long non-coding RNAs (lncRNAs) has been associated with progression and decreased survival, suggesting that these elements may play a more important role in this disease than previously expected. Nevertheless, an extensive high-throughput analysis that characterizes the deregulation of lncRNAs in MM has not yet been performed. To characterize the transcriptome, including all genomic types of lncRNAs, of MM we performed a paired end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients, as well as PC samples from tonsils (TPCs, n=5) and bone marrow (BMPCs, n=3) of healthy donors as controls. Principal component analysis (PCA) demonstrated that normal PC samples from tonsil and bone marrow cluster separately, suggesting that in spite of being the same cell type, their coding and non coding transcriptomes are very different. Therefore, we selected BMPCs as the normal counterparts for comparison with BM of MM samples. PCA analysis also demonstrated that the well known heterogeneity of MM patients rely not only on the coding transcriptome but also on the lncRNA expression profile. Comparison of MM to BMPCs samples showed 70 previously annotated lncRNAs that were deregulated in MM patients, with 3 lncRNAs showing higher and 67 lower expression than normal BMPCs. Moreover, we identified 40.552 novel MM-specific lncRNAs that were present in at least 3 of the 38 patients, highlighting the magnitude of the deregulation of these non coding elements in MM. To determine the functional role of altered lncRNAs in the biology of MM plasma cells we focused on the study of LINC-MSL1 (Myeloma-Specific LncRNA 1). Analysis of the expression of this lncRNA at different stages of B-cell differentiation (Naïve, Germinal Center, Memory and PC) indicated that it is not expressed at any stage, except for a modest expression in BMPCs. Interestingly, its overexpression was detected in 40% of MM specimens when compared to normal BMPCs which was validated by qPCR in an independent cohort of MM patients. To determine whether the expression of this lncRNA is regulated by epigenetic mechanisms, we studied the DNA methylation state of this gene. DNA methylation analysis in MM demonstrated that the CpGs located upstream of LINC-MSL1 were differentially methylated in comparison with normal counterpart BMPC. These CpGs showed 70% DNA methylation in control samples, about 40% in MGUS, whereas the average of MM was about 20%, showing a remarkable hypomethylation. We validated these results by pyrosequencing, which showed a significantly lower DNA methylation at the promoter region in comparison with B cell populations from tonsil, normal BMPCs and cell lines that do not overexpress LINC-MSL1. We also have observed a gain of active chromatin states analyzed by ChiP-seq in the promoter region of LINC-MSL1 in MM patient samples. These data suggest that epigenetic mechanisms, namely the progressive hypomethylation and the gain of active histone modifications, are the cause of the overexpression of LINC-MSL1 in MM. To analyze the role of the overexpression of LINC-MSL1 in MM, we engineered two MM cell lines that show high levels of LINC-MSL1, MM.1S and MM.1R, to express shRNAs against this lncRNA. Knockdown of LINC-MSL1 by two different shRNAs resulted in a reduced proliferation of the cell lines over time. This effect was not associated with a cell cycle arrest but with a marked increased in the percentage of Annexin V-positive apoptotic cells, indicating that the overexpression of LINC-MSL1 is necessary for the survival of MM cells. All together, these data demonstrate that the alteration of lncRNAs is an important an unexplored feature that contributes to MM pathogenesis. The overexpression of LINC-MSL1 is essential for MM survival and is very specific of MM BMPCs, suggesting it could be a relevant therapeutic target. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Melnick:Janssen: Research Funding.

Genome Wide DNA Methylation Profiling In Patients with Multiple Myeloma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3622-3622
Author(s):  
Yang Liu ◽  
Shenghua Duan ◽  
Xavier Leleu ◽  
Yong Zhang ◽  
Abdel Kareem A. Azab ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Cell Lines ◽  
Promoter Region ◽  
Genomic Dna ◽  
Research Funding ◽  
Plasma Cells ◽  
Genome Wide

Abstract Abstract 3622 Introduction: Epigenetic factors such as DNA methylation have been shown to play a crucial role in the pathogenesis and progression of multiple myeloma (MM), yet studies of DNA methylation in MM are still limited. Therefore, in order to better understand the role of DNA methylation and identify specific genes that may be affected by differential methylation in MM patients, we conducted genome-wide DNA methylation profiling in cd138+ plasma cells purified from bone marrow of the patients with MM and normal donors. Methods: Genomic DNA of CD138+ Plasma cell selected from both MM patients and normal primary bone marrow was extracted using QIAGEN genome isolation kit. Following extraction, methylated DNA was isolated by Chip and hybridized to Affymetrix Human 2.0 tiling arrays. Chip assay and array hybridization was performed by Genepathway Inc. CEL files were processed and normalized using the MAT program, and methylation peaks were called from the resulting MAT scores using a custom segmentation method. Peak annotation and characterization of different genomic regions was done with custom tools and using genome annotation files from the UCSC genome database. All peaks were visualized by IGB online software. Medip-PCR was done in human MM cell lines to validate the methylation status. Methylated gene expression was determined by both Semi-quantitative PCR and real-time PCR. 5′aza was used for demethylation in human MM cell lines. Methylated gene expression with or without 5′aza treatment was determined by both Semi-quantitative PCR and real-time PCR. Results: Genomic DNA from CD138+ plasma cells from bone marrow of MM patients showed a significant increase in methylation levels compared to normal controls. We demonstrated that the hypermethylated sites were distributed across the genome in the following proportions: 3.2% in the promoter region; 45.6% in the intragenic region; 5.4 % in the 3′ end region; and 46.8 % in the intergenic region. Furthermore, around 9 % promoter CpG islands (CGIs); 11% intragenic CGIs; 15 % CGIs in 3′end region; and 14.3 % intergenic CGIs of patients genomic DNA were methylated. Moreover 2.1% promoter CGIs; 2.3 % intragenic CGIs; 2.5% CGIs in 3′end region; and 4.7% intergenic CGIs were methylated for the normal control. Medip-PCR showed that the identified methylation pattern in MM patients showed similar results in MM cell lines. Expectedly, we also observed that suppressor of cytokine signaling 1 (SOCS1) was hypermethylated at the promoter region (MAT score=19.986) as has been reported in human cell lines. Importantly, another member of SOCS family SOCS3 showed much stronger signal in the promoter region with CpG island (MAT score=31.707) in MM patients compared to normal control. Notably, the expression of two members of TNFR superfamily TNFRSF18 and TNFRSF4 which play an important role in development and programmed cell death of lymphocyte significantly have increased 283 and 141-fold after treatment with 5′aza in MM cell lines. Conclusion: These findings enhance our understanding of the role of DNA methylation in MM, as one of the epigenetic changes that may contribute to the pathogenesis of this disease. The identification and functional characterization of novel key molecules affected by DNA methylation will provide deeper insight into the molecular basis of MM disease. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The Biologic Relevance of PIM Kinases in the Context of Multiple Myeloma and Their Potential As Therapeutic Targets in Combination Drug Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2992-2992
Author(s):  
Janani Ramachandran ◽  
Loredana Santo ◽  
Homare Eda ◽  
Dharminder Chauhan ◽  
Ka Tat Siu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Dna Damage ◽  
Drug Therapy ◽  
Cell Viability ◽  
Cell Lines ◽  
Research Funding ◽  
Doxorubicin Treatment ◽  
Pim Kinases

Abstract The proto-oncogene proviral integration sites for moloney murine leukemia virus (PIM) are serine/threonine kinases currently under investigation as therapeutic targets in hematologic cancers, where they have important functional roles as mediators of apoptosis, cell migration and homing. Thus far in Multiple Myeloma (MM), the PIM Kinases have been shown to mediate cap-dependent and cap-independent translation through the PI3K/AKT pathway and the activation of mTOR signaling (Lu J et al. 2013). In the context of the bone marrow microenvironment, PIM kinase expression in MM cells is known to be elevated in the presence of bone marrow stromal cells, and is mediated by the IL-6/STAT3 pathway, as well as the TNFα/NFΚB pathway (activated by Osteoclast secretion) (Hiasa M et al. 2014). Here, we have further studied the role of PIM kinases in MM. We observed elevated expression of PIM 1, 2 and 3 in patient derived myeloma cells (CD138+) as opposed to the stromal compartment (CD138-), confirming that PIM expression is predominantly hematologic lineage-specific. Inhibition of all three kinases by a pan PIM inhibitor results in reduced cell viability in tested human-derived MM cell lines, reinforcing the importance of the kinases as targets in drug therapy, as shown previously. Because the expression of Pim2 is consistently higher in primary tumor cells as well as in human MM lines, compared to Pim1 and Pim3, we sought to identify their distinct biologic significance. Single knock down (KD) of each kinase resulted in varying effects on cell viability, suggesting, together with the PIM expression profile, that the three kinases play different roles in the biology of MM. As the Pim2 KD resulted in the most profound decrease in cell viability, we focused our efforts to dissect the mechanistic importance of Pim2. Since Pim1 and Pim2 regulate the DNA damage response (DDR) via checkpoint kinase 1 (Chk1) in other hematologic malignancies, we investigated the effect of Pim2 KD on major signaling factors involved in the DDR. Transient KD resulted in phosphorylation of DDR pathway markers including ATR, CHK1/2, P21 and H2AX, and mimicked the effects of Doxorubicin treatment (a known DNA Damage causing agent). Furthermore, Doxorubicin treatment downregulated Pim2 expression, suggesting that Pim2 functions as an upstream regulator of the DDR pathway in MM. Pim2 appears to be the most relevant target in MM; however, because of a lack of a specific PIM2 inhibitor we used the pan-PIM inhibitor as a tool compound. Although the pan PIM kinase inhibitor showed single agent activity, combination approaches were more efficacious. Combining the pan-PIM inhibitor with bortezomib shows a significant synergistic effect on cell viability in multiple MM cell lines (MM1S, U266, KMS-12BM). Based on our KD experiments Pim2 predominantly mediates cell viability, suggesting that specifically targeting Pim2 in combination with Bortezomib will have a more direct effect on MM cell survival. To confirm the role of Pim2, ongoing experiments aim to focus on the effects of ectopically expressing Pim2 in the context of myeloma. We propose that Pim2 overexpression will not only serve anti-apoptotic purposes, but will further protect against DNA Damage in human MM cell lines. Disclosures Chauhan: Stemline Therapeutics: Consultancy. Huszar:Astra Zeneca: Employment. Raje:AstraZeneca: Research Funding; Onyx: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Millenium: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Eli Lilly: Research Funding; Celgene Corporation: Consultancy.

Methylation-Dependent Epigenetic Silencing Of Mir-152 and Mir-10b-5p Plays a Crucial Role In Modulating Tumor Progression In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3751-3751
Author(s):  
Wenjing Zhang ◽  
Yu Zhang ◽  
Yong Zhang ◽  
Yuji Mishima ◽  
Michaela Reagan ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Promoter Region ◽  
Research Funding ◽  
Healthy Donor ◽  
Advisory Board ◽  
Epigenetic Silencing ◽  
Newly Diagnosed ◽  
Pcr Products

Abstract Background Epigenetic modifications including changes in DNA methylation lead to inhibited gene expressions and consequent phenotypic alterations. MicroRNAs (miRNAs) are a class of small non-coding RNAs (19∼25 nucleotides) which functions as endogenous silencers of target genes. In general, most miRNAs are downregulated in many cancers and specifically in multiple myeloma (MM). We hypothesized that the mechanism of low expression of tumor suppressor miRNAs in MM is through epigenetic silencing, specifically through CpG island hypermethylation. The aim of this study is to identify methylation-silenced miRNAs and clarify their contribution to MM development and progression. Methods MicroRNA microarray analysis was performed in a panel of six MM cell lines (MM1S, RPMI 8266, OPM2, U266, H929 and IMI9) with or without the treatment of 5 μM of 5'-aza-2'-deoxycytidine (5-Aza-CdR, DNA demethylating agent) to screen for the most commonly upregulated miRNAs in response to DNA demethylation. These results were compared to a global methylation data of patients with MM (GEO GSE21304). Of these, we further examined the role of miR-152 and miR-10b-5p in MM. Induced expression of miR-152 and miR-10b-5p with 5-Aza-CdR-treatment was validated with real-time PCR. The DNA methylation status of CpG islands of these two candidates was further evaluated by methylation specific PCR (MSP) and bisulfate sequencing PCR (BSP). The expression levels of miR-152 and miR-10b-5p in both newly diagnosed MM patients (N = 60) and normal healthy donor (N = 5) were further analyzed (GEO GSE16558). MiR-152 and miR-10b-5p mimics were transiently transfected into H929 cells, respectively and in vitro functional validation including cell proliferation, cell apoptosis and adhesion assays. Results We identified miR-152 as the most common upregulated miRNA (in all of the applied six cell lines); 7 miRNAs (miR-10b-5p, -320b, -4521, -548b-3p, -584-5p, -616-3p and -497-5p) and 77 miRNAs were upregulated by 1.5-fold or more in any four or three applied cell lines. Among them, the methylation of miR-152 and miR-10b-5p was reported in other hematologic malignancies, but little is known in MM. We first focused our attention on miR-152 and miR-10b-5p; and validated their significant upregulation in MM cell lines with 5-Aza-CdR treatment by real-time PCR. With specific methylated- or unmethylated primers for miR-152 or miR-10b-5p, MSP results indicated that miR-152 methylated PCR products had high levels in all control cells, whereas the unmethylated PCR products were significantly increased in the 5-Aza-CdR-treated cells. The BSP results of the promoter region of miR-152 showed extensive methylation throughout its promoter region (∼1000 bp upstream) in both H929 and IM9 cells, which was reversible following 5-Aza-CdR treatment. The average methylation levels of miR-152 were 77% and 84% in H929 and IM9 cells, however, after 5-Aza-CdR treatment, the methylation level decreased to 19% and 10%, respectively. Similar results were found for miR-10b-5p in both cell lines. Together, these findings confirmed that miR-152 and miR-10b-5p were suppressed through CGI methylation in MM cell lines. Compared with healthy donor, the expression of miR-152 and miR-10b-5p were significantly decreased in newly diagnosed MM patients. Moreover, overexpression experiments demonstrated that the cell proliferation of H929 cells, as detected by MTT assay, was significantly reduced after the restoration of miR-152 or miR-10b-5p (p< 0.05). Western blot results showed that cleaved PARP, caspase 9 and caspase 3 proteins were all significantly increased after miR-152 or miR-10b-5p mimics transfection. Adhesion assays showed that the adhesion capacity to stroma or fibronectin of H929 cells with miR-152- or miR-10b-5p-overexpression was significantly reduced compared with the negative control cells (p< 0.05). Conclusion As methylation-sensitive miRNAs, miR-152 and miR-10b-5p may play an important role as tumor suppressors in MM, targeting methylation of miRNAs could be a promising approach for the treatment of MM. Disclosures: Ghobrial: BMS: Advisory board Other, Research Funding; ONYX: Advisory board, Advisory board Other; NOXXON: Research Funding; Sanofi: Research Funding.

Potential Therapeutic Role of the Selective Adhesion Molecule (SAM) Inhibitor Natalizumab in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1850-1850 ◽  
Author(s):  
Klaus Podar ◽  
Alexander Zimmerhackl ◽  
Ursula Hainz ◽  
Mariateresa Fulciniti ◽  
Sonia Vallet ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Research Funding ◽  
Therapeutic Role ◽  
Potential Therapeutic Role

Abstract Abstract 1850 Poster Board I-876 Multiple Myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells in the bone marrow. Despite current therapeutic approach and prolongation of the median survival, new therapies are urgently needed. Integrins are cell surface receptors which mediate both cell-cell adhesion and cell-extracellular matrix (ECM) protein adhesion. beta1-integrins, including very-late antigen-4 (VLA-4;á4β1), are typically expressed on MM cells. In MM, VLA-4-mediated binding to ECMS and bone marrow stromal cells (BMSCs) confers protection against drug-induced apoptosis and triggers transcription and secretion of IL-6, the major MM growth and survival factor. In addition to up-regulation of cell surface-clustering, integrin activity can also be triggered by multiple agonists through ‘inside-out’ signaling, independent of changes in integrin expression levels. Importantly, VEGF-induced migration of MM cells on fibronectin is also associated with β1-integrin- and PI3-kinase- dependent PKC activation. Targeting VLA-4 is therefore of potential high therapeutic interest in MM. Indeed, an antibody against murine á4 induces inhibition of MM growth in a murine model. Natalizumab is a recombinant humanized IgG4 monoclonal antibody, which belongs to a new class of molecules known as selective adhesion molecule (SAM) inhibitors and binds to á4-integrin. Clinically, Natalizumab has demonstrated activity in patients with multiple sclerosis and Crohn's disease. Here we tested the potential therapeutic role of Natalizumab on MM cell survival, and migration in the BM microenvironment. VLA-4 is expressed by all MM cell lines investigated (NCIH929, RPMI8226, INA-6, MM.1S, and OPM2). Functionally, Natalizumab but not a control antibody, triggered dose-dependent inhibition of MM cell adhesion to fibronectin, BMSCs, and endothelial cells (ECs). Importantly, inhibition of adhesion to fibronectin, BMSCs, or ECs was observed in MM cells pretreated with Natalizumab. Moreover, inhibition of MM cell adhesion to fibronectin, BMSCs, or ECs was also observed when Natalizumab was added to already adherent MM cells. Taken together, Natalizumab decreases adhesion of non-adherent MM cells as well as binding of already adherent MM cells to non-cellular and cellular components of the microenvironment. Given the protective role of the microenvironment on MM cell survival, we next sought to evaluate the chemosensitizing activity of Natalizumab. Specifically, we investigated dose- and time- dependent effects of Natalizumab, alone and when combined with conventional and novel therapies, on MM cells. Our results show that Natalizumab alone did not inhibit growth or survival of MM cells when cultured without components of the microenvironment. However, Natalizumab enhanced sensitivity of tumor cells to both bortezomib and dexamethasone in MM-BMSC and, MM-EC co-cultures. These data indicate a potential role of Natalizumab in bortezomib- and dexamethasone-containing treatment regimens including MPV. Moreover, Natalizumab decreases IL-6 and VEGF secretion triggered in MM-BMSC co-cultures. Consequently, angiogenesis triggered by supernatants of Natalizumab- treated MM-BMSC co-cultures was inhibited. Moreover, Natalizumab blocked MM cell migration on fibronectin triggered by both VEGF and IGF-1. Finally, our previous results implicate an PKC signaling in MM cell migration on fibronectin, and our current results show that Natalizumab inhibits phosphorylation of á4 integrins and PKC induced by co-stimulation with VEGF/ fibronectin, IGF-1/ fibronectin, and patient serum. Taken together, our data indicate a potential therapeutic role of Natalizumab in MM. Ongoing studies evaluating the effect of Natalizumab in a SCID-hu murine model of MM will also be reported. Disclosures: Podar: Biogen Idec: Research Funding. Off Label Use: natalizumab, integrin inhibitor. Zimmerhackl:Biogen Idec: Research Funding. Olsen:Biogen Idec: Employment.

Frequent Dysregulation of Fc Gamma Receptor IIb (Fc γRIIb) and Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Signaling Occurs in Multiple Myeloma (MM) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2917-2917
Author(s):  
Jennifer Li ◽  
Andrew Leu ◽  
Mingjie Li ◽  
Ethan D Hobel ◽  
Kevin Delijani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Critical Role ◽  
Rt Pcr ◽  
Binding Ability ◽  
Downstream Signaling

Abstract Abstract 2917 The inhibitory Fc receptor, Fc γRIIb, is expressed on plasma cells, controls their persistence in the bone marrow (BM) and their ability to produce serum Ig. Activation of Fc γRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing inositol 5'-phosphatase (SHIP) in plasma cells. Immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposing signaling modules that duel for control of plasma cell activation. Fc γRIIb-mediated SHIP phosphorylation activates downstream ITAM or ITIM signaling. To determine whether multiple myeloma (MM) cells express Fc γRIIb, we performed immunohistochemical staining on bone marrow mononuclear cells from MM patients and controls. We found that not only CD20+ B cells expressed Fc γRIIb but more importantly CD138+ cells from MM patients also showed expression of this receptor. Next, we examined whether Fc γRIIb was present and expressed in CD138+ primary MM cells purified from fresh MM BM and the MM cell lines MM1s, RPMI8226, and U266 using PCR and RT-PCR on DNA and mRNA, respectively. We focused on the transmembrane domain of the Fc γRIIb gene with four primers from different parts of this domain since this portion plays a critical role in this receptor's function. The MM cell lines expressed different amounts of Fc γRIIb. Notably, we found that 17% (5/30) of MM patients showed absence of Fc γRIIb both using RT-PCR for mRNA and PCR for DNA. Moreover, use of these same primers on nonmalignant PBMCs from the MM patients also showed absence of this gene in the same five patients. As a result of these findings, we are currently sequencing Fc γRIIb in MM patients to determine if additional patients show mutational changes that affect the function of this receptor. We also further determined SHIP-1 phosphorylation using Western blot analysis since this protein mediates downstream signaling of Fc γRIIb. Following stimulation with Fc complexes, phosphorylation of SHIP-1 was markedly reduced in MM tumor cells compared to normal CD20+ B cells. Interestingly, the patients with missing Fc γRIIb expressed higher levels of SHIP-1 gene expression compared to patients with normal Fc γRIIb expression. We investigated the IgG-binding ability of MM patients (n=33) and normal donors (n=33) to Fc γRIIb. Each serum sample was incubated with cells from MHC1, a cell line that specifically expresses Fc γRIIb but not Fc γRI and Fc γRIIa. The results showed MM patients' serum IgG have much lower Fc γRIIb-binding ability than normal human IgG (P<0.05) by using both flow cytometric and immunofluorescence assays. Our findings suggest that the monoclonal protein produced by MM patients has a very low Fc γRIIb-binding ability and is incapable of signaling through the inhibitory ITIM pathway. Germline loss of Fc γRIIb in MM patients with variation in its expression level and its downstream signaling molecule SHIP and its phosphorylation as well as the inability of MM IgG to bind cells containing this receptor is a potential new mechanism that contributes to the uncontrolled growth of MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

Targeting Brouton's Tyrosine Kinase with PCI-32765 Blocks Growth and Survival of Multiple Myeloma and Waldenström Macroglobulinemia Via Potent Inhibition of Osteoclastogenesis, Cytokines/Chemokine Secretion, and Myeloma Stem-Like Cells in the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 883-883
Author(s):  
Yu-Tzu Tai ◽  
Betty Y Chang ◽  
Sun-Young Kong ◽  
Mariateresa Fulciniti ◽  
Guang Yang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Equity Ownership ◽  
Trap Staining

Abstract Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: 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. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Genome Wide Studies in Multiple Myeloma Identify XPO1/CRM-1 As a Critical Target Validated Using the Selective Inhibitor of Nuclear Export (SINE) KPT-276

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 573-573
Author(s):  
Jessica Schmidt ◽  
Esteban Braggio ◽  
Marta Chesi ◽  
Jan Egan ◽  
Yuan Xiao Zhu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Nuclear Export ◽  
Research Funding ◽  
Phase 1 ◽  
A549 Cells ◽  
B Cell Lymphoma ◽  
Anticancer Activities

Abstract Abstract 573 Using high throughput RNA interference screening on 6,722 druggable genes we previously identified XPO1/CRM1 as one of the 50 most vulnerable targets in Multiple Myeloma (MM)1. XPO1 knockdown proved lethal in MM cell lines, but had no effect on human embryonic kidney (293) cells or lung cancer (A549) cells, showing that XPO1 is a specific myeloma vulnerability, and that myeloma cell survival is dependent upon XPO1 expression. XPO1 encodes the protein exportin 1, a nuclear transport protein that exports tumor suppressor proteins from the nucleus, where they are active, to the cytoplasm, where they become inactive. We next analyzed XPO1 in MM via gene expression profiling (GEP). XPO1 expression is up-regulated as the disease progresses: patients with active MM have a higher level of XPO1 compared to normal plasma cells (p<0.04) and to patients with monoclonal gammopathy of undetermined significance or smoldering MM (p<0.0001). The highest levels were in human MM cell lines. TC classification revealed highest levels in t(11;14) and lowest levels in t(4;14) disease. Selective inhibitors of nuclear export (SINE) compounds have recently been developed that irreversibly inhibit XPO1/CRM1 and its nuclear export function. One such inhibitor, KPT-276, decreased the viability of all 12 MM cell lines tested in vitro, as shown by MTT assay. After 72 hours of drug treatment, a median IC50 value of approximately 175 nM (range 30–1000 nM) was observed. No synergy with other commonly used anti-MM therapeutics was observed in vitro. In contrast, the drug had little effect in 8 solid tumor cell lines with the exception of the B cell lymphoma line Ramos. KPT-276 was also consistently active in inducing apoptosis against MM primary patient samples. Using an IC80 dose of KPT-276, drug-treated samples had a reduced population of cells in S phase (8%) compared to cells treated with DMSO (21%). Using the vkappa*myc transgenic MM model, KPT-276 reduced monoclonal spikes (by a mean of 56%) in all mice treated orally with 150 mg/kg dose three times per week for 4 weeks. Furthermore, KPT-276 significantly reduced tumor growth in a xenograft MM1.S mouse model. GEP was performed in the presence or absence of drug in two different MM cell lines. Two genes of probable relevance, cell division cycle 25 homolog A (CDC25A) and Bromodomain-containing protein 4 (BRD4), were dysregulated by SINE treatment. Both are involved in cell cycle control and have been linked to MYC. RT-PCR and western blotting confirm that MYC, CDC25A and BRD4 are down-regulated, as soon as six hours, after treatment with KPT-276. KPT-276 has shown marked anticancer activities against B cell malignancies in vitro and is active and tolerated in Phase I canine studies. KPT-330, a close analog of KPT-276, is currently in Phase 1 studies in human with advanced hematological and solid tumors. Disclosures: Schmidt: Karyopharm: Research Funding. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

scholarly journals MYD88 L265P Augments Proximal B-Cell Receptor Signaling in Large B-Cell Lymphomas Via an Interaction with DOCK8

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1324-1324
Author(s):  
Elisa Mandato ◽  
Qingsheng Yan ◽  
Jing Ouyang ◽  
Julia Paczkowska ◽  
Yan Qin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
Genetically Engineered ◽  
Bcr Signaling ◽  
Myd88 L265p ◽  
Research Grant

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease comprised of five subtypes including a subset of poor-prognosis activated B cell (ABC)-enriched tumors with frequent MYD88L265P mutations, often in association with CD79B alterations (Cluster 5 DLBCLs) (Nat. Med. 2018; 24:679-690). Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) have similar genetic signatures including recurrent MYD88L265P mutations and concurrent CD79B alterations (Blood 2016; 127: 869-81). These findings prompted us to evaluate a potential role for MYD88L265P in proximal B-cell receptor (BCR) signaling, in addition to its defined function as an intermediary in the Toll-Like Receptor (TLR) pathway and downstream NF-kB activation. In previous studies by Jabara et al., wild-type (WT) MYD88 was found to be constitutively associated with the DOCK8 adapter and the PYK2 tyrosine kinase in normal B-cells (Nat. Immunol. 2012; 13:612-20). In this setting, physiologic ligation of TLR9 with CpG oligodeoxynucleotides (CpG) induced PYK2-mediated phosphorylation of DOCK8, recruitment of Src kinases, including LYN, and downstream activation of the proximal BCR pathway member, spleen tyrosine kinase (SYK) (Nat. Immunol. 2012; 13:612-20). We postulated that mutated MYD88L265P might similarly augment proximal BCR signaling in DLBCLs in the absence of physiologic (CpG-induced) TLR9 signaling. Using three DLBCL cell lines (OCI-Ly1, SU-DHL4 and OCI-Ly7) with intact BCR signaling and WT endogenous MYD88 and CD79B, we first established that physiologic CpG activation of TLR signaling induced the phosphorylation of PYK2 and the proximal BCR signaling components, SYK and Bruton's tyrosine kinase (BTK). Thereafter, we genetically engineered these three DLBCL cell lines to express MYD88 L265P or MYD88 WT, alone or in association with CD79B Y196F. In all three cell lines, the co-expression of MYD88 L265P and CD79B Y196F significantly increased magnitude and duration of SYK and BTK phosphorylation following BCR crosslinking. These findings highlight the likely role of MYD88L265P in CD79BY196F-associated proximal BCR signaling in DLBCL. To elucidate the potential role of the DOCK8 adapter in MYD88 L265P-augmented BCR signaling, we first assessed the colocalization of MYD88 WT or MYD88 L265P with DOCK8 in the same three genetically engineered DLBCL cell lines using proximity ligation assays (PLA), which detect protein-protein interactions at less than 40 nm in situ. In each of these cell lines, we detected significantly increased co-localized MYD88 L265P/DOCK8 signals in comparison to MYD88 WT/DOCK8 signals (p&lt;.0001, all). Additionally, there were significantly increased co-localized DOCK8/LYN signals in DLBCL cell lines that expressed MYD88 L265P rather than MYD88 WT (p&lt;.0001, all). These data provide the first direct evidence of an enhanced association between MYD88 L265P, DOCK8 and LYN in BCR-dependent DLBCLs and a basis for enhanced BCR signaling in primary tumors with concurrent MYD88L265P and CD79B genetic alterations. We next analyzed the consequences of MYD88 L265P-associated, DOCK8-dependent increased proximal BCR signaling by depleting DOCK8 in BCR-dependent DLBCL cells with endogenous MYD88L265P/CD79BY196F alterations (HBL1 and TMD8) or endogenous unmutated MYD88 WT/CD79B WT (OCI-Ly1 and SU-DHL4). ShRNA-mediated DOCK8 knockdown (KD) significantly decreased BCR-mediated phosphorylation of SYK and BTK in MYD88L265P/CD79BY196F DLBCL cell lines but not in lines with MYD88 WT/CD79B WT, highlighting the specific role of DOCK8 in MYD88 L265P-associated proximal BCR signaling. Of great interest, DOCK8 KD selectively decreased the proliferation of MYD88L265P/CD79BY196F, but not MYD88WT/CD79BWT, DLBCLs (p&lt;.004, HBL1 and p&lt;.009, TMD8; p = non sig., OCI-Ly1 and SU-DHL4). Additionally, DOCK8 KD significantly increased the efficacy of chemical PI3Kα/δ (copanlisib) and BTK (ibrutinib) inhibition in MYD88L265P/CD79BY196F DLBCLs (HBL1 and TMD8). Taken together, these data identify DOCK8 as an intermediary in MYD88L265P-driven proximal BCR signaling and a possible treatment target in LBCLs with co-occurring MYD88L265P/CD79BY196F mutations. Disclosures Shipp: AstraZeneca: Consultancy, Research Funding; Immunitas Therapeutics: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Research Funding; Bayer: Other: Institution: Research Grant/Funding; Abbvie: Other: Institution: Research Grant/Funding.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

scholarly journals Hypersialylation Protects Multiple Myeloma Cells from NK Cell-Mediated Immunosurveillance and This Can be Overcome By Targeted Desialylation Using a Sialyltransferase Inhibitor

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 138-138
Author(s):  
John Daly ◽  
Subhashis Sarkar ◽  
Alessandro Natoni ◽  
Robert Henderson ◽  
Dawn Swan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Sialic Acid ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Immune Evasion ◽  
Research Funding ◽  
Nk Cell ◽  
Advisory Committees

Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (&gt;85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p&lt;0.01 at 0.5:1, p&lt;0.001 at 1:1, p&lt;0.01 at 2.5:1). Furthermore, CD38 expression on H929 MM cells significantly increased after treatment with 300µM 3Fax-Neu5Ac for 3 days (p&lt;0.01, n=3). In a cytotoxicity assay, expanded PNK cell-mediated antibody dependent cellular cytotoxicity (ADCC) of H929 MM cells pre-treated with Daratumumab (anti-CD38 moAb) and 3Fax-Neu5Ac was significantly higher than H929 cells pre-treated with Dara (p&lt;0.05 at 0.5:1, p&lt;0.01 at 1:1) or 3Fax-Neu5Ac (p&lt;0.01 at 0.5:1, p&lt;0.01 at 1:1) alone (n=5). Using CRISPR/Cas9, over 50% complete KO of Siglec-7 was observed on expanded PNK cells, yet did not result in enhanced NK cell-mediated cytotoxicity against either H929 or JJN3 (n=7). Siglec-9 KO using CRISPR/Cas9 is ongoing. Discussion: Hypersialylation of MM cells facilitates immune evasion and targeted removal of sialic acid strongly enhances the cytotoxicity of NK cells against MM. However, to date the role of Siglecs remains inconclusive. Nevertheless, our data suggest that targeted desialylation is a novel therapeutic strategy worth exploring in MM. In particular, upregulation of CD38 provides a strong rationale for combinatory strategies employing targeted desialylation with CD38 moAbs such as Daratumumab, with the goal of maximizing ADCC. Disclosures Sarkar: Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy.

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