A20 Cells Research Articles

Increasing intratumoral dendritic cells to potentiate anti-tumor effects of Newcastle disease virus-based immunotherapy

Abstract Oncolytic viruses (OV) represent a novel, mechanistically distinct therapy with significant anti-tumor potential and minimal toxicity. Crucial to OV efficacy is their induction of immunogenic cell death resulting in tumor associated antigen (TAA) uptake by dendritic cells (DCs) and cross-presentation to TAA-reactive T cells. Therefore, we hypothesized that OV efficacy would be increased by increasing intratumoral DCs. Because DCs are potently activated by type I IFNs and Newcastle disease virus (NDV) is a potent type I IFN inducer, we tested our hypothesis by treating A20 lymphoma-bearing mice with intratumoral Flt3L and NDV. In vitro, NDV significantly enhanced the susceptibility of A20 cells to CD8 T cell killing, in a TAA-specific manner. In vivo, the growth of A20 tumors was significantly inhibited by NDV; however NDV alone failed to eradicate some tumors. The addition of Flt3L potentiated the effect of NDV and led to sustained complete remissions in the majority of mice. Ex vivo characterization showed that Flt3L induced a potent accumulation of immature DCs in the tumor, spleen and lymph nodes, and NDV induced the activation of these DCs, as well as activation of NK and T cells. Further, Flt3L enhanced the ability of APCs to cross-prime TAA-specific T cells upon NDV-induced tumor cell death, as shown by increased proliferation and production of IFN-γ and TNF. The therapeutic effect of the Flt3L and NDV combination was completely lost in Batf3−/− mice (which lack cross-presenting DCs), demonstrating the importance of cross-priming in vivo. These findings support the use of NDV and Flt3L as a combined OV-immunotherapy with the potential to activate a powerful anti-tumor immune response and promote long-term immunological memory.

MiR155 sensitized B-lymphoma cells to anti-PD-L1 antibody via PD-1/PD-L1-mediated lymphoma cell interaction with CD8+T cells

BackgroundMicroRNAs (miRs) are involved in lymphoma progression by regulating tumor cell interaction with microenvironment. MiR155 is overexpressed in diffuse large B-cell lymphoma (DLBCL) and its biological effect on tumor microenvironment needs to be futher investigated.MethodsMiR155 was detected by quantitative real-time PCR in patients with newly diagnosed DLBCL. The mechanism of action of miR155 on lymphoma progression and tumor microenvironment was examined in vitro in B-lymphoma cell lines and in vivo in a murine xenograft model.ResultsSerum miR155 was significantly elevated, correlated with tumor miR155 expression, and indicated poor disease outcome in DLBCL. MiR155 overexpression was associated with decreased peripheral blood CD8+T cells and inhibition of T-cell receptor signaling. Of note, EBV-positive patients showed higher serum miR155 than EBV-negative patients. In co-culture systems of B-lymphoma cells with immune cells, miR155 induced Fas-mediated apoptosis of CD8+T cells, which could be targeted by anti-PD-1 and anti-PD-L1 antibodies. Moreover, miR155 enhanced lymphoma cell PD-L1 expression, recruited CD8+T cells by PD-1/PD-L1 interaction and inhibited CD8+T cell function via dephosphorylating AKT and ERK. MiR155-induced AKT/ERK inactivation was more obvious in CD8+T cells co-cultured with EBV-infected B-lymphoma cells. In vivo in a murine xenograft model established with subcutaneous injection of A20 cells, PD-L1 blockade particularly retarded miR155-overexpressing tumor growth, consistent with maintenance of CD8+T cells and their function.ConclusionsAs a oncogenic biomarker of B-cell lymphoma, serum miR155 was related to lymphoma progression through modulating PD-1/PD-L1-mediated interaction with CD8+T cells of tumor microenvironment, indicating the sensitivity of B-cell lymphoma to PD-L1 blockade. Also CD8+T cells could be a therapeutic mediator of immune checkpoint inhibitors in treating EBV-associated lymphoid malignancies.

MiR155 Sensitized B-Lymphoma Cells to PD-L1 Blockade Via PD-1/PD-L1-Mediated Lymphoma Cell Interaction with CD8+T Cells

Diffuse large B-cell lymphoma (DLBCL) represents the most common neoplastic disorder of B-lymphocytes. Besides genetic aberration of lymphoma cells themselves, dysfunction in immune cells can lead to lymphoma cell resistance to immunochemotherapy. Immune checkpoint inhibitors have emerged as successful treatment strategies for DLBCL. However, the underlying mechanisms that lymphoma cells escape from anti-tumor immune responses need to be further investigated.MiR155 was assessed by quantitative RT-PCR in a large cohort of patients with newly diagnosed DLBCL. Serum miR155 was significantly elevated, correlated with tumor miR155, and indicated poor prognosis. In peripheral blood, miR155 overexpression was associated with decreased CD8+T cells, reduced IFN-γ level and inhibition of T-cell receptor signaling. RNA sequencing was performed on blood samples of 82 DLBCL patients, which indicated that 73 genes closely related with lymphoma were differentially expressed between high miR155 group and low miR155 group. Above genes were plotted by heatmap. Dysregulation of multiple signaling pathway enriched by KEGG pathway analysis. Gene set enrichment analysis (GSEA) analysis manifested that miR155 is closely related with T cell receptor signaling pathway, cell cycle pathway and cytokine-cytokine receptor interaction pathway.In co-culture systems of B-lymphoma cells with immune cells, miR155 modulated Fas-mediated apoptosis of CD8+T cells, which was targeted by PD-1 and PD-L1 antibodies. As mechanism of action, miR155 enhanced lymphoma cell PD-L1 expression, recruited CD8+T cells via PD-1/PD-L1 interaction, and inhibited AKT/ERK phosphorylation of CD8+T cells. Moreover, EBV-positive patients showed higher serum miR155 than EBV-negative patients. In vivo in a murine xenograft model established with subcutaneous injection of A20 cells, PD-L1 blockade particularly retarded the growth of miR155-overexpressing tumors, consistent with AKT/ERK phosphorylation and CD8+T cell persistence.Taken together, as a serum oncogenic biomarker of DLBCL, miR155 indicated the sensitivity of B-lymphoma cells to PD-L1 antibody via PD-1/PD-L1-mediated interaction with CD8+T cells. Persistence of CD8+T cells could be an alternative target of action of PD-L1 antibody in treating B-lymphoid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Effect of Chlorambucil on the Apoptotic Signaling Pathway in Lymphoma Cells

To study whether chlorambucil has apoptotic effect on the B cell lymphoma A20 cells and its exact mechanisms in apoptotic signaling pathway. The experimental cells were treated with 20 μmol/L chlorambucil, the control cells were treated with PBS. Annexin V-FITC Cell Apoptosis Detection Kit was used to examine cell apoptosis. Western blot was used to detect the expressions of active caspase-3, Survivin, NF-κB and pAKT. Real-time fluorescent quantitative PCR was performed to examine the mRNA expression of Survivin. Compared with the control group, the proportion of FITC+/PI+ apoptotic cells and the expression of active caspase-3 (t=7.384, P=0.000) in the chlorambucil treatment group was significantly elevated. However, the expression of Survivin mRNA (t=4.384, P=0.000), protein expressions of survivin (t=12.360, P=0.000), NF-κB (t=5.462, P=0.000) and pAKT (t=7.183, P=0.000) in the chlorambucil-treated group all significantly decreased. The chlorambucil can induce the apoptosis of lymphoma cells, its mechanism may related with inhibition of PI3K/AKT signaling pathway, and expression of NF-κB and survivin.

Abstract 3356: The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines

Abstract BACKGROUND: Ewing sarcoma (ES) is the second most common pediatric bone cancer. ES cells are dependent on the activity of the EWS/FLI1 transcription factor for cell survival. EWS/FLI1 drives proliferation, dysregulates the cell cycle and establishes a block in differentiation. Despite the known dependence of Ewing sarcoma on EWS/FLI1, the exact identity of all the downstream targets of EWS/FLI1 is not known. While gene signatures have been reported for EWS/FLI1, these fail to capture the marked heterogeneity in the EWS/FLI1 transcriptome or correlate these expression changes with cellular phenotype. METHODS: Next generation RNA sequencing with deep coverage was used to generate a comprehensive analysis of the EWS/FLI1 transcriptome by silencing EWS/FLI1 in 5 different ES cell lines at two different time points. These results were organized into gene expression networks and signaling nodes were identified by weighted correlation network analysis (WGCNA). The networks were validated by a 500-gene signature of EWS/FLI1 targets using a next generation sequencing capture assay and the Illumina platform. The cellular endpoint of EWS/FLI1 silencing was evaluated by caspase 3,7 cleavage to measure apoptosis, BGAL staining to measure senescence and scratch assays to capture migration. RESULTS: Next generation RNA sequencing with siRNA silencing of EWS/FLI1 revealed marked heterogeneity in the EWS/FLI1 transcriptome among cell lines. Targets of EWS/FLI1 that were common across cell lines exhibited small magnitude gene expression changes (log fold <2), while large magnitude gene expression changes were unique to individual cell lines (log fold>2). When the log fold change in expression was restricted to greater than 2, only 13 common repressed targets and 4 common induced targets were identified in all 5 cell lines. In addition, silencing of EWS/FLI1 induced dramatically different phenotypes across cell lines, where p53 wild-type cell lines underwent senescence and the p53 mutant cell lines did not. Most cell lines did not continue to proliferate with EWS/FLI1 silencing with the exception being the widely used A673 cell line. Cellular migration did not change with EWS/FLI1 silencing in many of the cell lines in contrast to A673 cells. CONCLUSIONS: EWS/FLI1 drives different gene expression signatures and different phenotypes in unique cell lines. This diversity needs to be considered as EWS/FLI1 directed therapies are developed. Citation Format: Susan M. Kitchen-Goosen, Megan J. Bowman, Marie Adams, Penny Berger, Patrick J. Grohar. The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3356.

Abstract 1017: Vinorelbine, cyclophosphamide, and 5-FU effects on the circulating and intratumoral landscape of immune cells improve anti-PD-L1 efficacy in preclinical models of breast cancer and lymphoma

Abstract Checkpoint inhibitors (CIs) are active in many types of cancer. However, only a minority of patients achieve complete and/or long-lasting responses. We studied the effects of 4 different doses of 3 widely-used, orally-active chemotherapeutics (vinorelbine, V, cyclophosphamide, C, and 5FU) over local and metastatic tumor growth, and over the landscape of circulating and tumor-infiltrating immune cells. V, C and 5-FU were given alone or with anti-PD-1 or anti-PD-L1 CIs. Immunocompetent Balb/c mice were used to generate orthotopic models of breast cancer (BC, 4T1 cells) and B-cell lymphoma (NHL, A20 cells). V, C and 5-FU were administered at low-dose metronomic, medium, or maximum tolerable dosages. V and and C increased circulating monocytes, 5-FU reduced circulating monocytes. C increased MDSC count. C and 5-FU reduced circulating APCs, V increased circulating APCs. V and C reduced circulating Tregs. C at low doses and 5-FU increased circulating Tregs. C reduced circulating CD3+CD4+ and CD3+CD8+ T cells. V slightly reduced CD3+CD4+ T cells. V, C, and 5-FU reduced circulating B cells, with C showing the most significant effect. V reduced NK cells, C and 5-FU increased circulating NKs. In both BC and NHL models, anti-PD-L1 was significantly more effective than anti-PD-1. In BC models V, C and 5-FU were effective in reducing local and metastatic tumor growth. The association of V, C and anti-PD-L1 was the most effective combinatorial regimen in terms of local and metastatic BC control. Anti-PD-L1 treatment alone increased BC-infiltrating immune cells, in particular B cells. Treatment with C alone was associated with the largest tumor infiltration by NK, CD3+CD4+ T cells and m-MDSC, and with the lowest B cell infiltration. Combinatorial treatment with C+V+anti-PD-L1 was associated with the largest intratumoral B cell count and the lowest number of CD3+CD8+PD-1+ T cells. In NHL models, the association of C and anti-PD-L1 was the most effective regimen. Anti-PD-L1 alone was associated with the largest increase in infiltrating immune cells, and in particular in B cells. C alone was associated with increased NK infiltration. V decreased the immune cell infiltrate. The addition of anti-PD-L1 to V and C increased B cell infiltrate. Taken together, our data indicate that chemotherapeutics have very complex effects on the circulating landscape of immune cells, with subtle but significant differences related to the dosage and duration of the administration of the drugs. Notably, these effects on circulating immune cells differ qualitatively and quantitatively from those observed in the intratumoral immune cell infiltrate. The present data also suggest that chemotherapy might add to the effect of CIs, even though different types and sites of cancer generate significantly different atlases of intratumoral infiltrates. Citation Format: Stefania Orecchioni, Giovanna Talarico, Valentina Labanca, Patrizia Mancuso, Francesco Bertolini. Vinorelbine, cyclophosphamide, and 5-FU effects on the circulating and intratumoral landscape of immune cells improve anti-PD-L1 efficacy in preclinical models of breast cancer and lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1017.

Immunomodulation with SA-FasL-Engineered Microgels Achieves Long-Term Survival of Allogeneic Islet Grafts

Introduction and Objective Allogenic islet transplantation has shown efficacy in the clinic for the treatment of type 1 diabetes. However, sustained survival of allogeneic islet grafts requires chronic immunosuppression that has significant adverse effects. T effector (Teff) cells recognizing and responding to allograft antigens are the major culprit of graft rejection. Upon activation, Teff cells upregulate Fas death receptor on their surface and become sensitive to FasL-mediated apoptosis. Fas pathway, therefore, presents an important target for immunomodulation to block alloreactive responses with significant therapeutic potential. Herein, we assessed the efficacy of PEG microgels engineered with a novel form of FasL chimeric with a modified form of core streptavidin, SA-FasL, in achieving sustained survival of allogeneic islet grafts in the absence of chronic immunosuppression. Materials and Methods Microgels presenting biotin on their surface were produced by reacting biotin-PEG-thiol with a maleimide-terminated 4-arm poly(ethylene) glycol macromer, and generating 200 μm diameter microgels crosslinked with dithiothreitol. Biotinylated microgels were engineered with SA-FasL (1 μg/103 microgels) taking the advantage of the high affinity interaction between biotin and streptavidin. The apoptotic activity of SA-FasL-engineered microgels was tested on mouse A20 B cell lymphocytes in vitro. The immunomodulatory function of microgels for the prevention of graft rejection was tested by mixing ~500 BALB/c naïve islets with 1000 SA-FasL-engineered microgels and transplanting under the kidney capsule of streptozotocin-induced diabetic C57BL/6 recipients. A group of recipients were also treated transiently with rapamycin (0.2 mg/kg daily for 15 days starting the day of transplantation). Results and Discussion Biotin-PEG microgels were efficiently coupled with SA-FasL, and showed a dose-dependent apoptotic activity in A20 cells. Co-transplantation of SA-FasL-engineered microgels with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified microgels) with 20% surviving for a 200-day observation period. Transient use of low dose rapamycin, enhanced survival to > 90% of the grafts. In marked contrast, only 20% of islets co-transplanted with PEG microgels, and short-course rapamycin, survived long-term. Importantly, CD4+CD25+FoxP3+ Treg cells were required for graft survival as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. Conclusion These results provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential. Funded in part by NIH (grants R21EB020107, R21AI113348, 185 R56AI121281, and F30AR069472) and the Juvenile Diabetes Research Foundation (2-SRA-186 2014-287-Q-R).

Abstract 5557: EnanDIM, a family of potent enantiomeric TLR9 agonists, modulate the tumor microenvironment and show single-agent antitumor effects in various syngeneic murine models

Abstract Introduction: TLR9 agonists, being recognized via non-methylated CG-motifs, target the Toll-like receptor 9 (TLR9). This way, they activate the innate and the adaptive immune system. Two different families of TLR9 agonists are currently used in preclinical and clinical studies: Covalently-closed dumbbell-shaped DNA molecules with natural phosphodiester backbone which are protected from degradation by their conformation (dSLIM®) and single-stranded, oligodeoxynucleotides (CpG-ODN) commonly stabilized by phosphorothioates (PTO). However, chemical PTO-modifications have resulted in off-target effects and an unfavorable risk-to-benefit ratio in clinical trials. EnanDIM® are a new family of TLR9 agonists. EnanDIM® molecules consist of linear single-stranded ODN. Protection against exonucleases is achieved through L-deoxyribose nucleotides instead of the naturally occurring D-deoxyribose nucleotides at their 3'-ends. EnanDIM® lead to pronounced induction of cyto- and chemokines, like IFN-alpha and IP-10, as well as broad activation of immune cells, like monocytes, NK cells and dendritic cells, within PBMC. Therefore, EnanDIM® were evaluated in various syngeneic murine tumor models for their anti-tumor effects. Methods: Mice were subcutaneously inoculated with either CT26 (colon carcinoma), MC38 (colon carcinoma), EMT-6 (breast cancer), B16F10 (melanoma) or A20 (lymphoma) cells for solid tumor growth. EnanDIM® (250 µg per dose, intratumoral) was used to treat established tumors. In addition, the capability of EnanDIM® to modulate the tumor micro-environment (TME) was analyzed via immunohistochemistry in a syngeneic tumor model. Results: Mice in all models showed clear reduction in tumor growth (TGI: 53 to 85%) and, therefore, an increased survival (p ≤0.001, logrank). Notably, in the EMT-6 tumor model 8/10 mice showed complete tumor regression after EnanDIM® injection. Those 8 mice rejected EMT-6 tumor cells in a subsequent re-challenge study, indicating a sustained immune memory against the tumor, which was in contrast to age-matched naïve mice. When analyzing the TME of mice from the CT26 model an increased immune cell infiltration into the tumor by CD3+, especially CD8+ T cells was detected, showing the ability of EnanDIM® to beneficially modulate the TME. Conclusions: The enantiomeric TLR9 agonists EnanDIM® show promising anti-tumor effects in a variety of syngeneic murine tumor models and a beneficial modulation of the TME towards an increase of anti-tumor effector cells. These data provide the basis for a further development of EnanDIM® in cancer. Citation Format: Barbara Volz, Kerstin Kapp, Detlef Oswald, Burghardt Wittig, Manuel Schmidt. EnanDIM, a family of potent enantiomeric TLR9 agonists, modulate the tumor microenvironment and show single-agent antitumor effects in various syngeneic murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5557.

Abstract 1043: Patient-derived and cell line xenograft growth in the B6;129-Rag2 tm1Fwa IL2rg tm1Rsky/DwlHsd (R2G2) mouse model

Abstract Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumor cell line xeno- (CDX) and allografts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data were also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models; however, the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, athymic nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in athymic nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in athymic nude mice. In study A, no tumor growth was seen in athymic nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models. Citation Format: Jamie L. Naden. Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1043.

A20 and ABIN-1 synergistically preserve intestinal epithelial cell survival.

A20 (TNFAIP3) and ABIN-1 (TNIP1) are candidate susceptibility genes for inflammatory bowel disease and other autoimmune or inflammatory diseases, but it is unclear how these proteins interact in vivo to prevent disease. Here we show that intestinal epithelial cell (IEC)-specific deletion of either A20 or ABIN-1 alone leads to negligible IEC loss, whereas simultaneous deletion of both A20 and ABIN-1 leads to rapid IEC death and mouse lethality. Deletion of both A20 and ABIN-1 from enteroids causes spontaneous cell death in the absence of microbes or hematopoietic cells. Studies with enteroids reveal that A20 and ABIN-1 synergistically restrict death by inhibiting TNF-induced caspase 8 activation and RIPK1 kinase activity. Inhibition of RIPK1 kinase activity alone, or caspase inhibition combined with RIPK3 deletion, abrogates IEC death by blocking both apoptosis and necroptosis in A20 and ABIN-1 double-deficient cells. These data show that the disease susceptibility proteins A20 and ABIN-1 synergistically prevent intestinal inflammation by restricting IEC death and preserving tissue integrity.

Ruthenium Arene Complexes with α‐Aminoacidato Ligands: New Insights into Transfer Hydrogenation Reactions and Cytotoxic Behaviour

The RuII arene complexes [Ru(η6‐p‐cymene)(κ2N,O‐aa)Cl] (aa = l‐glutaminato, 1; l‐homoserinato, 2; l‐tyrosinato, 3; l‐serinato, 4; l‐prolinato, 5; trans‐4‐hydroxyl‐l‐prolinato, 6; l‐threoninato, 7; glycinato, 8) were synthesized in high yields by the reactions of [Ru(η6‐p‐cymene)Cl2]2 with aa/MOH (M = Na, K). The salt [Ru(η6‐p‐cymene)(κ2N,O‐l‐serinato)(PPh3)][CF3SO3], 9[CF3SO3], was prepared in 68 % yield from 4 and AgSO3CF3/PPh3. All the products were fully characterized by analytical and spectroscopic methods, moreover the X‐ray structures of 1 and 2 were elucidated by X‐ray diffraction. Compounds 1–7 and 9[CF3SO3] were studied as catalytic precursors for the transfer hydrogenation reaction (THR) of acetophenone, using 2‐propanol or sodium formate (in water) as hydrogen sources, under variable experimental conditions. The catalytic process was investigated also under microwave irradiation. In general, all the catalytic reactions selectively afforded 1‐phenylethanol, and the best results were achieved with 5 and 6, in terms of both activity and enantioselectivity (enantiomeric excess up to 78 %). The in vitro cytotoxicity of 1, 4, 5 and 9[CF3SO3] was assessed towards the human pancreatic cancer cell line BxPC3 and the mouse embryo fibroblast Balb/3T3 Clone A31 cell line, all of the investigated compounds showing to be substantially inactive. The catalytic and biological results will be discussed in relation to the relevant chemical behaviour of the compounds in water solution.

PO-215 Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

Introduction Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumour cell line xeno- (CDX) and allo-grafts in the R2G2 immunodeficient mouse model. Material and methods The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumour growth data was also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Results and discussions Colorectal PDX tissue was comparable between the R2G2 and the NSG mouse models, however the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in into 4 sections of R2G2 mice each, and 100% of mice developed either one or two tumours. The human esophageal adenocarcinoma OE33 cells were implanted into both flanks of R2G2, Athymic Nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in Athymic Nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in Athymic Nude mice. In study A, no tumour growth was seen in Athymic Nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in R2G2 mice and take rate was 90%. Growth of two allogeneic tumour lines was also examined. The mouse colon carcinoma CT26 cells were implanted in R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. Conclusion These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumour models research, with high take rates and quick growth of allogeneic models.

In situ vaccination improves efficacy of PD-1 blockade in unresponsive lymphoma tumors through induction of a highly efficient cross-presenting dendritic cell subset expressing TLR3

Abstract B cell lymphomas are generally incurable, with low response rates to anti-PD1 therapy. Here, we tested the combination of in situ vaccination with PD-1 blockade in a pre-clinical mouse model. A20 lymphoma-bearing mice were treated with a PD1 blocking antibody with or without an in situ vaccine combining intratumoral (it) injections of FMS-like tyrosine kinase-3 ligand (Flt3L), poly-ICLC (pIC) and local irradiation (XRT) of the tumor. Untreated lymphoma tumors contained few dendritic cells (DC) and anti-PD1 treatment did not induce tumor regression. Flt3L treatment resulted in a dramatic increase of IRF8+TLR3+ DC in the tumor. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on TLR3+ DC. Local XRT also enhanced uptake of dying tumor cells by it DC. Importantly, tumor antigens were taken up mainly by CD103+ DC and not CD103-DC. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103-subsets. Combination of Flt3L with XRT and pIC induced tumor-reactive T cells, and delayed tumor growth and improved survival in 40% of mice. In situ vaccination also increased expression of PD-L1 on tumor cells and it DC. Consistently, combination of in situ vaccination with anti-PD1 led to complete tumor regression and increased long-term survival in the majority of mice. PD1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect. In conclusion, in situ vaccination combining Flt3L, local XRT and pIC improves efficacy of anti-PD1 in checkpoint-unresponsive lymphoma tumors through induction of a highly efficient cross-presenting DC subset.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Influences Sensitivity to Anti-IGF System Agents Through the Translational Regulation of IGF1R.

Insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IGF2BP3) is an oncofetal protein that binds RNA, thereby influencing the fate of target transcripts. IGF2BP3 is synthesized de novo in cancer, where it promotes proliferation, drug resistance, and metastasis via both IGF2-dependent and IGF2-independent mechanisms. Ewing sarcoma (ES) is a rare bone and soft tissue tumor in which the IGF system plays a pivotal role. This study aimed to investigate the effect of IGF2BP3 on the regulation of the IGF system in ES. Among the components of the IGF axis, a direct significant correlation was identified between IGF2BP3 and IGF1R at mRNA and protein levels in two independent series of clinical specimens from patients with localized ES. After the formal demonstration of a direct association between IGF2BP3 and IGF1R mRNA using ribo-immunoprecipitation assay, we performed in vitro studies using A673 and TC-71 ES cell lines to demonstrate that IGF2BP3 loss promotes the downregulation of IGF1R and a decreased biological response to IGF1, represented by reduced migration and cell growth. Additionally, the compensatory activation of insulin receptor (IR) and its mitogenic ligand IGF2 is triggered in some but not all cell lines in response to IGF2BP3-mediated IGF1R loss. These findings have therapeutic implications because cells with a decreased expression of IGF2BP3/IGF1R axis but an increased expression of the IR/IGF2 loop display higher sensitivity to the dual inhibitor OSI-906 than do control cells. Therefore, studies on IGF2BP3, which was confirmed as a post-transcriptional regulator of IGF1R, provide a step forward in the identification of new mechanisms regulating the IGF system. In addition, our results demonstrate that the detection of IGF2BP3 expression should be combined with the assessment of the IGF1R/IR ratio to predict cell responses to anti-IGF1R/IR agents.

Patient‐derived and cell line xenograft growth in the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL‐2, IL‐4, IL‐7, IL‐9 and IL‐15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient‐derived (PDX) and tumor cell line xeno‐ (CDX) and allo‐grafts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data was also collected from two allografts of murine colorectal cancer (CT26) and B‐cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models, however the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, Athymic Nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in Athymic Nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in Athymic Nude mice. In study A, no tumor growth was seen in Athymic Nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B‐cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gold nanoparticles stabilize peptide-drug-conjugates for sustained targeted drug delivery to cancer cells

BackgroundPeptide-drug-conjugates (PDCs) are being developed as an effective strategy to specifically deliver cytotoxic drugs to cancer cells. However one of the challenges to their successful application is the relatively low stability of peptides in the blood, liver and kidneys. Since AuNPs seem to have a longer plasma half-life than PDCs, one approach to overcoming this problem would be to conjugate the PDCs to gold nanoparticles (AuNPs), as these have demonstrated favorable physico-chemical and safety properties for drug delivery systems. We set out to test whether PEG coated-AuNPs could provide a suitable platform for the non-covalent loading of pre-formed PDCs and whether this modification would affect the bioavailability of the PDCs and their cytotoxicity toward target cancer cells.MethodsPeptides specifically internalized by A20 murine lymphoma cells were isolated from a phage library displaying 7mer linear peptides. Peptide specificity was validated by flow cytometry and confocal microscopy. PDCs were synthesized containing a selected peptide (P4) and either chlorambucil (Chlor), melphalan (Melph) or bendamustine (Bend). Gold nanoparticles were sequentially coated with citrate, PEG-6000 and then PDC (PDC-PEG-AuNP). The physico-chemical properties of the coated particles were analyzed by electrophoresis, TEM, UV–VIS and FTIR. Stability of free and PDC-coated AuNP was determined.ResultsBiopanning of the phage library resulted in discovery of several novel peptides that internalized into A20 cells. One of these (P4) was used to synthesize PDCs containing either Chlor, Melph or Bend. All three PDCs specifically killed A20 target cells, however they had short half-lives ranging from 10.6 to 15.4 min. When coated to PEG-AuNPs, the half-lives were extended to 21.0–22.3 h. The PDC-PEG-AuNPs retained cytotoxicity towards the target cells. Moreover, whereas pre-incubation for 24 h of free PDCs almost completely abolished their cytotoxic activity, the PDC-PEG-AuNPs were still active even after 72 h pre-incubation.ConclusionsPeptide-drug-conjugates hold potential for improving the target efficacy of chemotherapeutic drugs, however their short half-lives may limit their application. This hurdle can be overcome by easily conjugating them to gold nanoparticles. This conjugation also opens up the possibility of developing slow release formulations of targeted drug delivery systems containing PDCs.

Effect of dual PI3K-δ,γ inhibitor duvelisib on immunosuppressive Tregs and myeloid cells to enhance efficacy of checkpoint and co-stimulatory antibodies in a B cell lymphoma model.

33 Background: Duvelisib is a dual inhibitor of PI3K-δ and PI3K-γ which has shown clinical activity as monotherapy in CLL/SLL, FL and T cell lymphoma (O’Brien; Flinn; Horwitz, ASH 2014). Recent publications have demonstrated that PI3K-δ inhibition reduces immunosuppressive Tregs (Ali, Nature, 2014) whereas PI3K-γ inhibition reduces immunosuppressive myeloid cells (Kaneda, Nature, 2016; De Henau, Nature, 2016). Hence, we postulated that duvelisib may augment the efficacy of immune checkpoint or co-stimulatory antibodies. Methods: Mice bearing syngeneic A20 B cell lymphoma tumors (60-90 mm3) were treated with vehicle, duvelisib, anti-PD1, anti-PD1 + duvelisib, anti-OX40, or anti-OX40 + duvelisib. Tumor volumes were measured by caliper. Tregs, macrophages and MDSCs were quantified by flow cytometry from mice bearing A20 tumors after 8 days of treatment. Results: In the A20 model, duvelisib, anti-PD-1 and anti-OX40 treatments each induced tumor growth delay. When duvelisib and anti-PD-1 were combined in mice with pre-existing A20 tumors, strong anti-tumor synergy was observed. When anti-OX40 and duvelisib were combined, tumor regression was observed which correlated with strong reduction of tumor Tregs, M2 macrophages and MDSCs. To assess immune memory, tumor-free mice following anti-OX40 alone or anti-OX40 + duvelisib were injected with A20 cells in the contralateral flank with no further treatment. Whereas mice that had received anti-OX40 alone grew new tumors upon A20 re-challenge, all tumor-free mice that had received anti-OX40 + duvelisib did not grow tumors upon re-challenge and showed elevated memory T cells in blood and spleen. These findings indicate that anti-OX40 + duvelisib treatment established immune memory. Conclusions: The dual inhibition of PI3K-δ and PI3K-γ appears to make duvelisib especially effective in reducing both lymphoid and myeloid immuno-suppressive populations, enhancing the anti-tumor efficacy of immune checkpoint and co-stimulatory antibodies. These data support potential clinical exploration of duvelisib in combination with checkpoint or co-stimulatory antibodies.

Improving efficacy of PD-1 blockade in unresponsive lymphoma tumors with in situ vaccination through induction of a highly efficient cross-presenting dendritic cell subset.

76 Background: Low-grade non-Hodgkin’s B-cell lymphomas are generally incurable; preliminary results with anti-PD-1 therapy have yielded low response rates. Tumoral DC infiltration correlates with efficacy of checkpoint blockade and tumor-targeted vaccines represent promising, novel treatment strategies to induce anti-tumor T cells. Methods: A20 lymphoma-bearing mice were treated with a PD-1 blocking antibody with an in situ vaccine (ISV) consisting of intratumoral injections of FMS-like tyrosine kinase-3 ligand (Flt3L), local irradiation (XRT) of the tumor and intratumoral injections of the TLR3 agonist poly-ICLC (pIC). Results: Untreated lymphoma tumors contained very low numbers of DC and treatment with anti-PD1 alone did not induce tumor regression or increase survival. Flt3L treatment resulted in a dramatic increase of IRF8+TLR3+ DC at the tumor site, the draining lymph node and the spleen. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on infiltrating TLR3+ DC. Local XRT also enhanced uptake of dying tumor cells by DC. Interestingly, tumor antigens were taken up mainly by CD103+ DC and not CD103- subtypes. CD103+ expression distinguishes a subset of migratory cross-presenting DC. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103- subsets. The combination of Flt3L with XRT and pIC induced tumor-reactive, IFNg-producing T cells, but delayed tumor growth and improved survival only in 40% of mice. ISV also increased expression of PD-L1 on tumor cells and tumor infiltrating DC. Consistently, combination of ISV with PD-1 blockade led to complete tumor regression and increased long-term survival in the majority of mice. PD-1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect. Conclusions: In situ vaccination can improve efficacy of anti-PD-1 in checkpoint-unresponsive lymphoma tumors through induction of a cross-presenting DC subset leading to long-term regression of established lymphoma tumors.

Establishment of A20 Murine B Lymphoma Transplantation Model Labeled with Luciferase

To establish the animal model of luciferase-transfected A20 murine B cell lymphoma, so as to provide experimental tools to explore the effect of graft versus tumor. Luciferase- labeled A20 cells were cloned with puromycin selection. Transfected A20 cells and C57BL/6 bone marrow were inoculated into the irradiated BALB/c mice by injection in tailvein to establish the transplantation model. The bioluminescent imaging technique was used to monitor the tumor growth, and then the survival, body weight, tumor formation and pathological characteristics of target organs were observed. A20 cell line stably expressing luciferase gene was successfully obtained. The the bioluminicent imaging found that the tumor luminescence could be observed on day 8 of A20 cell inoculation, and the mean fluorescent intensity was increased along with the tumor growth. Compared with the BMT group, the survival rate and body weight of BMT+A20-Luc+ mice were decreased significantly. General anatomy showed the tumor mainly formed in the liver and spleen. A mouse transplantation model with luciferase- transfected A20 cells has been successfully established, thus laying a foundation for investigation of graft-versus-tumor.

External signals regulate germinal center fate-determining transcription factors in the A20 lymphoma cell line

Due to the apoptosis-prone nature of primary germinal center B (GCB) cells, it remains a huge challenge to dissect signals that guide their differentiation towards memory B cells and plasma cells in vitro. Here we show that the murine lymphoma cell line A20 resembles primary GCB cells in expression of GC-specific surface markers and the master transcription factor BCL6 and may serve as a useful system to model certain GCB cell behaviors in vitro. Using these cells, we found that both CD40 and B cell receptor (BCR) signaling are able to drive BCL6 downregulation, which is a prerequisite of post-GC B-cell differentiation. Under the steady state, BCL6 is constantly and rapidly degraded in A20 cells by the proteasome in a strictly FBXO11-dependent manner. This process can be further enhanced by signals downstream of the BCR. Both CD40 and BCR stimulation can upregulate IRF4, a transcription factor that suppresses BCL6 expression. However, only BCR signaling downregulate PAX5 and BACH2, two transcription factors that help maintain the GCB identity. Together, these results validate the A20 cell line as an experimental system suitable for studying regulation of BCL6 and potentially other transcription factors relevant to post-GC fate determination, and they support that combined signaling from BCR and CD40 receptors would drive termination of the GC program.