scholarly journals The Limitations of Collagen/CPP Hybrid Peptides as Carriers for Cancer Drugs to FaDu Cells

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 676 ◽  
Author(s):  
Kevin Ho ◽  
Cristobal Morfin ◽  
Katarzyna Slowinska
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Cellular Uptake ◽  
Hypopharyngeal Carcinoma ◽  
Uptake Mechanism ◽  
Microscopy Imaging ◽  
Fadu Cell ◽  
Hybrid Peptides ◽  
Fadu Cells

The in vitro efficacy of cancer prodrugs varies significantly between malignant cell lines. The most commonly identified problems relate to delivery: uptake mechanism, endosomal entrapment, and drug release. Here we present the study of collagen/cell penetrating hybrid (COL/CPP) peptide carriers intended to deliver paclitaxel to the hypopharyngeal carcinoma (FaDu) cells. Confocal microscopy imaging revealed the surprising response of FaDu cell to COL/CPP in comparison to previously studied cancer cell lines: hybrid peptides that carry both COL and CPP domain adsorb on the FaDu cell surface. While the CPP domain was design to facilitate the cellular uptake, in the case of FaDu cells, it also induced detrimental interactions with the cell membrane. Despite surface adsorption, the colocalization study with endosomal markers EEA1 and LAMP1 reveals that COL/CPP is internalized via endosomal pathway, peptides are able to escape before lysosome formation and release paclitaxel. Therefore, the main obstacle for paclitaxel delivery to FaDu cells appears to be related to cell surface properties. This behavior seems specific to FaDu cells, and could be linked to previously reported overexpression of T5, heparanase splice variants that produces protein lacking enzymatic activity of heparanase. This results in increased concentration of HSPG on FaDu cell surface, and possibly creates a barrier for cellular uptake of highly charged COL/CPP.

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

scholarly journals Ricin binding and protein synthesis inhibition in human hematopoietic cell lines

Blood ◽  
1988 ◽  
Vol 72 (4) ◽  
pp. 1357-1363 ◽  
Author(s):  
JE Leonard ◽  
CD Grothaus ◽  
R Taetle
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Close Correlation ◽  
Hematopoietic Cell ◽  
Protein Synthesis Inhibition ◽  
Human Blood Cells ◽  
Kd Values ◽  
Membrane Bound ◽  
Hematopoietic Cell Lines

Abstract Previous studies showed that human blood cells exhibited varying sensitivities to ricin. To investigate the basis for these differences, ricin binding to human hematopoietic cell lines was assessed and correlated with in vitro ricin sensitivities. Resistant mutants were also isolated and characterized. Ricin binding to CEM cells was rapid, time-dependent, and blocked by unlabeled ricin, but not albumin; ricin binding approached saturation at 3 mumol/L. Scatchard analyses showed multiple classes of binding sites, with maximum and minimum Kd values estimated at 1.5 x 10(-8) mol/L and 2.5 x 10(-7) mol/L. At 4 degrees C, membrane-bound ricin dissociated slowly from the cell surface in the presence of unlabeled ricin, but greater than 95% of the surface-bound ricin was removed with 0.1 mol/L lactose. At 37 degrees C, ricin dissociated from the cell surface with biphasic kinetics. Ricin uptake at 37 degrees C increased linearly for 15 to 30 minutes and plateaued at levels representing 12% to 29% of the amount of ricin bound at 4 degrees C, depending on the cell line. Ricin binding at 4 degrees C varied two- to fivefold among hematopoietic cell lines and was reduced approximately tenfold by incubation with lactose. When compared with parent CEM cells, ricin-resistant CEM variants showed a greater than 95% reduction in ricin binding and showed no detectable binding with lactose added. However, these cells were as sensitive as parent CEM cells to an anti-T-cell ricin immunoconjugate. For all cells examined, there was a close correlation (r = +.9) between ricin bound per cell and in vitro ricin sensitivity. Human hematopoietic cells show widely varying ricin binding, indicating major differences in the carbohydrate content or structure of surface glycoproteins and glycolipids. These variations are probably the major determinant of nonspecific toxicity of ricin immunoconjugates.

GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12 ◽  
Author(s):  
Nikhil Hebbar ◽  
Rebecca Epperly ◽  
Abishek Vaidya ◽  
Sujuan Huang ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Cells ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Pediatric Aml

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p<0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p<0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Vaidya:St. Jude: Patents & Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

2008 ◽  
Vol 295 (1) ◽  
pp. G16-G26 ◽  
Author(s):  
Mubeen Jafri ◽  
Bryan Donnelly ◽  
Steven Allen ◽  
Alex Bondoc ◽  
Monica McNeal ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Biliary Atresia ◽  
Cell Lines ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Newborn Mice ◽  
A Cell

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as α2β1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express α2β1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether α2β1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the α2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the α2β1-integrin. Newborn mice were pretreated with a monoclonal antibody against the α2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed α2β1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-α2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the α2β1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

scholarly journals HDAC Inhibition Increases HLA Class I Expression in Uveal Melanoma

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3690
Author(s):  
Zahra Souri ◽  
Aart G. Jochemsen ◽  
Mieke Versluis ◽  
Annemijn P.A. Wierenga ◽  
Fariba Nemati ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Uveal Melanoma ◽  
Hdac Inhibitor ◽  
Mrna Level ◽  
Hla Class I ◽  
Class I ◽  
In Vitro Tests ◽  
Hla Class I Antigens

The treatment of uveal melanoma (UM) metastases or adjuvant treatment may imply immunological approaches or chemotherapy. It is to date unknown how epigenetic modifiers affect the expression of immunologically relevant targets, such as the HLA Class I antigens, in UM. We investigated the expression of HDACs and the histone methyl transferase EZH2 in a set of 64 UMs, using an Illumina HT12V4 array, and determined whether a histone deacetylase (HDAC) inhibitor and EZH2 inhibitor modified the expression of HLA Class I on three UM cell lines. Several HDACs (HDAC1, HDAC3, HDAC4, and HDAC8) showed an increased expression in high-risk UM, and were correlated with an increased HLA expression. HDAC11 had the opposite expression pattern. While in vitro tests showed that Tazemetostat did not influence cell growth, Quisinostat decreased cell survival. In the three tested cell lines, Quisinostat increased HLA Class I expression at the protein and mRNA level, while Tazemetostat did not have an effect on the cell surface HLA Class I levels. Combination therapy mostly followed the Quisinostat results. Our findings indicate that epigenetic drugs (in this case an HDAC inhibitor) may influence the expression of immunologically relevant cell surface molecules in UM, demonstrating that these drugs potentially influence immunotherapy.

Reversion of physiological drug resistance of weakly basic drugs: The discovery of a new mechanism of PEG-PCL nanoparticles

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e13528-e13528
Author(s):  
R. Li ◽  
L. Xie ◽  
X. Li ◽  
Q. Liu ◽  
X. Qian ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Cellular Uptake ◽  
Antitumor Effect ◽  
In Vitro Cytotoxicity ◽  
Extracellular Ph ◽  
Fluorescent Particle ◽  
Tumor Ph

e13528 Background: A number of studies have reported the superior antitumor effect of nanoparticles loading chemotherapeutics than the free agents, yet the underlying mechanism has not attract enough attention. The extracellular pH of cancer cells is lower than that of the intracellular pH. Due to this pH gradient, weakly basic drug will protonated extracellularly and display decreased intracellular concentration. In this study, we aimed to reveal a new mechanism of PEG-PCL nanoparticles, namely the reversion of physiological drug resistance. Methods: Tetradrine (Tet), an alkaloid isolated from traditional Chinese medicine, was incorporated into the diblock copolymer methoxy poly(ethylene glycol)-polycaprolactone (mPEG-PCL). In vitro cytotoxicity of free Tet and Tet-loaded nanoparticles at pH7.4 and pH6.8 was compared on four different cancer cell lines. Fluorescent particle cellular uptake study was also used. To evaluate the antitumor effect of the nanoparticles in a more complex model rather than monolayer cell culture, we used Histoculture Drug Resistance Assay (HDRA). The in vivo antitumor effect of the nanoparticles was also studied in ICR mice bearing H22 tumor with different in vivo pH values. Results: In vitro cytotoxicity study in four tumor cell lines showed that the cytotoxicity of free Tet decreased significantly (P<0.05) when the extracellular pH decreased from 7.4 to 6.8, while the cytotoxicity of Tet-loaded nanoparticles increased or didn’t change significantly. The possible mechanism may mainly be the endocytosis of nanoparticles, which was proven by fluorescent particle cellular uptake study. HDRA indicated better tissue penetration of nanoparticles over free Tet. As to in vivo study, the mice with in vivo tumor pH 6.8 and treated with Tet-loaded nanoparticles exhibited best tumor inhibit rate and mildest side effect, suggesting that the use of nanoparticles was more preferable than the manipulation of tumor pH by the use of basic water. Conclusions: Our study clearly demonstrated that the mPEG-PCL nanoparticles could overcome the drug resistance caused by low extracellular pH and enhance drug penetration in the tumor tissue, thus increasing the antitumor efficacy of weakly basic agents. No significant financial relationships to disclose.

Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements

1988 ◽  
Vol 8 (12) ◽  
pp. 5358-5368
Author(s):  
C F Barth ◽  
E H Humphries
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Light Chain ◽  
Lymphoid Cells ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Chain Locus ◽  
Light Chain Locus

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.

Heterogeneity and modulation of tumor-associated antigens in human glioblastoma cell lines

1989 ◽  
Vol 71 (3) ◽  
pp. 388-397 ◽  
Author(s):  
Marco Colombatti ◽  
Bruno Dipasquale ◽  
Lorena Del-l'Arciprete ◽  
Massimo Gerosa ◽  
Giuseppe Tridente
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Cell Lines ◽  
Glioblastoma Cell ◽  
Heterogeneous Distribution ◽  
Human Glioblastoma ◽  
Human Glioblastoma Cell ◽  
Tumor Associated Antigens ◽  
Glioblastoma Cell Lines

✓ Seven human glioblastoma cell lines established in vitro from primary tumor explants were studied. A marked heterogeneity of glial fibrillary acidic protein was observed whereas vimentin was uniformly expressed by all cell lines. Indirect immunofluorescence and flow cytofluorometry revealed a heterogeneous distribution of surface GE 2 and CG 12 tumor-associated antigens (TAA's): three cell lines were positive (> 69% TAA-positive cells) and three cell lines were negative (< 9% TAA-positive cells). One cell line (Hu 228) was moderately positive at early culture passages and subsequently acquired a TAA-negative phenotype. The difference in the relative amounts of surface TAA's of the three positive cell lines was less than twofold. In spite of the heterogeneous distribution of surface TAA's, all cell lines exhibited considerable amounts of intracellular TAA. Treatment with phorbol esters and density-dependent growth arrest decreased the percentage of the TAA-positive cells and the amount of cell-surface TAA's in one cell line (Hu 195). Interferon-γ treatment in vitro increased the percentage of CG 12-positive cells by 12% and the amount of cell-surface CG 12 antigens by 38% as compared to untreated cells. The percentage of TAA-positive cells among phorbol ester-treated cells of the Hu 195 cell line was lowest 48 hours after treatment, but returned to normal values within the next 48 hours. Reduction of 3H-thymidine incorporation preceded the decrease in number of TAA-positive cells by about 18 hours. Two-color fluorescence analysis performed in positive cell lines for simultaneous determination of surface TAA's and deoxyribonucleic acid content or reactivity with the proliferation-associated Ki67 intracellular marker indicated that GE 2 and CG 12 antigens are expressed preferentially by actively proliferating glioma cells. The results of this study indicate the existence of two different phenotypes in cultured human glioblastoma cells: surface TAA-positive/cytosol TAA-positive and surface TAA-negative/cytosol TAA-positive cell populations. In addition, modulation of TAA expression was dependent on the cell-cycle differentiation stage, culture conditions, and proliferative state of the cells.

scholarly journals Pre-Clinical Combination of AO-176, a Highly Differentiated Clinical Stage CD47 Antibody, with Either Azacitidine or Venetoclax Significantly Enhances DAMP Induction and Phagocytosis of Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Mike J Donio ◽  
W. Casey Wilson ◽  
Isra Darwech ◽  
Gabriela Andrejeva ◽  
Benjamin J Capoccia ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Surface ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Innate Immune ◽  
Private Company ◽  
Acute Myeloid

While T cell checkpoint inhibitors are mainstays of cancer immunotherapy, therapies that direct innate immune responses against cancer are lacking. CD47, a "don't eat me" signal, is an innate immune cell checkpoint which binds SIRPα on macrophages and dendritic cells to limit phagocytosis and its upregulation on tumor cells leads to evasion of immune detection and clearance. Therapeutic antibodies have previously been developed to block CD47 and induce phagocytosis of tumor cells, thus validating the pathway. AO-176, a next generation humanized IgG2 anti-CD47 antibody, was developed to block the CD47/SIRPα interaction and induce tumor cell phagocytosis. Moreover, AO-176 directly kills tumor cells through a non-ADCC-dependent mechanism via induction of programmed cell death type III. In addition to these tumor eliminating properties, AO-176 has the potential for a strong safety profile as a result of its preferential binding to tumor versus normal cells, lack of RBC binding, and enhanced binding to tumor cells at acidic pH. CD47 has previously been shown to be upregulated on acute myeloid leukemia (AML) leukemic stem cells (LSC), enabling their expansion through evasion from phagocytic clearance. As a result, patients with increased CD47 on AML LSCs have worse overall survival. In this study, AO-176 efficacy was evaluated in AML cell lines as a single agent and in combination with azacitidine and venetoclax which are approved therapies for AML. Azacitidine is a cytosine analogue which acts to inhibit DNA methylation, and venetoclax is a potent Bcl-2 inhibitor. Previous studies have shown that azacitidine induces apoptosis of tumor cells and increases cell surface exposure of calreticulin, a DAMP (Damage Associate Molecular Pattern) which provides a strong pro-phagocytic signal. From these findings, it was hypothesized that azacitidine would enable increased tumor cell phagocytosis when combined with AO-176. The potential for a similar enhancement with a combination of AO-176 and venetoclax was also explored. The ability of AO-176, with or without azacitidine or venetoclax, to induce DAMPs on the surface of AML cells was assessed. Cell surface expression of DAMPs, calreticulin and PDIA3, were measured by flow cytometry. AO-176, azacitidine, and venetoclax as single agents potently increased both calreticulin and PDIA3 in a dose-dependent manner on AML cell lines such as HL60 This is the first time, to our knowledge, that venetoclax has been shown to induce DAMPs. To better understand the functional implications of these findings, in vitro phagocytosis assays were performed. Azacitidine and venetoclax significantly enhanced AO-176-mediated phagocytosis of AML cells compared to any of the agents alone. Moreover, when AO-176 was combined with azacitidine in direct tumor cell killing assays, enhanced activity was observed in a subset of AML cell lines. In conclusion, AO-176 combined with either azacitidine or venetoclax, resulted in significant enhancement of phagocytic AML cell clearance in vitro which also correlated with the ability of these agents to induce DAMPs. In vivo treatment with AO-176 in combination with these agents is in progress. AO-176 is being evaluated in phase 1 clinical trials for the treatment of patients with solid tumors (NCT03834948) and multiple myeloma (NCT04445701). Disclosures Donio: Arch Oncology: Current Employment, Current equity holder in private company. Wilson:Arch Oncology: Current Employment, Current equity holder in private company. Darwech:Arch Oncology: Current Employment, Current equity holder in private company. Andrejeva:Arch Oncology: Current Employment, Current equity holder in private company. Capoccia:Arch Oncology: Current Employment, Current equity holder in private company. Puro:Arch Oncology: Current Employment, Current equity holder in private company. Kashyap:Arch Oncology: Current Employment, Current equity holder in private company. Pereira:Arch Oncology: Current Employment, Current equity holder in private company.

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