Primary Normal Human Cells Research Articles

TROCEPT: An oncolytic virus platform engineered for αvß6 integrin targeted tumor-localised expression of an immune checkpoint inhibitor following intravenous delivery.

e14579 Background: TROCEPT is a novel tumor-selective replicating, oncolytic adenovirus type 5 (Ad5) rationally engineered to remove all natural tissue tropisms. This engineering addresses the main limitation of other viral therapies which infect normal tissues and are rapidly removed by the liver and are therefore limited to local delivery. TROCEPT has been further engineered to specifically bind to αvβ6 integrin which is expressed at high frequency in the majority of epithelial cancers, so that following intravenous delivery the virus targets tumor tissue where it replicates and amplifies. In addition, the TROCEPT platform encodes transgenes which enables in-tumor production of powerful therapeutic drugs. The lead programme, TROCEPT-01, encodes a fully human, full length immune checkpoint inhibitor (ICI) antibody. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications. Methods: Proof of concept studies, both in vitro and in vivo, to demonstrate TROCEPT-01’s tumor selectivity and toxicity profile have been performed. Results: In vitro experiments demonstrate that viral replication, transgene expression and oncolytic cell death following infection with TROCEPT-01 is highly selective for αvβ6 integrin positive tumor cells compared to a panel of normal human primary cells. Intravenous delivery in in vivo SKOV3 ovarian human tumor xenograft mouse models demonstrate virus delivery, replication, and transgene expression in the tumor. In comparison to the parental Ad5 virus, the engineering of TROCEPT-01 leads to more viral delivery to the tumour, less delivery to normal tissue including liver, reduced markers of liver toxicity and reduced inflammatory cytokine release, all of which validate the tumour selectivity and safety profile of the virus. Conclusions: We have demonstrated that TROCEPT engineering enables highly selective tumor targeting and significant tumor exposure following intravenous delivery. TROCEPT-01’s in-tumor selective expression of ICIs enables high local drug concentration in the tumor, potentially reducing systemic toxicity and increasing efficacy. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications.

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1338.

Induction of Aryl Hydrocarbon Receptor-Mediated Cancer Cell-Selective Apoptosis in Triple-Negative Breast Cancer Cells by a High-Affinity Benzimidazoisoquinoline.

Triple-negative breast cancer (TNBC) remains a disease with a paucity of targeted treatment opportunities. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in a wide range of physiological processes, including the sensing of xenobiotics, immune function, development, and differentiation. Different small-molecule AhR ligands drive strikingly varied cellular and organismal responses. In certain cancers, AhR activation by select small molecules induces cell cycle arrest or apoptosis via activation of tumor-suppressive transcriptional programs. AhR is expressed in triple-negative breast cancers, presenting a tractable therapeutic opportunity. Here, we identify a novel ligand of the aryl hydrocarbon receptor that potently and selectively induces cell death in triple-negative breast cancer cells and TNBC stem cells via the AhR. Importantly, we found that this compound, Analog 523, exhibits minimal cytotoxicity against multiple normal human primary cells. Analog 523 represents a high-affinity AhR ligand with potential for future clinical translation as an anticancer agent.

PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies.

In spite of impressive success in treating hematologic malignancies, adoptive therapy with chimeric antigen receptor modified T cells (CAR T) has not yet been effective in solid tumors, where identification of suitable tumor-specific antigens remains a major obstacle for CAR T-cell therapy due to the “on target off tumor” toxicity. Protein tyrosine kinase 7 (PTK7) is a member of the Wnt-related pseudokinases and identified as a highly expressed antigen enriched in cancer stem cells (CSCs) from multiple solid tumors, including but not limited to triple-negative breast cancer, non-small-cell lung cancer, and ovarian cancer, suggesting it may serve as a promising tumor-specific target for CAR T-cell therapy. In this study, we constructed three different PTK7-specific CAR (PTK7-CAR1/2/3), each comprising a humanized PTK7-specific single-chain variable fragment (scFv), hinge and transmembrane (TM) regions of the human CD8α molecule, 4-1BB intracellular co-stimulatory domain (BB-ICD), and CD3ζ intracellular domain (CD3ζ-ICD) sequence, and then prepared the CAR T cells by lentivirus-mediated transduction of human activated T cells accordingly, and we sequentially evaluated their antigen-specific recognition and killing activity in vitro and in vivo. T cells transduced with all three PTK7-CAR candidates exhibited antigen-specific cytokine production and potent cytotoxicity against naturally expressing PTK7-positive tumor cells of multiple cancer types without mediating cytotoxicity of a panel of normal primary human cells; meanwhile, in vitro recursive cytotoxicity assays demonstrated that only PTK7-CAR2 modified T cells retained effective through multiple rounds of tumor challenge. Using in vivo xenograft models of lung cancers with different expression levels of PTK7, systemic delivery of PTK7-CAR2 modified T cells significantly prevented tumor growth and prolonged overall survival of mice. Altogether, our results support PTK7 as a therapeutic target suitable for CAR T-cell therapy that could be applied for lung cancers and many other solid cancers with PTK7 overexpression.

Re-assessing the toxicity of particles from biodiesel combustion: A quantitative analysis of in vitro studies

Biofuels may reduce road transport carbon intensity; however, it is uncertain whether displacing fossil diesel would alter the engine-derived particulate toxicity. The primary objective of this work was to determine whether there is a fuel effect on the comparative in vitro toxicity of biodiesel exhaust particulates relative to those from fossil diesel. A secondary aim was to determine qualitatively whether the observed outcome is related to the organic phase, namely Polycyclic Aromatic Hydrocarbons (PAHs). In vitro and acellular exposure studies were recovered from a literature survey following the PRISMA framework. Biological responses attributable to biodiesel and paired fossil diesel particles, including particle-extracts were selected. To qualify for inclusion, either of the paired responses must differ statistically significantly (p < 0.05) from the control or each other. Paired responses were assigned to one-of-five categories which best represents the pathophysiological role of the biomarker: inflammation, oxidative stress, cytotoxicity, genotoxicity and mutagenicity. Biodiesel reduced particle toxicity in two-thirds of paired responses, however, there were large differences between biodiesels for category-specific biomarkers. Particles derived from Rapeseed oil Methyl Ester (RME) were less inflammatory, whereas Soybean oil Methyl Ester (SME) particles were more inflammatory than fossil diesel on average. Conversely, SME reduced oxidative stress while few trends emerged for mutagenicity and genotoxicity. The largest fuel effect was observed for cytotoxicity: Waste Cooking Oil Methyl Ester (WCOME) increased and Palm oil Methyl Ester (PME) decreased particle cytotoxicity. Particle-phase PAH emissions compiled on a mass-of-soot basis also followed this trend, however, literature focusing on both these aspects is limited; careful consideration of fuel composition and use of normal primary human cell types and omics technologies, could resolve this open question. This assessment systematically compares biological responses from particulate only exposure, with co-exposure necessarily excluded due to an absence of understanding of how gaseous components modify particulate toxicity.

Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Acute Myeloid Leukemia.

Feline McDonough Sarcoma-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T-cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with 2 bispecific T-cell engager molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells, whereas only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.

CAR T Cells Targeting MISIIR for the Treatment of Ovarian Cancer and Other Gynecologic Malignancies.

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor β (TGF-β) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) Tcell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR Tcell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. Tcells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity invitro and eliminated MISIIR-overexpressing tumors invivo. MISIIR CAR Tcells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens invitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.

Postbiotic metabolites produced by Lactobacillus plantarum strains exert selective cytotoxicity effects on cancer cells

BackgroundLactobacillus plantarum, a major species of Lactic Acid Bacteria (LAB), are capable of producing postbiotic metabolites (PM) with prominent probiotic effects that have been documented extensively for rats, poultry and pigs. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on cytotoxic and antiproliferative activity of PM produced by L. plantarum. Therefore, the cytotoxicity of PM produced by six strains of L. plantarum on various cancer and normal cells are yet to be evaluated.MethodsPostbiotic metabolites (PM) produced by six strains of L. plantarum were determined for their antiproliferative and cytotoxic effects on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. The toxicity of PM was determined for human and various animal red blood cells via haemolytic assay. The cytotoxicity mode was subsequently determined for selected UL4 PM on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic observation using AO/PI dye reagents and flow cytometric analyses.ResultsUL4 PM exhibited the lowest IC50 value on MCF-7, RG14 PM on HT29 and RG11 and RI11 PM on HL60 cell lines, respectively from MTT assay. Moreover, all tested PM did not cause haemolysis of human, dog, rabbit and chicken red blood cells and demonstrated no cytotoxicity on normal breast MCF-10A cells and primary cultured cells including human peripheral blood mononuclear cells, mice splenocytes and thymocytes. Antiproliferation of MCF-7 and HT-29 cells was potently induced by UL4 and RG 14 PM respectively after 72 h of incubation at the concentration of 30% (v/v). Fluorescent microscopic observation and flow cytometric analyses showed that the pronounced cytotoxic effect of UL4 PM on MCF-7 cells was mediated through apoptosis.ConclusionIn conclusion, PM produced by the six strains of L. plantarum exhibited selective cytotoxic via antiproliferative effect and induction of apoptosis against malignant cancer cells in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells. This reveals the vast potentials of PM from L. plantarum as functional supplement and as an adjunctive treatment for cancer.

Abstract OT2-07-06: Antibody-coupled T cell receptor (ACTR) engineered autologous T cells in combination with trastuzumab for the treatment of HER2-positive malignancies

Abstract Antibody-Coupled T cell Receptor (ACTR) is an autologous engineered T cell therapy developed to combine with tumor-targeting antibodies to exert potent anti-tumor immune responses and tumor cell killing. The ACTR construct is composed of the extracellular domain of CD16 fused to CD3ζ signaling and T cell co-stimulatory domains. ACTR-expressing T cells are universal in that they can be paired with a therapeutic antibody to target specific antigens on tumors. Unum has two ACTR constructs, ACTR087 and ACTR707, currently in clinical testing. ACTR087 and ACTR707 are being tested in combination with rituximab in subjects with CD20+ B cell lymphoma in two separate trials (NCT02776813 and NCT03189836, respectively). Preliminary data with ACTR087 + rituximab has demonstrated clinical proof-of-concept and a dose-response relationship in subjects with relapsed/refractory B cell lymphoma. ACTR087 is also being tested in combination with a novel BCMA-targeting antibody in subjects with multiple myeloma (NCT03266692). While T cell therapies, such as chimeric antigen receptor (CAR) T cells, have demonstrated clinical activity in hematological cancers, the therapeutic potential of this approach has yet to be established in solid tumors. Challenges associated with targeting solid tumors with CAR-T cells include tumor antigen heterogeneity and antigen expression on normal tissues. HER2 is a well-established therapeutic target that is over-expressed in a number of cancer indications. HER2 is also expressed at low levels on normal epithelial cells, creating a risk for on-target/off-tumor toxicities of HER2-targeted CAR-T cells. Here we present nonclinical studies demonstrating that ACTR T cells in combination with trastuzumab have antigen density-dependent activity on HER2-expressing tumor cell lines, while trastuzumab-based CAR-T cells do not. We observed that ACTR + trastuzumab had robust activity against HER2-amplified tumor cells and more modest activity against non-amplified tumor cells, whereas HER2-targeting CAR-T cells had comparable activity against HER2-amplified and non-amplified tumor cells. On normal human primary cells, ACTR + trastuzumab had minimal activity in comparison to HER2 CAR-T cells, suggesting that ACTR + trastuzumab may exhibit a superior clinical therapeutic index. Furthermore, the activity of ACTR T cells against HER2-amplified tumor cells was titratable with antibody concentration, allowing for control of ACTR activity by modulation of trastuzumab concentration. Together, these data demonstrate the specificity of the ACTR T cell therapeutic approach to target HER2-amplified tumors and support clinical testing in combination with trastuzumab. A phase 1, multicenter, single-arm, open-label dose escalation study, ATTCK-34-01, is proposed to evaluate ACTR T cells in combination with trastuzumab in subjects with advanced HER2-positive malignancies. The primary study objectives are to assess the safety and tolerability of the combination, and to define the recommended phase 2 dose combination for further study. Additional objectives include assessment of anti-tumor activity, ACTR T cell persistence and trastuzumab pharmacokinetics. Enrollment is expected to commence in early 2019. Citation Format: O'Callaghan KM, Shin J, Cheung AS, Cheema T, Judge C, Ranger A, Huet HA, Ettenberg SA, Sachs J, Vasconcelles M, Motz G. Antibody-coupled T cell receptor (ACTR) engineered autologous T cells in combination with trastuzumab for the treatment of HER2-positive malignancies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-07-06.

Synthesis and biological evaluation of pyrimidine bridged combretastatin derivatives as potential anticancer agents and mechanistic studies

A number of pyrimidine bridged combretastatin derivatives were designed, synthesized and evaluated for anticancer activities against breast cancer (MCF-7) and lung cancer (A549) cell lines using MTT assays. Most of the synthesized compounds displayed good anticancer activity with IC50 values in low micro-molar range. Compounds 4a and 4p were found most potent in the series with IC50 values of 4.67 µM & 3.38 µM and 4.63 µM & 3.71 µM against MCF7 and A549 cancer cell lines, respectively. Biological evaluation of these compounds showed that selective cancer cell toxicity (in vitro using human lung and breast cancer cell lines) might be due to the inhibition of antioxidant enzymes instigating elevated ROS levels which triggers intrinsic apoptotic pathways. These compounds were found nontoxic to the normal human primary cells. Compound 4a, was found to be competitive inhibitor of colchicine and in the tubulin binding assay it showed tubulin polymerization inhibition potential comparable to colchicine. The molecular modeling studies also showed that the synthesized compounds fit well in the colchicine-binding pocket.

The NAD+ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation

NAD+ is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD+ consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD+ levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes.We observed that nicotinic acid supplementation increased NAD+ levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD+ concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD+ levels may be limiting under conditions of genotoxic stress and that boosting the NAD+ system with nicotinic acid can improve genomic stability.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

510. Treating Epithelial and Metastatic Malignancies with Glycoform-Specific Chimeric Antigen Receptors

Cancer-specific glycosylation deficiencies generating Tn or sTn O-linked glycans are found in >80% of the most lethal cancers (lung, prostate, breast, colon, ovary, pancreas). These hypoglycosylated cancer glycoantigens decrease cell-cell interactions and increase the mobility and metastatic potential of tumor cells. We developed a CAR specific to the Tn/sTn antigen of the oncogene Mucin-1 (MUC1), which is highly upregulated in many epithelial cancers and is heavily O- and N-glycosylated. Our CAR uses the scFv sequence derived from the 5E5 mAb and the endodomains of CD3zeta and 4-1BB that promote T cell activation and co-stimulation, respectively. We have shown that the 5E5 mAb and the 5E5 CAR are specific to the cancer-specific glycoform of MUC1 but not normal MUC1 through in vitro binding and cytokine assays and that the 5E5 CAR is non-reactive to normal human primary cells. We demonstrated that the 5E5 CAR T cells are responsive to ovarian, breast, lung, pancreatic, and leukemia cancers and developed NSG mouse xenograft models using Jurkat leukemia cell line and Hs766T metastatic pancreatic cell line that show rapid clearance of established tumors by 5E5 CAR T cells and prolonged survival of tumor-bearing mice. Restoring normal glycosylation in Jurkat leukemia cells abolishes the expression of the hypoglycosylated form of Muc1 and also prevents 5E5 CAR T cell cytotoxicity, further highlighting the specificity of the 5E5 CAR for the cancer-specific glycoform of MUC1. Infusion of human MUC1-transgenic mice with murine 5E5 CAR T cells showed an exquisite safety profile, while infusion of normal glycoform MUC1-specific murine CAR T cells (HMFG1 CAR, a previously described MUC1-specific CAR) demonstrate extreme safety concerns. These results demonstrate that targeting the cancer-specific glycoform of MUC1 provides a safe, yet potent cancer-specific immunotherapy for treating most epithelial malignancies and holds the potential to eradicate metastatic disease.

RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity

Semi-synthetic triterpenoids are antioxidant inflammation modulator (AIM) compounds that inhibit tumor cell growth and metastasis. Compounds in the AIM class bind to Keap1 and attenuate Nrf2 degradation. In the nucleus, Nrf2 increases antioxidant gene expression and reduces pro-inflammatory gene expression. By increasing Nrf2 activity, AIMs reduce reactive oxygen species and inflammation in the tumor microenvironment, which reverses tumor-mediated immune evasion and inhibits tumor growth and metastasis. AIMs also directly inhibit tumor cell growth by modulating oncogenic signaling pathways, such as IKKβ/NF-κB. Here, we characterized the in vitro antioxidant, anti-inflammatory, and anticancer activities of RTA 408, a novel AIM that is currently being evaluated in patients with advanced malignancies. At low concentrations (≤ 25 nM), RTA 408 activated Nrf2 and suppressed nitric oxide and pro-inflammatory cytokine levels in interferon-γ-stimulated RAW 264.7 macrophage cells. At higher concentrations, RTA 408 inhibited tumor cell growth (GI50 = 260 ± 74 nM) and increased caspase activity in tumor cell lines, but not in normal primary human cells. Consistent with the direct effect of AIMs on IKKβ, RTA 408 inhibited NF-κB signaling and decreased cyclin D1 levels at the same concentrations that inhibited cell growth and induced apoptosis. RTA 408 also increased CDKN1A (p21) levels and JNK phosphorylation. The in vitro activity profile of RTA 408 is similar to that of bardoxolone methyl, which was well-tolerated by patients at doses that demonstrated target engagement. Taken together, these data support clinical evaluation of RTA 408 for cancer treatment.

Analysis by live imaging of effects of the adenovirus E4orf4 protein on passage through mitosis of H1299 tumor cells.

The adenovirus E4orf4 protein expressed at high levels kills cancer cells but not normal human primary cells. Previous studies suggested that disruption of processes that regulate mitosis may underlie E4orf4 toxicity. Here we have used live imaging to show that E4orf4 induces a slowed defective transit through mitosis, exhibiting a delay or often failure in cytokinesis that may account for an accumulation of G1 tetraploids in the population of dying E4orf4-expressing cells.

Abstract 2028: A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro

Abstract The significance of bacterial infections in the initiation of human cancers is increasingly being recognized. A well established example is Helicobacter pylori, a causative agent of gastric adenocarcinoma. Current research largely relies on the use of transformed human cell lines as in vitro models of these infections. Obviously, this concept is largely artificial and especially inappropriate when it comes to investigations on the mechanisms of cancerogenesis. A primary cell system would be preferable in order to approximate the in vivo situation, however, previous attempts to establish such models are hampered by the limited growth and reproducibility of normal human primary cell cultures. Here, we report on the establishment of specific long-term culture conditions that allow the formation of 3-dimensional human gastric organoids with epithelial domains that have the capacity to self-renew and can be maintained in culture indefinitely. Interestingly, these 3-dimensional structures can be transferred into 2-dimensional primary cell layers, which provide ideal conditions to study infections in vitro. We expect that our novel human gastric primary cell model will facilitate investigations on the mechanisms underlying infection and is suited to open up avenues of approach to investigations on the initial processes of cellular transformation from normal to a transformed cell stages including the tumor-promoting capacity of the gastric pathogen H. pylori. Citation Format: Philipp Schlaermann, Thomas F. Meyer. A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2028. doi:10.1158/1538-7445.AM2014-2028

Metabolic alterations accompanying oncogene-induced senescence

Senescence is defined as a stable cell growth arrest. Oncogene-induced senescence (OIS) occurs in normal primary human cells after activation of an oncogene in the absence of other cooperating oncogenic stimuli. OIS is therefore considered a bona fide tumor suppression mechanism in vivo. Indeed, overcoming OIS-associated stable cell growth arrest can lead to tumorigenesis. Although cells that have undergone OIS do not replicate their DNA, they remain metabolically active. A number of recent studies report significant changes in cellular metabolism during OIS, including alterations in nucleotide, glucose, and mitochondrial metabolism and autophagy. These alterations may be necessary for stable senescence-associated cell growth arrest, and overcoming these shifts in metabolism may lead to tumorigenesis. This review highlights what is currently known about alterations in cellular metabolism during OIS and the implication of OIS-associated metabolic changes in cellular transformation and the development of cancer therapeutic strategies.

Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity

Oncolytic viruses (OVs) are attractive avenues of cancer therapy due to the absence of toxic side effects often seen with current treatment modalities. Bovine herpesvirus 1 (BHV-1) is a species-specific virus that does not induce cytotoxicity in normal primary human cells but can infect and kill various human immortalized and transformed cell lines. To gain a better understanding of the oncolytic breadth of BHV-1, the NCI panel of established human tumor cell lines was screened for sensitivity to the virus. Overall, 72% of the panel is permissive to BHV-1 infection, with corresponding decreases in cellular viability. This sensitivity is in comparison to a sensitivity of only 32% for a herpes simplex virus 1 (HSV-1)-based oncolytic vector. Strikingly, while 35% of the panel supports minimal or no BHV-1 replication, significant decreases in cellular viability still occur. These data suggest that BHV-1 is an OV with tropism for multiple tumor types and is able to induce cytotoxicity independent of significant virus replication. In contrast to other species-specific OVs, cellular sensitivity to BHV-1 does not correlate with type I interferon (IFN) signaling; however, mutations in KRAS were found to correlate with high levels of virus replication. The knockdown or overexpression of KRAS in human tumor cell lines yields modest changes in viral titers; however, overexpression of KRAS in normal primary cells elicits permissivity to BHV-1 infection. Together, these data suggest that BHV-1 is a broad-spectrum OV with a distinct mechanism of tumor targeting. Cancer remains a significant health issue, and novel treatments are required, particularly for tumors that are refractory to conventional therapies. Oncolytic viruses are a novel platform given their ability to specifically target tumor cells while leaving healthy cells intact. For this strategy to be successful, a fundamental understanding of virus-host interactions is required. We previously identified bovine herpesvirus 1 as a novel oncolytic virus with many unique and clinically relevant features. Here, we show that BHV-1 can target a wide range of human cancer types, most potently lung cancer. In addition, we show that enhanced KRAS activity, a hallmark of many cancers, is one of the factors that increases BHV-1 oncolytic capacity. These findings hold potential for future treatments, particularly in the context of lung cancer, where KRAS mutations are a negative predictor of treatment efficacy.

The human secretome atlas initiative: Implications in health and disease conditions

Proteomic analysis of human body fluids is highly challenging, therefore many researchers are redirecting efforts toward secretome profiling. The goal is to define potential biomarkers and therapeutic targets in the secretome that can be traced back in accessible human body fluids. However, currently there is a lack of secretome profiles of normal human primary cells making it difficult to assess the biological meaning of current findings. In this study we sought to establish secretome profiles of human primary cells obtained from healthy donors with the goal of building a human secretome atlas. Such an atlas can be used as a reference for discovery of potential disease associated biomarkers and eventually novel therapeutic targets. As a preliminary study, secretome profiles were established for six different types of human primary cell cultures and checked for overlaps with the three major human body fluids including plasma, cerebrospinal fluid and urine. About 67% of the 1054 identified proteins in the secretome of these primary cells occurred in at least one body fluid. Furthermore, comparison of the secretome profiles of two human glioblastoma cell lines to this new human secretome atlas enabled unambiguous identification of potential brain tumor biomarkers. These biomarkers can be easily monitored in different body fluids using stable isotope labeled standard proteins. The long term goal of this study is to establish a comprehensive online human secretome atlas for future use as a reference for any disease related secretome study. This article is part of a Special Issue entitled: An Updated Secretome.

Abstract 3252: A novel small molecule, LY5, selectively inhibits STAT3 phosphorylation and activities and exhibits potent growth suppressive activity in cancer cells.

Abstract In response to cytokines and growth factors, Signal Transducers and Activators of Transcription 3 (STAT3) signaling is activated which plays a critical role in oncogenesis, proliferation, survival, and invasion of various human cancers and cancer cell lines. Numerous evidences have demonstrated that inhibition of STAT3 signaling pathway results in decreased growth and apoptosis in human cancer cells. This makes STAT3 an excellent molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable, small molecule, named as LY5, which selectively targets STAT3. LY5 inhibited constitutive STAT3 activation in various human pediatric sarcoma and medulloblastoma cell lines expressing elevated levels of STAT3 phosphorylation. LY5 also blocked STAT3 phosphorylation induced by Interleukin-6, IGF-1, and OSM in SJSA human osteosarcoma cells, but did not inhibit STAT1, STAT2, STAT4, and STAT6 phosphorylation stimulated by interferon (IFN)-γ,IFN-α, and IL-4, respectively. Furthermore, LY5 suppressed the induction of STAT3 nuclear localization by IL-6, which could prevent STAT3 function as a transcription factor, but not STAT1 nuclear localization induced by IFN-γ. In addition, LY5 induced apoptosis in sarcoma and medulloblastoma cells as indicated by the increases of cleaved caspase-3, but LY5 did not induce apoptosis in normal primary human cells. The inhibition of STAT3 signaling by LY5 was further confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, Bcl-XL, and survivin. Taken together, these findings indicated LY5 selectively inhibited constitutive STAT3 activation and suggested that LY5 is a promising therapeutic candidate for pediatric sarcoma and medulloblastoma through inhibiting STAT3 signaling. Citation Format: Hui Xiao, Wenying Yu, Veronica R. Olson, Chenglong Li, Peter Houghton, Jiayuh Lin. A novel small molecule, LY5, selectively inhibits STAT3 phosphorylation and activities and exhibits potent growth suppressive activity in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3252. doi:10.1158/1538-7445.AM2013-3252