Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 275-275
Author(s):  
Siobhan Glavey ◽  
Salomon Manier ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Myeloma Cells ◽  
Sialyl Lewis ◽  
And Migration ◽  
Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Plasma Levels of SDF-1 and Expression of SDF-1 Receptor on Multiply Myeloma Cells of Human Multiply Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4874-4874
Author(s):  
Caixia Li ◽  
De Pei Wu ◽  
Junjie Cao ◽  
Xiaojin Wu ◽  
Xiao Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Levels ◽  
Myeloma Cell ◽  
Migration Ability ◽  
Myeloma Cells ◽  
And Migration ◽  
Cxcr4 Axis ◽  
Healthy Persons

Abstract Multiple myeloma(MM) is a monoclonal expansion of malignant cells with a plasmablast-plasma cell morphology that is almost exclusively localized to the bone marrow, except at the final stages of disease, when they proliferate in the extramedullary area. The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 contribute to stem cell homing and play a role in trafficking of leukemic cells. In this study we have investigated expression and biological behavior of SDF-1/CXCR4 in MM-derived cell lines and primary MM cells. FACS and RT-PCR analysis was used to study the expression of CXCR4 and ICAM-1(CD54) on the surface of MM cells from 4 IL-6 dependant cell lines (XG1,XG2,XG6 and XG7) and 25 freshly isolated tumor samples from patients with diagnosed MM. Mononuclear cells were purified by positive selection of magnetical and FACS sorting. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the SDF-1, IL-6, and sICAM-1 levels. We found that[circ1]Fresh MM cells and MM cell lines expressed various levels of functional CXCR4 ranging from 23.1% to 77.7%,which was correlated with the in vitro migration ability of MM cells[(23.2±1.08)%, P<0.01]; [circ2]SDF-1 levels in the bone marrow(BM) of MM patients were significantly higher than the those of healthy persons (3489.23±651.63)pg/ml, (2818.57±597.79)pg/ml, P<0.05; but plasma levels of SDF-1 in peripheral blood of MM patients were lower than those of healthy persons[(1973±133)pg/ml, (2334.857±574.92), P=0.062]; [circ3]Plasma levels of PCL(4097.14±680.71) were significantly higher than those of healthy persons, P<0.01. The results firstly demonstrated abnormal expression of SDF-1 and its receptor CXCR4 on Human MM cells, which is closely correlated with the migration of MM cells. Furthermore, we discovered that SDF-1 could up-regulate the expression of ICAM-1 on MM cells; the plasma level of soluble ICAM-1 was correlated with the expression of CXCR4 on MM cells. These findings suggested that SDF-1/CXCR4 axis play a key role on the trafficking of MM cells via mediating the effect of adhesion molecules. Moreover, we observed higher plasma levels of IL-6 in PB of 60% MM patients compared with those of healthy individuals. Finally, the levels of IL-6 were closely correlated with SDF-1 levels (γ=0.8, P<0.01), These data indicated that in the IL-6-dependent myeloma cell lines or fresh myeloma samples and myeloma cell growth triggered by SDF-1 maybe due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. The results suggested that the expression of CXCR4 have an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.In conclusion, MM cells expressed various levels of functional CXCR4, which were correlated with the migration ability of MM cells in vitro; SDF-1/CXCR4 axis plays a key role in the trafficking of MM cells via mediating the effect of adhesion molecules; The plasma levels of IL-6 closely correlated with SDF-1 plasma levels, myeloma cell growth triggered by SDF-1 may be due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. All these suggested that the expression of CXCR4 play an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (&gt;20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

Improved Survival and Recovery of Hematopoiesis Following Lethal Radiation by Chloroquine-Mediated Activation of ATM

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2228-2228
Author(s):  
Yiting Lim ◽  
Mohammad Hedayati ◽  
Akil Merchant ◽  
Yonggang Zhang ◽  
Theodore DeWeese ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dose Rate ◽  
Cell Lines ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Chloroquine Treatment ◽  
Radiation Induced

Abstract Abstract 2228 Irreversible bone marrow damage and impaired blood formation is the primary cause of death following exposure to high doses of radiation. Moreover, the rate at which radiation is delivered may have a profound impact on cytotoxicity; prolonged exposure at a low dose-rate (LDR; 9.4 cGy/hr) has been found to induce greater cell death than the same total dose given at a high dose-rate (HDR; 4500 cGy/hr). Few non-toxic agents are presently available that can offer substantial protection against radiation induced bone marrow failure and death, especially during LDR exposure. We previously demonstrated that chloroquine, a commonly used agent in the treatment of malaria and rheumatologic diseases, can prevent LDR radiation induced cytotoxicity of cell lines in vitro and studied its effects on hematopoiesis in vivo. We initially quantified the effects of LDR radiation on C57/B6 mice and found that 9 Gy delivered at 9.4 cGy/hr for 95.7 hrs induced death in 13/19 (68%) of animals at 15–35 days after radiation. The administration of syngeneic bone marrow cells (1 × 106 cells) immediately after LDR radiation completely rescued animals (10/10) demonstrating that bone marrow failure was responsible for LDR radiation induced death similar to HDR radiation. Next we treated mice with chloroquine (0.0594 mg/17g body weight, i.p.) 24 hrs and 4 hrs prior to exposure to LDR radiation and found that it significantly improved survival (80%, p < 0.05) compared to untreated animals exposed to LDR radiation (32%). We examined hematopoietic recovery following LDR radiation and found that the peripheral WBC was significantly greater in mice treated receiving chloroquine (3.4 × 106/ml vs 1.1 × 106/ml at day 16, p<0.05). Similarly, we found that in vivo chloroquine treatment significantly increased the recovery of bone marrow myeloid CFC (p=0.02), suggesting that it impacted myeloid progenitors. To further validate this finding, we transplanted bone marrow from LDR irradiated mice into lethally irradiated CD45 congenic recipient mice, and found a significant improvement in early engraftment (4.2% vs. 0.4% engraftment at 6 weeks post-transplant, p=0.015). Chloroquine has been found to protect cancer cell lines from LDR radiation in vitro by activating ATM, an essential DNA damage sensor. We examined the effect of chloroquine on ATM and treated unradiated lin- bone marrow cells with chloroquine in vitro (35 ug/ml, 2 hr). Compared to control cells, chloroquine treated cells expressed 2.5-fold more phosphorylated ATM suggesting that the activation of ATM by chloroquine abrogated the lethal effects of LDR radiation in hematopoietic progenitors. We confirmed that ATM was required for chloroquine-mediated radioprotection by studying ATM null mice. In contrast to wild type mice, chloroquine treatment failed to protect ATM null mice from LDR radiation (9 Gy total) with 8/13 (62%) and 9/13 (69%) of animals surviving in treated or non treated mice, respectively (p=0.86). These data suggest that chloroquine exerts a radioprotective effect from LDR radiation by activating ATM in vivo, and may represent a novel means of limiting acute bone marrow failure in the event of widespread environmental LDR radiation exposure. Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.

Inhibition of Constitutively Activated JAK/STAT3 Pathway in Multiple Myeloma Cells Leads to Induction of Protective Autophagy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4914-4914
Author(s):  
Huihui Ma ◽  
Caisheng Lu ◽  
Judith Ziegler ◽  
Rentian Feng ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Specific Inhibitor ◽  
Janus Kinase ◽  
Signal Pathways ◽  
Myeloma Cells

Abstract Abstract 4914 Constitutive activation of Janus Kinase-2 (JAK-2)/signal transducer and activator of transcription-3 (STAT3) has been implicated to play a crucial role in the pathogenesis of Multiple Myeloma (MM). Therefore, targeted inhibition of STAT3 is an attractive therapy for MM patients. JSI-124 is a natural product isolated from various plant families and has been recently described as a specific inhibitor of JAK2/STAT3. It has been shown to exert strong apoptosis-inducing effects against STAT3+ tumor cell lines in vitro and in vivo. However, there is little information available about the anti-tumor effects of JSI-124 on myeloma cells. We therefore tested the role of JSI-124 as a potential novel anti-MM compound and were able to determine that JSI-124 has potent anti-myeloma properties against human MM cell lines. Our results revealed that JSI-124 could inhibit both constitutively expressed and IL-6 induced phosphorylation of Tyr705 STAT3 as well as other important cell signal pathways that regulate cell proliferation in MM cell lines. However, the anti-myeloma effects were not restricted to STAT3+ MM cells. In STAT3+ cells JSI-124 induced cell death, which was predominantly caspase dependent. In contrast, JSI-124 induced both caspase-dependent and caspase-independent cell death in STAT3-MM cells. Furthermore, JSI-124 induced DNA damage only in STAT3- MM cells. Surprisingly, STAT3- cell lines were more sensitive to JSI-124-induced growth inhibition and induction of cell death than STAT3+ MM cell lines. Further analysis revealed that JSI-124 led to induction of autophagy only in STAT3+ MM cells as determined by upregulation of LC3B. A dramatic increase of JSI-124-dependent apoptosis was observed when the autophagy pathway was blocked by Chloroquine suggesting that STAT3-inhibition in STAT3+ MM cells leads to protective autophagy, which can be overcome by Chlorquine treatment. Interestingly, inhibition of autophagy by Chloroquine or 3-mA in STAT3+ cell lines U266 or ARH 77 resulted in AIF release and well as caspase-independent cell death. The results in this study suggest that JSI-124 could be an effective anti-myeloma agent regardless of STAT3 activation status and that combination with Chloroquine may enhance its anti-MM effect. Disclosures Lentzsch: celgene: Consultancy, Honoraria, Research Funding; cephalon: Consultancy, Research Funding. Mapara:Resolvyx: Consultancy, Honoraria, Research Funding; Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees; GENTIUM: STOCK OWNERSHIP.

A Novel Activating Mutation Of CXCR4 Plays a Crucial Role In Waldenstrom Macroglobulinemia Biology

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 272-272 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Cristina Jimenez ◽  
Patricia Maiso ◽  
Michele Moschetta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Tumor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Advisory Board ◽  
Beta Catenin ◽  
Activating Mutation

Abstract Background The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating the biology of B-cell lymphoproliferative disorders. Recent whole genome sequencing studies have identified unique CXCR4 mutations in 29% of the 55 evaluated patients with Waldenstrom Macroglobulinemia (WM). In this study, we sought to better define the mutation status of CXCR4 in B-cell malignancies and define the functional role of this mutation in the progression of WM in vivo. Methods Allele-specific(AS) PCR has been performed on bone marrow (BM)-derived tumor cells of patients with WM (n: 131); IgM monoclonal gammopathy of undetermined significance (MGUS; n: 40); as well as in patients with diffuse large cell lymphomas (DLBCL; n: 75), splenic marginal zone lymphoma (SMZL; n: 14), B-chronic lymphocytic leukemia (B-CLL; n: 37), hairy cell leukemia (HCL; n: 35), multiple myeloma (MM; n: 36), IgA/IgG MGUS (n: 22), lymphoplasmacytic lymphoma without WM criteria (n: 13), and amyloidosis (n: 6). CXCR4-loss and -gain of function studies have been performed on WM cells stably expressing either shRNA-CXCR4, CXCR4-ORF-GFP-tagged or scramble-RFP-tagged (generated via lentivirus-based infection). A mutagenesis kit has been used to generate the C1013GCXCR4 mutant protein (C1013GCXCR4) in WM cells, via lentivirus-based infection. CXCR4-knock-in or C1013GCXCR4-mutated cells and the corresponding controls have been injected i.v. into SCID/Bg mice and tumor dissemination has been evaluated ex vivo by immunohistochemistry IHC (human-CD20; -CXCR4). C1013GCXCR4-mutated cells have been characterized at mRNA levels (U133 plus2) using GSEA. A novel human anti-CXCR4 mAb (BMS-936564/MDX-1338; Bristol Myers Squibb, NY) has been tested in vitro (cell proliferation, MTT, adhesion and migration to primary WM BM mesenchymal stromal cells) and in vivo (10mg/kg i.p. x3-4/week). Tumor growth has been evaluated by IHC ex vivo (hCD20; hCXCR4) and by immunofluorescence. Results We examined the mutational status of C1013GCXCR4 and confirmed the presence of this specific mutation in 28% of the 131 cases evaluated. The mutation was also detected at the stage of IgM-MGUS (20%); while it was present in a minority of patients with DLBCL (1%) and SMZL (7%). Remarkably, it was absent in all MM (n=36) and IgA/IgG MGUS patients (n=22), and it was not detected in healthy subjects (n=32). The functional relevance of the C1013G-CXCR4 variant was next examined in vivo. Mice injected with C1013GCXCR4-cells presented with a significant dissemination of tumor cells, demonstrating involvement of liver, bone marrow, lymph nodes, kidney and lung. IHC showed the presence of CXCR4+ and CD20+ cells in all the tissues examined; and quantification of CXCR4 and CD20 positivity was higher in C1013GCXCR4-cells-, compared to parental(p)-WM cell-injected mice (NIS Elements software, Nikon, Melville, NY; P<0.05). In addition, C1013GCXCR4-cells were further characterized in vitro, showing increased adhesion and cell proliferation in the presence of primary WM BM-MSCs. These findings were also confirmed using CXCR4-overexpressing cells. In contrast CXCR4-knock-down cells presented the opposite behavior, where reduced adhesion and proliferation in presence of primary WM BM-MSCs were observed. By performing GSEA we demonstrated that genes related to invasiveness, cell proliferation, anti-apoptosis, and oncogenesis were all enriched in C1013GCXCR4-cells compared to the parental-WM cells. These findings let us hypothesize that C1013GCXCR4 may act as an activating mutation in WM cells. Indeed, in a different mouse model, CXCR4 over-expressing cells and scramble infected cells were injected into mice, showing similar phenotype to the one observed upon C1013GCXCR4-WM cell-injected-mice. Finally, the novel antibody BMS-936564/MDX-1338 exerted anti-WM activity both in vitro and in vivo, with anti-tumor effects observed also against the mutated variant. This was supported by inhibition of pro-survival pathways (p-ERK; pAKT); induction of pro-apototic proteins (cleaved-PARP and -caspase-9); up-regulation of p-GSK3beta, p-beta catenin and relative beta catenin degradation. Conclusion These findings demonstrate that C1013GCXCR4 acts as an activating mutation in WM; and it is targetable by using MDX936564/1338 thus providing the basis for translating these observations into clinical trials for WM patients. Disclosures: Kuhne: BMS: Employment. Cardarelli:BMS: Employment. Ghobrial:BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other; Noxxon: Research Funding; Sanofi: Research Funding.

scholarly journals JAM-A as a Prognostic Factor and New Therapeutic Target in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 307-307 ◽  
Author(s):  
Antonio Solimando ◽  
Andreas Brandl ◽  
Mattenheimer Katharina ◽  
Carolin Graf ◽  
Miriram Ritz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Healthy Subjects ◽  
Plasma Cells ◽  
Cell Membrane Protein ◽  
And Migration ◽  
Rpmi 8226

Abstract Cell adhesion in the multiple myeloma (MM) microenvironment is a mechanism by which MM plasma cells escape the effects of therapy and survive. To improve clinical strategies and overcome drug resistance, approaches directed to both MMPCs and bone marrow microenvironment are under investigation. Here, we examined the cell membrane protein Junctional adhesion molecule-A (JAM-A) as a clinical biomarker and novel therapeutic target for MM. We evaluated JAM-A expression by real time PCR (RT-PCR), flow cytometry and immunofluorescence microscopy in 132 MM patients at different stages and various MM cell lines. Next, we measured the concentrations of soluble JAM-A from MM and healthy subjects sera by enzyme linked immune assay (ELISA). We investigated JAM-A functionally in vitro and in vivo by transient gene silencing (siRNA) and with blocking antibodies. Patient-derived plasma cells (MMPCs) expressed increased JAM-A expression levels when compared to control PC from healthy individuals. Elevated JAM-A expression correlated with poor prognosis (Figure 1A,B). Furthermore, soluble JAM-A was significantly increased in MM patient sera when compared to healthy subjects. Additionally, MM cell lines showed high expression of both membrane and cytoplasmic JAM-A. Consequently, inhibition of JAM-A using specific siRNA treatment resulted in diminished tumorigenic potential, including decreased colony formation, chemotaxis and migration. Importantly, treatment of luciferase+RPMI-8226 MM bearing NSG with a JAM-A blocking monoclonal antibody reduced significantly MM progression and dissemination in vivo when compared to MM bearing mice that received an non-specific isotype control antibody (Figure 1C). Conclusively, our data suggest that JAM-A can serve as a biomarker of malignancy in MM patients. Soluble plasma JAM-A could contribute to serum-based clinical stratification. Furthermore, therapeutic targeting of JAM-A appears attractive for clinical translation. Figure 1 Figure 1. Disclosures Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria.

scholarly journals Interactions with a "Humanized" Mesenchymal Bone Marrow Stromal Niche In Vivo Modify the Patterns of Essential Genes for Myeloma Cells: Therapeutic Implications

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-40
Author(s):  
Ryosuke Shirasaki ◽  
Sondra L. Downey-Kopyscinski ◽  
Ricardo De Matos Simoes ◽  
Olga Dashevsky ◽  
Sara Gandolfi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Broad Spectrum ◽  
Research Funding ◽  
Advisory Committees ◽  
Stromal Compartment ◽  
Oncology Research

Background: The biology and treatment response of human multiple myeloma (MM) cells in vivo is influenced by interactions with mesenchymal bone marrow stromal cells (BMSCs). For several key BMSC-derived cytokines (including IL-6) only the human, not murine, form optimally interacts with their respective receptor(s) on human MM cells. To better simulate the treatment responses of human MM cells in the BM, "humanized" BM-like niches in vivo have been engineered with biocompatible ceramic scaffolds "functionalized" via osteogenic differentiation of human mesenchymal BMSCs and implanted subcutaneously in immunocompromised mice. Aim: To determine if the patterns of genetic dependencies elucidated through in vitro CRISPR-based functional genomic studies are recapitulated when human MM cells are grown in mice within BM-like scaffolds with "humanized" mesenchymal stromal compartment. Methods: Cas9+ MM cell lines KMS11 and XG7 were transduced with a library of 1372 single-guide RNAs (sgRNAs) targeting 184 genes of interest (4 sgRNAs/gene), including 89 genes preferentially essential for MM cell lines compared to other neoplasms in vitro; broad-spectrum oncogenic targets (e.g. KRAS, MYC); tumor suppressor genes (e.g. PTEN); genes with limited in vitro essentiality in MM cells, but significantly higher expression vs. non-MM lines (e.g. ZFP91, ZBP1); and 155 olfactory receptor (OR) genes (typically not expressed or biologically inactive in tumor cells) as "DNA cutting" control sgRNAs. MM cells transduced with this focused CRISPR knockout (KO) library were injected into "humanized" scaffolds implanted subcutaneously (s.c.) in NSG mice. Tumors were collected when mice reached criteria for euthanasia and next-generation sequencing quantified the changes in sgRNA distribution at the end vs. start of experiment. Results: A large majority of genes defined by in vitro CRISPR KO screens as MM-preferential dependencies were also essential for MM cells in BM-like scaffolds. Among 57 MM-preferential dependencies in vitro which were plausible dependencies for KMS11 cells (e.g. CERES scores &lt;-0.4), 50 genes were essential for KMS11 cells in BM-like scaffolds in vivo (average log2fold change&lt;-1.0 and depletion of 3+ of 4 sgRNAs relative to the 99% confidence interval for control sgRNAs). These genes included key transcription factors/cofactors (e.g. IRF4, PRDM1, POU2AF1, RELB, MAF); epigenetic regulators (e.g. CARM1, MTA2, DOT1L); kinases upstream of NFkappaB (CHUK, IKBKB); ER regulators (e.g. SYVN1). Most "core-essential" or broad-spectrum oncogenic dependencies (e.g. MYC, CFLAR, CDK7 on both lines; KRAS in XG7) of this sgRNA library remained essential in vivo; while PTEN KO cells were enriched. Notably, several genes had more pronounced essentiality in vivo vs. invitro (e.g. BCL2, PIM2); or converted from non-essential in vitro to essential in vivo. For instance, among 95 genes of this library which are not likely dependencies in vitro (CERES scores &gt;-0.4) for KMS11 cells, 29 genes exhibited in vivo essentiality for both KMS11 and XG7 cells: several of these latter "in vivo dependencies" are recurrently essential for other MM lines in vitro (e.g. ZBTB38, UBE2J1, TCF3, DNAJB11), while also others have limited if any in vitro essentiality (e.g. ZBP1, PYGO2) across MM despite significantly higher transcript levels vs. other neoplasias. Notably, several genes with increased essentiality in the "humanized" BM scaffolds vs. in vitro also had stronger in vivo dependency in the BM scaffolds vs. when growth of the same MM cells as s.c. plasmacytomas (e.g. BCL2, PIM2, UBE2J1, SYVN1, ALG9, AMFR). Co-culture with BMSCs or IL-6 treatment induces several of these transcripts in MM cells suggesting that increased dependency of MM on these genes in the "humanized" BM model is due at least partly to its human cytokines. Conclusions: This study provides evidence for significant impact of the "humanized" BM-like niche on the patterns of genetic dependencies for human MM cells. Most genes preferentially essential for MM cells in vitro remain essential for their in vivo growth in the "humanized" BM model. Notably, several genes that do not meet criteria for dependency in vitro show such metrics in "humanized" BM scaffolds, but not sc plasmacytomas. These observations highlight important implications of the "humanized" BM-like in vivo model for current and future efforts to define and prioritize therapeutic targets for MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Janssen/Johnson & Johnson: Research Funding; Arch Oncology: Research Funding; TEVA: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; EMD Serono: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

ARRY-797, a Potent and Selective Inhibitor of p38 Map Kinase, Inhibits LPS-Induced IL-6 and In Vivo Growth of RPMI-8226 Human Multiple Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3478-3478
Author(s):  
Dale Wright ◽  
Shannon L. Winski ◽  
Deborah Anderson ◽  
Patrice Lee ◽  
Mark Munson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Small Molecule ◽  
Single Agent ◽  
Tnf Alpha ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells within the bone marrow. Their growth, survival, and migration are mediated in part via cytokines. Interleukin 6 (IL-6) is necessary for sustaining the in vitro growth of many MM cell lines and enhancing the proliferation of explanted human myeloma cells. The mitogen-activated protein kinase family member, p38, is activated by cytokines and growth factors and plays a significant role in inflammatory diseases. However, its role in the pathogenesis of multiple myeloma is poorly understood. Specific p38 inhibitors inhibit paracrine MM cell growth which is associated with IL-6 and VEGF secretion from bone marrow stromal cells (BMSCs). Furthermore, p38 inhibition blocks TNF-alpha-induced IL-6 secretion in BMSCs, thereby further inhibiting MM cell growth and survival. Although these data suggest an important role for p38 in MM, the direct effects of p38 inhibiton on MM has not been extensively explored. Therefore, we investigated the effects of p38 inhibition on in vitro and in vivo IL-6 production and MM cell growth in vivo after lipopolysaccaride (LPS) stimulation. LPS has been shown to induce various cytokines, including TNF-alpha and IL-6, via the p38 pathway. ARRY-797, an orally bioavailable, small molecule inhibitor of p38 directly inhibited LPS-induced IL-6 production from RPMI-8226 (IC50 = 100 pM) in vitro. In SCID-beige mice, LPS (3 μg/kg) induced IL-6 (7897 ± 827 pg/mL) and TNF-alpha (1922 ± 282 pg/mL) after 2 hours and these cytokines were inhibited by oral administration of ARRY-797 (30 mg/kg) by 91% and 95%, respectively. In MM xenograft models, ARRY-797 (30 mg/kg, BID, PO) inhibited RPMI 8226 tumor growth by 72% as a single agent and by 56% when LPS was administered to stimulate growth in vivo. In addition, ARRY-797 inhibited LPS-induced phosphorylation of p38 in RPMI-8226 xenografts. Together, these data support a role for p38 in IL-6-mediated growth of multiple myelomas. To our knowledge, ARRY-797 is the first small molecule p38 inhibitor to demonstrate single agent activity in a MM xenograft model and it has been advanced into preclinical development.

The Antibody-Drug Conjugate (ADC) IMGN779 Is Highly Active in Vitro and in Vivo Against Acute Myeloid Leukemia (AML) with FLT3-ITD Mutations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2321-2321 ◽  
Author(s):  
Kathleen R Whiteman ◽  
Paul Noordhuis ◽  
Russell Walker ◽  
Krystal Watkins ◽  
Yelena Kovtun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Research Funding ◽  
Xenograft Model ◽  
Hematopoietic Stem ◽  
Highly Active ◽  
Ic50 Values

Abstract IMGN779 is a CD33-targeted ADC utilizing DGN462, a novel DNA-alkylating agent consisting of an indolino-benzodiazepine dimer containing a mono-imine moiety. CD33 is expressed on the surface of about 90% of AML cases, with elevated levels of CD33 found in cases having molecular markers associated with poor prognosis, including mutations in FMS-like tyrosine kinase 3 (FLT3). The internal tandem duplication mutation (FLT3-ITD) is the most common FLT3 mutation, present in about 20-25% of AML cases. Patients with FLT3-ITD AML have a worse prognosis than those with wild-type (WT) FLT3, with an increased rate of relapse and a shorter duration of response to induction chemotherapy. IMGN779 was found to demonstrate targeted activity against AML cell lines in vitro, with IC50 values ranging from 2-3,000 pM. The MV4-11 cell line, which has a FLT3-ITD mutation, was the most sensitive to IMGN779 of the cell lines tested, with an IC50 of 2 pM. We evaluated the in vivo activity of IMGN779 against MV4-11 xenografts in SCID mice; IMGN779 was highly active (T/C = 1 %) at a single dose of 0.6 mg/kg (conjugate dose, 10 µg/kg DGN462 dose), resulting in complete tumor regressions (CR) in 3/6 animals and partial regressions (PR) in 6/6 animals. A DGN462-ADC to a non-relevant target was inactive (T/C = 95%) at the same dose, demonstrating that the activity of IMGN779 was due to its CD33 targeting. IMGN779 has previously been shown to be highly active against AML xenograft models without FLT3-ITD mutations, at minimally efficacious doses of 0.6 mg/kg (10 µg/kg DGN462), demonstrating that the presence of FLT3-ITD does not confer resistance to IMGN779 treatment. IMGN779 was also highly active in vitro against primary patient AML cells isolated from peripheral blood or bone marrow samples. Patient AML cells with FLT3-ITD were more sensitive to IMGN779 compared with FLT3 WT AML samples. IC50 values in FLT3-ITD samples ranged from 10 to 300 pM. CD33 expression was generally greater on FLT3-ITD leukemic blast cells than on FLT3 WT blasts, which likely contributed to their increased sensitivity to IMGN779. In long term cultures, IMGN779 showed a dose dependent decrease in leukemic stem cell (LSC) colony formation using an AML patient sample with both FLT3-ITD and NPM1 mutations, which are an even worse prognostic marker than FLT3-ITD alone. In contrast, colony formation increased in normal bone marrow, indicating that normal hematopoietic stem cells (HSCs) were spared. The differential expression of CD33 on LSC compared to HSCs makes CD33 an attractive target for treatment of AML, with the potential to eliminate LSCs and, thus, minimal residual disease in FLT3-ITD AML. The potent in vitro activity of IMGN779 against FLT3-ITD AML cell lines and primary patient FLT3-ITD AML progenitor cells and LSCs and its high level of CD33-targeted in vivo activity in a FLT3-ITD AML xenograft model support the advancement of IMGN779 as a potential treatment for AML, including FLT3-ITD AML. Disclosures Whiteman: ImmunoGen, Inc.: Employment. Noordhuis:ImmunoGen, Inc.: Research Funding. Walker:ImmunoGen, Inc.: Employment. Watkins:ImmunoGen, Inc.: Employment. Kovtun:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Johnson:ImmunoGen, Inc.: Employment. Schuurhuis:ImmunoGen, Inc.: Research Funding. Ossenkoppele:ImmunoGen, Inc.: Research Funding. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

scholarly journals Metabolic Profiling Reveals Cellular Reprogramming of Acute Myeloid Leukemia By Omipalisib through Serine Synthesis Pathway

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3296-3296
Author(s):  
Liang-In Lin ◽  
Tseng Chiyang ◽  
Yu-Hsuan Fu ◽  
Hsin-An Hou ◽  
Wen-Chien Chou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Enrichment Analysis ◽  
Advisory Board ◽  
Mtor Signaling ◽  
Cycle Arrest

Abstract Background: More than 50% of AML patients had hyperactivation of PI3K-AKT-mTOR signaling. Those patients are supposed to be associated with poor prognosis and chemotherapy resistance. The PI3K-AKT-mTOR signaling involves many cellular processes, including mRNA translation, cellular metabolism, and protein turnover. Omipalisib is a dual PI3K/mTOR inhibitor that exhibits anti-tumor activity in several cancers. However, the precise metabolic consequences in response to PI3K/mTOR dual inhibitor are still not fully studied in AML. Aims: To evaluate the efficacy and to elucidate the metabolic alteration of the anti-cancer effects of omipalisib on leukemic cells from both in vitro and in vivo aspects. Materials and Methods: HL60, THP1, and OCI-AML3 myeloid leukemia cell lines were used in this study. Omipalisib (GSK2126458) was used for in vitro and in vivo experiments. Cell proliferation was measured by Cell Titer 96 AQueous One Solution Cell Proliferation Assay. The protein expression and phosphorylation status were analyzed by immunoblotting. Flow cytometry was used for cell cycle and mitochondrial analysis. The metabolomics profiles were analyzed by Agilent 1290 UHPLC system coupled with 6540-QTOF. RNA-seq was performed using an Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) between control and omipalisib groups were identified by EBseq. A threshold of fold change ≥2 (or ≤0.5) and p ≤ 0.05 was used to select the DEGs. The mRNA quantification was measured by QuantStudio 3 Real-Time PCR Systems. The oxygen consumption rate (OCR) was analyzed by the XFe 24 extracellular flux analyzer. The CAnN.Cg-Foxn1 nu/CrlNarl mice were used for evaluating in vivo efficacy of omipalisib in murine model. Results: We demonstrated the anti-proliferation effect of omipalisib on AML cell lines with different genetic background. The IC 50 of OCI-AML3, THP1, and HL60 were 16.97nM, 9.35 nM, and 18.69 nM, respectively. Omipalisib could significantly induce G 0/G 1 cell cycle arrest in all three cell lines. As expected, omipalisib could significantly down-regulate the phosphorylation of AKT, mTOR, S6K and 4E-BP1. Metabolomics profiling analysis revealed that 24 of the 137 tested metabolites were significantly different between the control group and the omipalisib-treated groups in OCI-AML3 cells. Further metabolic pathway enrichment analysis demonstrated that metabolites related to amino acid metabolisms were significantly reduced following omipalisib treatment. In addition, we identified 300 DEGs between control and omipalisib-treated OCI-AML3 cells; of these, 251 were upregulated and 49 were downregulated. Further gene set enrichment analysis (GSEA) of hallmark gene sets indicated omipalisib treatment was significantly negatively associated with E2F targets, Myc targets, G2M checkpoint, mTORC1 signaling pathway, and oxidative phosphorylation. Joint-Pathway analysis (MetaboAnalyst 5.0) revealed that 'glycine, serine and threonine metabolism' was the most downregulated pathway in the omipalisib-treated group with p-value of 1.0076E-5 and impact value of 0.86567. qRT-PCR confirmed that several important genes, PHGDH, PSAT1, PSPH, SHMT1/2 and MTHFD1/2 in the serine and glycine synthesis pathway were significantly decrease in the OCI-AML3 cells following treated with omipalisib. OCR analysis indicated that the capacity of the mitochondria to produce energy was reduced after omipalisib treatment. Mitochondrial analysis showed that mitochondria mass and membrane potential decreased after omipalisib treatment, indicating the biosynthesis and functions of mitochondrial may be affected by omipalisib. In vivo studies showed that oral administration of 0.2 or 1 mg/kg omipalisib in mice could significantly retard tumor growth without obvious changes in body weight. Summary: We found that nanomolar levels of omipalisib could significantly inhibit cell growth and induce G 0/G 1 cell cycle arrest in myeloid leukemia cells. Joint-Pathway analysis of RNA-seq and metabolomics data revealed that omipalisib mainly altered serine and glycine metabolism. Further experiments indicated that serine synthesis pathway could be suppressed by omipalisib at least in part through disrupting PI3K-AKT-mTOR signaling. In vivo xenograft model, omipalisib could retard tumor growth at as low as 0.2 mg/kg. This information may be potentially suitable for future clinical application. Figure 1 Figure 1. Disclosures Chou: Kirin: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; IQVIA: Honoraria, Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding. Tien: AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria.

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