scholarly journals Evaluation of Cytotoxicity of Perfluorocarbons for Intraocular Use by Cytotoxicity Test In Vitro in Cell Lines and Human Donor Retina Ex Vivo

2019 ◽  
Vol 8 (5) ◽  
pp. 24 ◽  
Author(s):  
Mario R Romano ◽  
Mariantonia Ferrara ◽  
Claudio Gatto ◽  
Barbara Ferrari ◽  
Laura Giurgola ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ex Vivo ◽  
Cytotoxicity Test ◽  
Human Donor

scholarly journals A decellularized human corneal scaffold for anterior corneal surface reconstruction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Naresh Polisetti ◽  
Anke Schmid ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Stefan J. Lang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Biological Properties ◽  
Host Cells ◽  
Human Cornea ◽  
Human Donor ◽  
Short Period

AbstractAllogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

scholarly journals AR-42, a novel HDAC inhibitor, exhibits biologic activity against malignant mast cell lines via down-regulation of constitutively activated Kit

Blood ◽  
2010 ◽  
Vol 115 (21) ◽  
pp. 4217-4225 ◽  
Author(s):  
Tzu-Yin Lin ◽  
Joelle Fenger ◽  
Sridhar Murahari ◽  
Misty D. Bear ◽  
Samuel K. Kulp ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Ex Vivo ◽  
Histone Deacetylation ◽  
Down Regulation ◽  
Biologic Activity ◽  
Promising Therapeutic Approach

Histone hypoacetylation occurs in many cancers and inhibition of histone deacetylation is a promising approach to modulate these epigenetic changes. Our laboratory previously demonstrated that the histone deacetylase inhibitors (HDACis) vorinostat and AR-42 reduced the viability of a canine malignant mast cell line. The purpose of this study was to further investigate the mechanisms of pan-HDAC inhibition in normal and malignant mast cells. Mouse and canine malignant mast cell lines expressing various Kit mutations, normal canine mast cells, and primary canine malignant mast cells were treated with AR-42 (a novel HDACi) and effects on cell viability, cycling, and signaling were evaluated. Treatment with AR-42 induced growth inhibition, cell- cycle arrest, apoptosis, and activation of caspases-3/7. AR-42 promoted hyperacetylation of H3, H4, and alpha-tubulin, and up-regulation of p21. Down-regulation of Kit occurred after AR-42 treatment via inhibition of Kit transcription. Disassociation between Kit and heat shock protein 90 (HSP90) and up-regulation of HSP70 were observed after AR-42 treatment, suggesting potential loss of HSP90 chaperone function. Lastly, AR-42 down-regulated the expression of p-Akt, total Akt, phosphorylated STAT3/5 (pSTAT3/5), and total STAT3/5. In summary, AR-42 exhibits in vitro and ex vivo biologic activity against malignant mast cells, representing a promising therapeutic approach for malignant mast cell disease.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3773-3773
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Solid Cancer ◽  
Dependent Manner

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

scholarly journals Combined Targeting of Distinct c-Myc and JunB Transcriptional Programs Inducing Synergistic Anti-Myeloma Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2644-2644
Author(s):  
Judith Lind ◽  
Sonia Vallet ◽  
Karoline Kollmann ◽  
Osman Aksoy ◽  
Vincent Sunder-Plassmann ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Cell Lines ◽  
Ex Vivo ◽  
Mapk Signaling ◽  
Loss Of Function ◽  
Tumor Cell Death ◽  
Expression Levels ◽  
Protein Levels

Abstract INTRODUCTION Transcription factors (TFs) are convergence points of signaling cascades that coordinate cell differentiation, proliferation and survival and are commonly deregulated in cancer, including multiple myeloma (MM). They contribute to the initiation of MM and promote tumor cell growth and drug resistance. Both cMyc, a merging point of the PI3K-, and JunB, a merging point of the MEK/MAPK-signaling pathway, play pivotal roles in MM. Exciting novel approaches to inhibit TFs like proteolysis-targeting-chimera (PROTAC) promise to lead to selective tumor cell death with little/no consequence for normal cells. However, redundancy phenomena of transcriptional programs are likely to challenge their efficacy. Here, we report our final results on combined targeting of distinct c-Myc & JunB transcriptional programs for MM therapy. METHODS MM cell lines and patient MM cells were analyzed. Following CRISPR-loss-of-function screens for cMyc & JunB across MM cell lines and correlation analyses in MM patient datasets, the functional relevance of BRD4/c-Myc- and MEK/JunB-induced TF programs was delineated using genomic and chemical approaches in 2D and 3D models of the bone marrow (BM) microenvironment. Specifically, effects of single or combined targeting of cMyc- and JunB-induced TF-programs were analyzed by flow cytometry, western blot, RNAseq, qPCR and luciferase assays. In vitro and ex vivo results were finally verified in a MM xenograft mouse model. RESULTS While CRISPR loss-of-function screens across various MM cell lines confirmed their growth dependency on cMyc and JunB, we did not observe correlative expression levels among these TFs, neither in the publicly available GSE6477 nor in the CoMMpass dataset. In contrast, a significant positive correlation was observed between Brd4 and cMyc, and MEK and JunB expression levels, respectively. The existence of two distinct Brd4/cMyc and MEK/JunB transcriptional programs in MM cells was subsequently supported by a lack of changes in cMyc mRNA/protein levels and resultant transcriptional activity upon JunB knockdown, and vice versa. Likewise, MZ-1, a novel PROTAC which targets Brd4, resulted in the inhibition of BMSC/IL-6- induced cMyc- but not JunB- upregulation. Conversely, neither the MEK inhibitor trametinib nor doxycycline-induced knockdown of BMSC/IL-6- induced JunB upregulation in TetshJunB/MM.1S cells reduced Brd4/c-Myc mRNA/protein levels. Importantly, the activity of MZ-1 and trametinib was predicted by Brd4 and JunB expression levels using mathematical models, respectively. Further, combination of MZ-1 with trametinib or JunB knockdown synergistically inhibited tumor cell proliferation, and induced cell death in a 2D and a dynamic 3D model of the MM-BM milieu. Finally, our in vitro and ex vivo results were confirmed in vivo, utilizing BMSC:TetshJunB/MM.1S vs. BMSC:TetshSCR/MM.1S-carrying NSG mice treated with MZ-1 with/without doxycycline or trametinib. CONCLUSION In summary, our data demonstrate for the first time the existence of non-overlapping cMyc and JunB-regulated TF programs providing a rationale for combined cMyc:JunB targeting treatment strategies in MM. Disclosures Vallet: Pfizer: Honoraria; MSD: Honoraria; Roche Pharmaceuticals: Consultancy. Podar: Celgene: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; Amgen Inc.: Consultancy, Honoraria.

scholarly journals A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257770
Author(s):  
Kazuyo Watanabe ◽  
Mikio Yoshiyama ◽  
Gaku Akiduki ◽  
Kakeru Yokoi ◽  
Hiroko Hoshida ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Honey Bee ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Plasmid Vector ◽  
Simple Method

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

scholarly journals Effect of structure in ionized albumin – based nanoparticle: Characterisation, Emodin interaction, and in vitro cytotoxicity

2019 ◽  
Author(s):  
Macarena Siri ◽  
Maria Julieta Fernandez Ruocco ◽  
Estefanía Achilli ◽  
Malvina Pizzuto ◽  
Juan F. Delgado ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
In Vitro Cytotoxicity ◽  
Haemolytic Activity ◽  
Hydrodynamic Diameter ◽  
Kinetic Properties ◽  
Experimental Conditions

AbstractA γ–irradiated bovine albumin serum based nanoparticle was characterised structurally, and functionally. The nanoparticle was characterised by A.F.M, D.L.S, zeta potential, T.E.M., gel-electrophoresis, spectroscopy (UV-Vis, Fluorescence, FT-IR, and CD). Its stability was studied under adverse experimental conditions: pH values, chaotropic agents, and ionic strength and stability studies against time were mainly carried out by fluorescence spectroscopy following the changes in the tryptophan environment in the nanoparticle. Its function was studied by the interaction of the NP with the hydrophobic drug Emodin was studied. The binding and kinetic properties of the obtained complex were tested by biophysical methods as well as its toxicity in tumour cells.According to its biophysics, the nanoparticle is a spherical nanosized vehicle with a hydrodynamic diameter of 70 nm. Data obtained describe the nanoparticle alone as nontoxic for cancer cell lines. When combined with Emodin, the bioconjugate proved to be more active on MCF-7 and PC-3 cancer cell lines than the nanoparticle alone. No haemolytic activity was found when tested against ex vivo red blood cells. The stability of the albumin nanoparticle is based on a competition between short-range attraction forces and long-range repulsion forces. The nanoparticle showed similar behaviour as albumin against pH while improving its stability in urea and tween 80. It was stable up to 15 days and presented no protein degradation in solutions up to 2 M salt concentration. Significantly, the albumin aggregate preserves the main activity-function of albumin and improved characteristics as an excellent carrier of molecules.Graphical Abstract

scholarly journals Phospho-Proteomic Profiling of T-Cell Acute Lymphoblastic Leukemia Identifies Targetable Kinase Activities and Novel Treatment Combination Strategies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Valentina Cordo' ◽  
Rico Hagelaar ◽  
Sander Piersma ◽  
Richard Goeij-de Haas ◽  
Thang V Pham ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Src Family Kinases ◽  
Signaling Networks ◽  
Combination Strategies ◽  
Effective Combination ◽  
Cell Acute Lymphoblastic Leukemia

Introduction Intensive multi-agent treatment has boosted survival up to 80% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Nevertheless, relapsed patients have a poor prognosis due to acquired therapy resistance while most survivors have detrimental chemotherapy-induced side effects. Therefore, novel targeted therapies are urgently needed since further intensification of the current standard treatment regimen is not feasible for refractory/relapsed cases. Protein kinase inhibitors (PKIs) are amongst the most successful cancer treatments. Targetable kinases activated by gene fusions are rare in T-ALL and include subclonal NUP214-ABL1 fusion in 6% of cases or other rare clonal ABL1 fusions. Nevertheless, leukemic blasts rely on enhanced kinase signaling to sustain their dysregulated proliferation. Protein kinases can be hyper-activated even in the absence of defects in their genes. Thus, together with the identification of genomic aberrations, phospho-proteomics can provide information on pathway activation, signaling networks and aberrant kinase activities that offer important opportunities for targeted therapies. Aim Here, we aimed to identify and quantify kinase activation in T-ALL cell lines that may yield differential sensitivity to PKIs in vitro. This approach could pinpoint targetable leukemia vulnerabilities and provide effective (combination) treatment strategies. Methods Protein extracts from 11 T-ALL cell lines were enriched for phospho-peptides by titanium dioxide enrichment and anti-phospho tyrosine immunoprecipitation followed by liquid chromatography - tandem mass spectrometry (MS). Subsequently, the Integrative Inferred Kinase Activity (INKA) pipeline was used to rank activated kinases in our panel (Beekhof et al., 2019). Based on these data, selected kinase inhibitors were tested in vitro as single treatment or in combinations. Eventually, drugs of interest were further tested ex vivo in a cohort of T-ALL patient-derived xenografts (PDXs). Results MS-based phospho-proteome profiling of 11 T-ALL cell lines identified about 3700 tyrosine phospho-sites and more than 13300 serine/threonine phosphorylation sites. We found SRC-family kinases including LCK, SRC, FYN, and YES1 as most activated kinases in many T-ALL cell lines while ABL1, ZAP70, LYN, and FGR were detected only in specific lines. Additionally, other kinases including CDK1/2 and PAK1/2 were found to be activated in all the cell lines while activation of the INSR/IGF-1R axis was detected only in a subset of lines. We then tested cellular response to multiple clinically relevant PKIs based on predicted kinase activities. The in vitro drug screening showed an effective response and G1-arrest following treatment with the CDK1/2 inhibitor milciclib in all the cell lines tested, with IC50 values between 10nM and 1uM. Despite a general SRC-family kinases activation profile, dual SRC/ABL inhibitors like dasatinib reduced cellular viability only in cell lines with ABL1 fusions or LCK translocations (IC50 < 10nM) while other lines lacking ABL or LCK rearrangements were affected at much higher drug concentration (IC50 > 3uM, which is beyond the clinical achievable plasma concentration). Thus, PKIs were tested in combination with other relevant inhibitors based on additional kinase activities detected. Interestingly, the concomitant inhibition of the SRC-family kinases by dasatinib and the INSR/IGF-1R axis by BMS-754807 led to a drastic reduction of cell viability at nanomolar concentrations even in cell lines that did not respond to dasatinib, identifying a novel possible effective combination strategy for T-ALL. Eventually, we tested clinically relevant PKIs in 50 PDXs ex vivo and identified various T-ALL samples with a high sensitivity to dasatinib single treatment (IC50 < 100nM) as previously reported by others (Frismantas et al., 2017). Moreover, 70% of our PDXs efficiently responded to the broad spectrum kinase inhibitor midostaurin (IC50 < 1uM), highlighting the importance of targeting multiple signaling nodes simultaneously to tackle T-ALL vulnerabilities. Conclusions Ranking kinase activities and signaling networks from phospho-proteomic data can guide the use of PKIs as treatment option for T-ALL patients. Moreover, kinase activity profiling can provide insights for efficient treatment combination strategies to develop personalized medicine. Disclosures No relevant conflicts of interest to declare.

scholarly journals TBK1/Ikkε Inhibitor Amlx Blocks Multiple Myeloma Cell Growth in Vitro and In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4504-4504
Author(s):  
Quanhong Sun ◽  
Peng Zhang ◽  
Juraj Adamik ◽  
Konstantinos Lontos ◽  
Valentina Marchica ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Growth ◽  
Cell Lines ◽  
Bone Disease ◽  
Research Funding ◽  
Ex Vivo ◽  
Dominant Negative ◽  
Induction Of Apoptosis

Abstract Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients. Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support. These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis. Disclosures Giuliani: Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding. Roodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

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