bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-184: Core circadian clock component, PERIOD2, regulates adaptive radioprotection via PER2-β-Catenin controlled mitochondrial bioenergetics
Authors: Alexandrou Aris , Fan Ming , Juma Shuaib , Perks Julian , Vaughan Andrew , Tepper Clifford , Fu Loning , Woloschak Gayle , Grdina David
Published: August 2018

Circadian clocks are intimately involved in the homeostatic maintenance of metabolic and physiological processes that may have increasingly important roles to enhance bone marrow transplantation success in cancer patients; to protect normal tissue surrounding tumors from the harmful effects of radio/chemotherapy; and to enhance the quality of diet and sleep for astronauts during space exploration. Herein, we report that expression of PERIOD2 (PER2; a core circadian clock component and “the inherent driver” of radioprotection) is required for adaptive protection against environmental radiation stress. PER2 expression is induced by exposure to low dose radiation (LDR; 10cGy) and siRNA blockade of PER2 ablates LDR-induced adaptive radioprotection. In addition, melatonin (N-acetyl-5-methoxytryptamine), a natural anti-oxidant and hypothalamic circadian synchronizer, stimulates PER2 expression and confers mice radioprotection through weight maintenance and increased survival. LDR-induced PER2 transcription is regulated by NF-κB and β-catenin and is released from phospho-glycogen synthase kinase-3ß (p-GSK3ß) in the WNT/β-catenin pathway. Unbound β-catenin interacts with the TCF/LEF domain on the Per2 promoter to promote feed-forward PER2-pGSK3β complex formation. Mitochondrial bioenergetics measured by oxygen consumption and ATP generation was attenuated in bone marrow isolated from PER2 mutant (Per2m/m) mice. Furthermore, RNA-seq profiling of bone marrow-derive progenitor hematopoietic stem cells (BM-pHSCs; Lin-/Sca1+/cKit+; LSK) cells isolated form LDR-treated wild-type (WT) versus Per2m/m mice showed a cluster of genes involved in mitochondrial bioenergetics and DNA repair capacity. These results demonstrate that a core circadian regulator plays an indispensable role in defending mammalian cells against environmental genotoxic stress through the PER2/β-catenin pathway, a potential therapeutic target to enhance cell survival under radiation.

Citation Format: Aris Alexandrou, Ming Fan, Shuaib Juma, Julian Perks, Andrew Vaughan, Clifford Tepper, Loning Fu, Gayle Woloschak, David Grdina. Core circadian clock component, PERIOD2, regulates adaptive radioprotection via PER2-β-Catenin controlled mitochondrial bioenergetics [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 LB-184.
Abstract LB-190: CDC25A suppression of apoptosis in cutaneous squamous cell carcinoma depends on 14-3-3ϵ
Authors: Holmes Thomas R. , Al-Matouq Jenan , Hammiller Brianna , Nicholson Yawah T. , Holmes Matti J. , Hansen Laura A.
Published: August 2018

Cytoplasmic relocalization of the dual-specificity phosphatase, CDC25A, occurs during nonmelanoma skin cancer development and progression, leading to suppression of cell death. Cytoplasmic CDC25A is phosphorylated at Ser178, which allows for its association with 14-3-3ϵ. 14-3-3ϵ is a member of the 14-3-3 family of proteins that bind phosphoserine/threonine-containing proteins and are involved in various processes including scaffolding, nuclear transport, protein degradation, cell cycle regulation, apoptosis and cell signaling. We hypothesized that CDC25A's antiapoptotic function depends upon 14-3-3ϵ binding to trigger activation of prosurvival signaling pathways in SCC cells. Co-immunoprecipitation of CDC25A from the SCC cell line SCC12B.2 followed by immunoblotting for 14-3-3ϵ confirmed an association between CDC25A and14-3-3ϵ. CDC25A over-expression in SCC12B.2 cells reduced apoptosis by approximately 50%, as assessed by a caspase 3/7 glo assay. However, inhibition of 14-3-3ϵ with the peptide R18, or silencing of 14-3-3ϵ, blocked the anti-apoptotic effect of CDC25A in SCC cells. Treatment with serial dilutions of R18 increased cell death with an IC50 of 8.9 μM in SCC12B.2 cells. Interestingly, over-expression of CDC25A significantly increased P-Akt (p=0.011), P-BAD (p=0.034) and Survivin (p=0.036) when compared to control. Consistent with our hypothesis that 14-3-3ϵ facilitates CDC25A's antiapoptotic activity, silencing of 14-3-3ϵ decreased P-Akt (p=0.044), P-BAD (p=0.034) and Survivin (p=0.008). Taken together, these results indicate that cytoplasmic CDC25A and 14-3-3ϵ interact in skin carcinomas to suppress apoptosis through a mechanism involving activation of Akt/BAD/Survivin signaling, rendering SCC cells sensitive to 14-3-3ϵ or CDC25A inhibition, a mechanism that requires further study.

Citation Format: Thomas R. Holmes, Jenan Al-Matouq, Brianna Hammiller, Yawah T. Nicholson, Matti J. Holmes, Laura A. Hansen. CDC25A suppression of apoptosis in cutaneous squamous cell carcinoma depends on 14-3-3ϵ [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 LB-190.
Abstract LB-192: 2,2'-Methylenebis (6-tert-butyl 4-methylphenol) enhances the antitumor efficacy of belotecan, a derivative of camptothecin, by inducing autophagy
Authors: Park Junsoo , Lee Eun-Ju
Published: August 2018

Autophagy regulation is important for tumor cell survival. Activation and inhibition of autophagy can sensitize tumor cells to anticancer drugs. However, few autophagy-regulating small molecules are available to increase the efficacy of anticancer drugs. Here, we report that 2,2'-methylenebis (6-tert-butyl 4-methylphenol), hereafter referred to as methylenebis, is a novel autophagy-regulating small molecule that sensitizes tumor cells to belotecan, which is a derivative of camptothecin, a topoisomerase I inhibitor. Methylenebis activates autophagic flux by increasing the level of LC3-II and forming autolysosome puncta. Moreover, methylenebis enhances the antitumor efficacy of belotecan by activating both autophagy and apoptosis. Interestingly, methylenebis increased the level of LC3-II and belotecan independently decreased the level of p62, suggesting that methylenebis and belotecan target different steps of autophagy. Finally, we searched for compounds that are structurally similar to methylenebis. Our results imply that the specific structure of methylenebis contributes to its ability to activate autophagy.

Citation Format: Junsoo Park, Eun-Ju Lee. 2,2'-Methylenebis (6-tert-butyl 4-methylphenol) enhances the antitumor efficacy of belotecan, a derivative of camptothecin, by inducing autophagy [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 LB-192.
Abstract LB-216: Protein pathway activation analysis of a panel of patient-derived epithelial tumor models identifies signatures of response to ErbB1-3 co-targeting
Authors: Calvert Valerie S. , Fröhlich Camilla , Poulsen Thomas T. , Pedersen Mikkel W. , Kragh Michael , Horak Ivan D. , Petricoin Emanuel F.
Published: August 2018

Plasticity of the ErbB family of receptors and ligands is a hallmark of many human tumors and drives resistance to EGFR and HER2 targeting agents such as cetuximab or trastuzumab. Pan-HER is a unique antibody mixture designed to counter ErbB family plasticity by simultaneously targeting EGFR, HER2 and HER3. Pan-HER contains pairs of antibodies against each of the receptors, six antibodies in total, and induces efficient internalization and degradation of all three receptors. Here, the anti-tumor activity of Pan-HER was investigated in a panel of 57 patient-derived epithelial tumor models (PDXs) of various origin including pancreatic (18), colorectal (11), triple negative breast (11), lung (6), squamous head and neck (6), ovarian (3), and gastric (2) cancer, and the level of activity was correlated to baseline protein and phosphoprotein status using laser capture microdissection (LCM) and Reverse Phase Protein Arrays (RPPA). This panel has been preselected to represent hard-to-treat cancers and that are not overtly HER family positive. Pan-HER demonstrated anti-tumor activity across all indications except the gastric models and induced tumor regression in 26 of the 57 PDX models tested. The LCM-RPPA analysis of over 150 proteins and phosphoproteins that are known to be central for receptor tyrosine kinase signaling, survival, apoptosis, growth, inflammation, motility and autophagy identified both broad and indication specific signatures of response to Pan-HER and points to a key role for the KRAS/BRAF/ERK and PIK3CA/AKT pathway activation that associate with therapeutic sensitivity. In conclusion, our results demonstrate that Pan-HER has broad anti-tumor activity across indications and identify phosphoprotein pathway activation signatures predicting for sensitivity or resistance that should be explored clinically.

Citation Format: Valerie S. Calvert, Camilla Fröhlich, Thomas T. Poulsen, Mikkel W. Pedersen, Michael Kragh, Ivan D. Horak, Emanuel F. Petricoin. Protein pathway activation analysis of a panel of patient-derived epithelial tumor models identifies signatures of response to ErbB1-3 co-targeting [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 LB-216.
Abstract LB-229: Mesothelin expression is a predict factor for peritoneal recurrence in gastric cancer
Authors: Shin Su-Jin , Kim Hyunki , Choi Yoon Young , Park Sejung , Nam Chung Mo , Chung Hyun Cheol
Published: August 2018

Background: Mesothelin is overexpressed in many solid tumors. Recent studies showed that mesothelin expression is associated with poor outcome in several malignant tumors and it may play a role of cancer cell proliferation, local invasion and metastasis. Clinical trials of mesothelin-targeted immunotherapies are currently in progress. However, the correlation between mesothelin expression and prognosis of gastric cancer is still conflicting.

Patients and Methods: Mesothelin expression in tumour cells were immunohistochemically evaluated in 964 advanced gastric cancer patients, and their associations with clinical outcome were analysed. The prediction model for recurrence was developed by cox proportional hazard models.

Results: High mesothelin expression was observed in 49.6% and significantly associated with a high stage (P = 0.022) and the presence of recurrence with peritoneal carcinomatosis (P = 0.002). In univariate survival analysis, high mesothelin expression was significantly associated with shorter recurrent-free survival (RFS) (P=0.003) and overall survival (OS) (P=0.006). Multivariate survival analysis revealed that high mesothelin expression was independently associated with poor RFS and OS (P=0.001; P=0.002, respectively) in addition to stage, lymphovascular invasion and Lauren classification (all, P<0.001). In multivariate analysis for recurrence with peritoneal carcinomatosis, high mesothelin expression was also independent prognostic factors in all advanced gastric cancer patients (P<0.001) and in intestinal (P=0.017) and diffuse/mixed type (P=0.005) subgroups. The overall discrimination ability of prediction model for recurrence, the model with mesothelin was better than model without mesothelin.

Conclusions: High mesothelin expression was correlated with poor outcome. And, mesothelin expression, along with Lauren classification was meaningful predict factor for peritoneal recurrence.

Citation Format: Su-Jin Shin, Hyunki Kim, Yoon Young Choi, Sejung Park, Chung Mo Nam, Hyun Cheol Chung. Mesothelin expression is a predict factor for peritoneal recurrence in gastric cancer [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 LB-229.
Abstract LB-241: Genomic characterization of breast cancer cells resistant to the histone deacetylase inhibitor romidepsin
Authors: Ali-Rahmani Fatima G. , Robey Robert , Lack Justin , Tebase Bethelihem , Bates Susan , Gottesman Michael
Published: August 2018

Overexpression of histone deacetylases (HDACs) has been reported in many cancers. As such, histone deacetylase inhibitors (HDIs) have emerged as potent antitumor agents that prevent HDACs from removing acetyl groups from both histone and non-histone proteins, thereby maintaining transcriptionally active DNA. Romidepsin (Rd) is an HDI that targets primarily class I HDACs and has shown efficacy in T-cell lymphomas. However, many solid tumors have been found to be intrinsically resistant to Rd. To investigate potential mechanisms of resistance, we performed a stepwise selection of MCF7 breast cancer cells in romidepsin in the presence of 2.5 μg/ml verapamil to prevent the emergence of P-glycoprotein, an ATP-binding cassette transporter, as a mechanism of resistance. Resistant cells were stable in 300 ng/ml romidepsin. Romidepsin-resistant (RdVp) cells were more than 100-fold resistant to Rd relative to untreated control cells. In addition, measurement of cell viability with CellTiterGlow showed that RdVp cells exhibited cross-resistance to other HDIs such as panobinostat, belinostat, and vorinostat. We performed RNA-Seq to identify genes responsible for the observed resistance to Rd. Downstream data analysis was performed using Partek Genomics Suite, followed by network analysis using Ingenuity Pathway Analysis (IPA) software. Only expression changes with p< 0.01 were considered significant for pathway analysis. Out of 47788 total transcripts identified, only 1910 transcripts were significantly altered in RdVp cells relative to untreated controls. Transcription of several genes involved in the cholesterol and lipid biosynthesis pathway was increased in RdVp cells, indicating increased cholesterol synthesis to support increased cell growth. Several members of the sonic hedgehog pathway were elevated in cells resistant to Rd. The GLI1 gene that encodes a zinc finger protein was upregulated in RdVp cells by 14-fold. Treatment with a GLI1 inhibitor resulted in decreased survival of cells resistant to Rd. The sequencing data were analyzed to identify single nucleotide variations and short insertions/deletions, and only those present in coding sequences of RdVp cells were further confirmed with Sanger sequencing. We identified novel mutations in MCCC2, HIPK3, ABR, SIRT6, TRAK2, and ZNF100 in RdVp cells. Our results suggest potential targets for combination therapy to sensitize cancer cells resistant to romidepsin and enhance its antitumor efficacy in the clinic.

Citation Format: Fatima G. Ali-Rahmani, Robert Robey, Justin Lack, Bethelihem Tebase, Susan Bates, Michael Gottesman. Genomic characterization of breast cancer cells resistant to the histone deacetylase inhibitor romidepsin [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 LB-241.
Abstract LB-253: Suppression of clear cell ovarian carcinoma growth by glutaminase-1 inhibitor as single agent and in combination with PARP-1 inhibitor
Authors: Yi Tianjin , Christodoulou Danos , Okazaki Arimichi , Birrer Michael , Iliopoulos Othon
Published: August 2018

Tumor hypoxia and cancer-associated mutations reprogram central carbon metabolism and enhance the dependence of cancer cells on glutamine for growth. Clear cell ovarian cancer (CCOC) is a unique epithelial ovarian cancer histotype that is less responsive to chemotherapy and has poorer prognosis than serous histotype. Whole genomic analysis identified major pathways activated in clear cell involving in hypoxic cell growth and carbohydrate metabolism, which were driven by hypoxia-inducible factors HIF1a and/or 2a. We demonstrated before that treatment of HIF-expressing cancer cells with glutaminase 1 (GLS1) inhibitor CB-839, restricts nucleotide synthesis and causes DNA replication stress, which sensitizes cells to the PARP-1 inhibitor. Herein, we observed a range of sensitivities to CB-839 treatment in vitro in CCOC cell lines, from which we selected 4 most sensitive cell lines (ES2, SMOV2, TOV21G and RMG1) and 4 most resistant cell lines (OVSAYO, KK, HAC2 and OVISE) for further study. The synergistic effect of combination treatment of CB-839 and olaparib was achieved in all eight cell lines in vitro. To identify more molecular and metabolic biomarkers of CCOC sensitivity to GLS1 inhibitors, we are currently profiling the gene expression signatures of the most sensitive and resistant CCOC cell lines by RNAseq as well as and the metabolic profile of the CCOC lines. To test the sensitivity of the ES2 cells in vivo to a GLS1 as a single agent or in combination with PARP-1 inhibitors we injected them orthotopically in nude mice. Thirty mice bearing growing ES2 derived tumors were randomly assigned into four groups: vehicle control (n=8), treatment with CB-839 (200mg/kg, gavage, twice daily, n=7), olaparib (75mg/kg, gavage, once daily, n=7) or their combination (n=8). We monitored tumor growth by serial bioluminescence imaging, animal body weight as well as animal survival. To date, after 15-day treatment, we detected a synergistic effect between the 2 agents CB-839 and olaparib in controlling ovarian tumor growth in vivo. Our work discovered a range of sensitivities of CCOC cell lines to GLS1 inhibitor and supports the therapeutic strategies of combining GLS1 inhibitor and PARP inhibitor for targeting HIF-driven CCOC.

Citation Format: Tianjin Yi, Danos Christodoulou, Arimichi Okazaki, Michael Birrer, Othon Iliopoulos. Suppression of clear cell ovarian carcinoma growth by glutaminase-1 inhibitor as single agent and in combination with PARP-1 inhibitor [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 LB-253.
Abstract LB-260: Development and validation of propeptide therapeutics for treating bone metastatic prostate cancer
Authors: Salameh Janelle W. , Umbaugh Charles S. , Figueiredo Marxa L.
Published: August 2018

Prostate cancer (PCa) is the second leading cause of cancer-related death in men in the United States. Most prostate cancer related fatalities are not due to tumors at origination sites, but rather due to prostate cancer metastasis throughout the body. Although the relative 5-year survival rate for localized prostate cancer nears 100%, unfortunately, upon metastasis the 5-year survival rate drops to 29.8%. Bone metastases disrupt tissue homeostasis and weaken the skeleton, resulting in an increased risk of severe bone pain and fracture. This is due to increased formation of immature unmineralized bone by osteoblasts, and increased osteolysis by osteoclasts, which induce high levels of bone resorption. Thus, a primary therapeutic objective for malignant bone disease is to eliminate tumors while promoting bone homeostasis. Current therapies are very limited and include palliative radiotherapy, and biphosphate or anti-RANK treatments, which target osteoclasts and decrease the rate of bone resorption. However, these treatments only delay, but do not prevent, skeletal related injuries. Our laboratory has recently developed a strategy aimed at disrupting malignant cytokines signals using inhibitory peptides targeting IL-6 receptors. I here present a novel strategy to express these peptides in vivo following gene delivery as targeted and multifunctional “propeptides.” These propeptides target bone metastases via EGFR antagonist (tumor-homing peptide) and contain dual therapeutic domains consistent of IL-6Ra antagonistic peptides and a pro-osteogenesis peptide, osteostatin. The propeptides are activated by metalloproteases (MMP2/9), which are overexpressed in metastatic prostate tumors and associated bone lesions. We can detect expression of the propeptides following C2C12 muscle cell transfection via real time quantitative PCR and are currently optimizing biochemical strategies for detecting propeptide products via western blot. We hypothesize that optimized secretion and targeting of the therapeutic propeptides will be effective in eliminating prostate tumor metastases, and restoring bone homeostasis. Future studies will examine the expression of propeptide in vivo following gene delivery to muscle and efficacy of propeptides in treating bone metastatic prostate tumors in a TRAMP-C2Ras tibial implantation model.

Citation Format: Janelle W. Salameh, Charles S. Umbaugh, Marxa L. Figueiredo. Development and validation of propeptide therapeutics for treating bone metastatic prostate cancer [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 LB-260.
Abstract LB-274: Overcoming colorectal cancer resistance to death receptor agonists: Design and delivery of treatment combinations
Authors: Manzari Mandana T. , Li Xinghai , Anderson Grace , Wood Kris , Chilkoti Ashutosh
Published: August 2018

Death Receptor 5 (DR5) is a key upregulated marker in colorectal cancer (CRC), overexpressed in over 90% of patients. There is a dire clinical need for drugs that can deliver targeted cell killing via this receptor. Over the past decade, various DR5 agonists have shown poor clinical efficacy, including both engineered antibodies and TRAIL, the natural ligand for this receptor. Comprehensive studies suggest that there are three major obstacles to success of these agents: 1) potency, 2) delivery, and 3) resistance. If these issues are addressed, DR5 will be an excellent therapeutic target for a variety of cancers, and provide a novel means for substantially advancing the clinical treatment of CRC. To improve delivery and overcome drug resistance, we have developed a platform that harnesses the power of genetic screening and protein engineering to design and deliver efficacious protein drug combinations. To pinpoint the genetic drivers of CRC resistance to a multivalent DR5 agonist (DRA), we used a CRISPR/Cas9 knockout screen. The screen identified genes that confer sensitivity to the DRA in resistant CRC cell lines. Over twenty small molecule drugs targeting pathways and proteins identified from the screens were then tested in combination with the DRA to identify highly synergistic combinations using cytotoxicity assays. Clonogenic, time-to-progression, and cell viability assays showed that pharmacological blockade of XIAP, Bcl-XL, and CDK4/6 strongly enhances antitumor activity of DRA in established human CRC cell lines and patient-derived CRC cells. To address the need for sustained delivery of therapeutic proteins, we developed injectable depots of anti-cancer proteins recombinantly fused to thermally responsive elastin-like polypeptide (ELP) biopolymers. The bioactive DRA-ELP fusions undergo temperature-driven phase transition upon subcutaneous injection in vivo, resulting in the formation of a gel-like depot suitable for sustained drug delivery. A single 30 mg/kg injection of the gel-like DRA-ELP depot induced significant tumor regression in Colo205 mouse xenografts. By addressing both delivery and resistance issues with our protein engineering and genomics platform, we have overcome key obstacles to clinical translation of a pro-apoptotic DRA.

Citation Format: Mandana T. Manzari, Xinghai Li, Grace Anderson, Kris Wood, Ashutosh Chilkoti. Overcoming colorectal cancer resistance to death receptor agonists: Design and delivery of treatment combinations [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 LB-274.
Abstract LB-289: Polarization of adipose-derived mesenchymal stem cells by toll-like receptors stimulation
Authors: Rivera-Cruz Cosette M. , Huff Krista , Figueiredo Marxa L.
Published: August 2018

Mesenchymal stem cells (MSCs) are a potential tool for cell therapy because of their ability to home to injury sites, modulate immune responses, promote tissue repair, among other features. MSCs can also home to tumors, similarly to how they home to injury sites, and exert modulatory functions in the tumor microenvironment. However, the mechanisms by which MSCs affect tumor cells is not well understood, and apparent contradictory effects of MSC-tumor interactions can be found in the literature, claiming that MSCs either promote or repress tumor growth and cell migration. In recent years, it was shown that MSCs derived from bone marrow (BM-MSCs) can be polarized by Toll-like receptor (TLR) signaling into two phenotypes with opposite effects on tumor development. TLR4 signaling induces an immune activating MSC1 population shown to inhibit tumor growth and migration, whereas TLR3 signaling induces an immune suppressive MSC2 population that promotes tumor progression. Adipose-derived mesenchymal stem cells (ASCs) are important constituents of tumor microenvironments. Their ability to migrate into tumors make these cells a potential candidate for delivering immunotherapy against tumor progression. Although the MSC1 and MSC2 duality has not been reported for ASCs, we hypothesized that due to their similarity to bone marrow MSCs, that ASC share the polarization phenotype. To test our hypothesis, we stimulated, or primed, hASCs with TLR3 and TLR4 agonists and evaluated gene expression changes expected to occur, in accordance to the previously described MSC1 and MSC2 phenotypes. Upregulation of IL-8 was observed for hASCs primed towards the MSC1 phenotype. CCL5 and IP10 were upregulated in cells primed towards the MSC2 phenotype. These results suggest that a polarization of hASCs may occur in a similar way as previously shown for BM-MSCs. On the other hand, no changes were observed in the expression of TGFβ, IL-6 and other associated genes, hinting to cell lineage phenotypical differences upon TLR stimulation. In addition, some phenotypical differences have been observed across different donor cells and priming conditions, which could contribute to the immunomodulatory inconsistencies reported in the literature.

Citation Format: Cosette M. Rivera-Cruz, Krista Huff, Marxa L. Figueiredo. Polarization of adipose-derived mesenchymal stem cells by toll-like receptors stimulation [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 LB-289.
Abstract LB-294: Synergy of an anti-HER2 ADC TAK-522 (XMT-1522) in combination with anti-PD1 monoclonal antibody (mAb) in a syngeneic breast cancer model expressing human HER2
Authors: Traore Tary , Khattar Mithun
Published: August 2018

Antibody-drug conjugates (ADCs) are a highly potent class of drugs that specifically target cancer cells expressing a tumor associated antigen (TAA). The ADC TAK-522 (XMT-1522) consists of a novel human IgG1 anti-HER2 monoclonal antibody and a novel, auristatin-based cytotoxic payload (Auristatin F-hydroxypropylamide, AF-HPA). An average DAR (drug-to-antibody ratio) of twelve AF-HPA molecules is achieved via a biodegradable polymer conjugation platform. We have characterized the ability of both the free payload AF-HPA and the ADC TAK-522 to induce immunogenic cell death (ICD) in vitro in multiple cell lines (NCI-N87, HT-29, SKBR3), as measured by cell surface expression of the ICD marker calreticulin (CRT) using microscopy and flow cytometry. CRT, usually contained in the lumen of the endoplasmic reticulum, translocated to the cell surface within a few hours after treatment with AF-HPA or TAK-522. Furthermore, we developed a novel syngeneic breast cancer (4T1) model expressing human HER2 at a relatively low antigen density. Treatment in this poorly immunogenic tumor model with TAK-522 but not Kadcyla showed significant inhibition of tumor growth in vivo. Importantly, a combination of anti-PD1 mAb and TAK-522 therapy substantially and synergistically enhanced the anti-tumor efficacy, resulting in complete responses in some mice. The frequency of complete responders was further increased when the two drugs were sequentially, rather than concurrently, administered such that TAK-522 administration was followed by anti-PD1 mAb therapy. These results suggest an immunological mechanism involving induction of immunogenic cell death by TAK-522, which in turn may activate the adaptive immune system by releasing tumor specific antigens. TAK-522 is currently being tested in a phase-1b clinical trial in patients with advanced breast, lung and gastric cancer expressing HER2. Based on our data, TAK-522 represents a potential candidate for combination therapies with immune checkpoint modulators in patients with poorly immunogenic HER2 expressing tumors.

Citation Format: Tary Traore, Mithun Khattar. Synergy of an anti-HER2 ADC TAK-522 (XMT-1522) in combination with anti-PD1 monoclonal antibody (mAb) in a syngeneic breast cancer model expressing human HER2 [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 LB-294.
Abstract LB-303: Novel peptide is synergistic with checkpoint inhibitor antiCTLA4
Authors: Leuschner Carola , Kern Clifford H.
Published: August 2018

Specific peptides are known to modulate cellular immune responses. Isolates of leukocytes containing tyrosine and glycine peptide sequences have been previously tested in HIV-positive, colon cancer, and other patients, where they were found to be safe and to demonstrate pleiotropic effects on CD4+ T cells, CD8+ T cells, and natural killer cells, as well as increased production of interferon gamma and other cytokines. We hypothesized, therefore, that Tyr-Gly peptides may enhance anti-tumor responses in combination with checkpoint inhibitors through stimulation of the immune system. INT-151 (synthetic Tyr-Gly), originally identified as the active component of a leukocyte isolate, was tested alone and in combination with the checkpoint inhibitor anti-CTLA4 in a murine syngeneic lymphoma model (E.G7-OVA) for anti-tumor effect and survival. Immune profiles in control and treatment groups were assessed for tumors and blood using FACs analysis to detect CD8+ T cells, CD4+ T cells, regulatory T cells (Treg), macrophages, and myeloid derived suppressor cells (MDSC). E.G7-OVA tumor bearing mice with an average tumor volume of 43 mm3, were treated 3x weekly for 17 days with INT-151 or anti-CTLA4 alone or in combination. The treatment group receiving INT-151 plus anti-CTLA4 had the lowest tumor volume (351 ± 38.9 mm3, N=6) compared with those treated with vehicle (876.7 ± 142.9 mm3, p < 0.04, N=6), INT-151 alone (821 ± 52.2 mm3, p = 0.8, N=6), and anti-CTLA4 alone (616 ± 117.4 mm3, p = 0.1, N=6). Tumor growth inhibition was highest in the combination group (60 %) compared to single agents anti-CTLA4 (30 %) and INT-151 (7 %). Immune cell populations increased in tumors were CD8+ T cells, CD4+ T cells and M1 macrophages in the combination group. Regulatory T cells, mMDSC or gMDSC remained unchanged in any of the treatment groups compared to controls. The highest ratio of CD8+T/Treg cells of 8.5±2.1 was found in the combination group (INT-151+anti-CTLA4, p<0.001) compared to 4.4±0.9 for anti-CTLA4 and 2.5±0.5 for vehicle and INT-151 (3.2±1.05). The E.G7-OVA mouse model also demonstrated increased survival on day 40 in groups treated with INT-151 plus anti-CTLA4 (55%, N=9) compared to single agent INT-151 (30 %, N=10), anti-CTLA4 (0%, N=8) and vehicle (0 %, N=10). These data demonstrate synergistic activity of INT-151 in a syngeneic murine lymphoma model. Combining a checkpoint inhibitor with INT-151 could be a safe and promising therapeutic strategy for lymphoma and other malignancies.

Citation Format: Carola Leuschner, Clifford H. Kern. Novel peptide is synergistic with checkpoint inhibitor antiCTLA4 [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 LB-303.
Abstract LB-304: Customizable, in vitro protein glycosylation for antibacterial and anti-cancer vaccines
Authors: Stark Jessica C. , Jaroentomeechai Thapakorn , Dubner Rachel S. , Hsu Karen J. , Glasscock Cameron , DeLisa Matthew P. , Jewett Michael C.
Published: August 2018

Changes in protein glycosylation are important hallmarks of cancer biology, serving as biomarkers for cancer progression and predictors of cancer prognosis. Moreover, tumor-associated carbohydrate antigens (TACAs) provide potential targets for therapeutic and prophylactic interventions. However, development of anti-cancer immunotherapies and vaccines directed against TACAs has been limited due to the significant challenges associated with synthesizing proteins bearing defined glycan structures in vivo. These challenges arise due to the complex, non-templated nature of the glycosylation process and the inability to control glycosylation components at precise ratios in living cells. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked (N-linked) protein glycosylation. The platform is flexible and modular, allowing the decoration of proteins with structurally diverse, yet homogeneous glycans, including the eukaryotic trimannosyl N-glycan (mannose3-N-acetylglucosamine2, Man3GlcNAc2), which forms the core of all tumor-associated N-glycans. We demonstrate the utility of this platform through the production of antibacterial glycoconjugate vaccines directed against the highly infectious pathogenic bacterium Franciscella tularensis. Importantly, we are able to synthesize and glycosylate a variant of E. coli maltose binding protein (MBP) that has been shown to elicit humoral and cell-based immunity to polysaccharide antigens in mice. Our work demonstrates a novel approach for the customizable glycosylation of immunogenic proteins with TACAs in a cell-free system. The ability of the CFGpS platform to synthesize defined glycan structures uniquely enables the production of novel antibacterial and anti-cancer vaccines.

Citation Format: Jessica C. Stark, Thapakorn Jaroentomeechai, Rachel S. Dubner, Karen J. Hsu, Cameron Glasscock, Matthew P. DeLisa, Michael C. Jewett. Customizable, in vitro protein glycosylation for antibacterial and anti-cancer vaccines [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 LB-304.
Abstract LB-328: Influence of radiation on the evolution of orthotopic xenografts initiated from glioblastoma stem-like cells
Authors: McAbee Joseph H. , Rath Barbara H. , Camphausen Kevin A. , Tofilon Philip J.
Published: August 2018

Glioblastoma (GBM) is the most common and malignant primary adult brain cancer with a median survival of 15 months despite treatment with surgical resection followed by chemo-radiotherapy. By examining changes that occur following irradiation of brain tumor xenografts initiated from glioma stem-like cells (GSCs), this study seeks to better understand the impact of radiotherapy on GBM evolution. Therefore, we intracranially implanted CD133+ NSC11 cells, a well-documented GSC line, into nude mice. After 21 days, bioluminescence imaging was performed to confirm the presence of tumor prior to randomization into control and radiation therapy groups (3x5Gy). Following treatment, brain samples were collected at various time points out to morbidity to investigate changes in tumor morphology and histology. Further, tumors from morbid mice (control and 3x5Gy) were collected for viral integration site analysis (VISA). Initial survival analysis demonstrated a significant survival advantage for mice undergoing radiation therapy (+34.2 days) compared to controls. On gross examination of morphology at morbidity, brains bearing irradiated tumors contained tumor tissue that was softer, more adherent to the skull, and more likely to efface olfactory bulb(s) than control tumor brains. H&E staining demonstrated that irradiated tumors were more dense/compact and did not grow as infiltrative as tumors from control mice. These histological changes were followed up with VISA to determine whether certain clones were responsible for this difference. VISA revealed that irradiated tumors were heterogeneously enriched for clones with different integration sites when compared to control tumors and in vitro tumor lines. Heat map analysis of the number of clones present in each sample demonstrated that control tumors harbor fewer clones than in vitro lines and that radiated tumors harbor the fewest clones of all. Initial data also revealed that various doses of radiation (3x5Gy or 10x5Gy) can provide very similar selection pressures to limit the number of clones. Our results demonstrate that radiation, a treatment component for almost all glioblastoma patients, can have wide-ranging effects on the evolution of this dynamic tumor. In particular, the pressures imposed by radiation treatment seem to lead to the selection of a reduced number of clones. This selection may have future implications for tumor evolution and the treatment of recurrent GBM.

Citation Format: Joseph H. McAbee, Barbara H. Rath, Kevin A. Camphausen, Philip J. Tofilon. Influence of radiation on the evolution of orthotopic xenografts initiated from glioblastoma stem-like cells [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 LB-328.
Abstract LB-348: Negative roles of C/EBPα in MDSC-mediated tumor angiogenesis and immune suppression
Authors: Qu Peng , Min Yongfen , Yang Li , Lin Charles
Published: August 2018

Myeloid-derived suppressor cells (MDSCs) are highly increased in peripheral blood of cancer patients, as well as tumor bearing animal models. These cells have long been known to suppress the host immune response to cancer. In the present study, we found that CCAAT/enhancer binding protein alpha (C/EBPα) expression was significantly reduced in MDSCs from tumor-bearing mice compared to non-tumor-bearing hosts. Tumor-conditioned medium down-regulated C/EBPα expression, suggesting tumor secreted factors inhibiting the gene expression. Consistent with the function of C/EBPα in regulating the balance between proliferation and growth arrest in hematopoietic progenitors, the specific deletion of C/EBPα on myeloid lineage resulted in distinctly enhanced the proliferation and expansion of MDSCs, as well as an increase of myeloid progenitors and a decrease of other mature cells. In addition, the deletion of C/EBPα in MDSCs enhanced the pro-angiogenic, immune suppressive and pro-tumorigenic behavior of these cells by upregulating the production of iNOS and arginase, as well as MMP-9 and VEGF. Accordingly, tumors growing in C/EBPα conditional null mice displayed greater MDSC infiltration, increased vascularization and accelerated tumor growth. Taken together, this study reveals dual negative roles of C/EBPα in the expansion as well as pro-angiogenic and immune suppressive functions in MDSCs.

Citation Format: Peng Qu, Yongfen Min, Li Yang, Charles Lin. Negative roles of C/EBPα in MDSC-mediated tumor angiogenesis and immune suppression [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 LB-348.
Abstract LB-363: Characterization of in vitro and in vivo models generated from disseminated tumor cells of patients without manifest metastasis
Authors: Werno Christian , Weidele Kathrin , Treitschke Steffi , Botteron Catherine , Scheitler Sebastian , Polzer Bernhard , Werner-Klein Melanie , Klein Christoph Andreas
Published: August 2018

We recently showed that dissemination of few melanoma cells to the lymph nodes is a quantitative risk factor for death from melanoma. However, the analysis of disseminated tumor cells (DTC) is limited by their extremely low frequency, resulting in a lack of in vitro/in vivo models for mechanistic studies of early systemic disease. Therefore, the objective of this study was to develop protocols to expand single DTC from lymph nodes of melanoma patients to establish better preclinical models for adjuvant cancer therapies. Sentinel lymph nodes from melanoma patients were cut in halves, disaggregated and analyzed for the presence of gp100-positive DTC. The other half was used for diagnostic assessment by histopathology. Single DTC within the lymph node-derived single cell suspension were then propagated under specific sphere forming conditions and expanded by transplantation in immunodeficient mice. DTC based pre-clinical in vitro/in vivo models were then characterized on molecular level and were used for functional analyses. To test drugs on DTC in presence of a human immune system we additionally generated humanized xenograft models. We successfully established cell lines and xenografts from DTCs of 17 melanoma patients. We confirmed the origin of the cells by genomic fingerprint and analyzed the genomic profile of the expanded cells over various culture and animal passages. DTC cell lines were used for functional analysis and for testing the efficacy of targeted therapies. In addition, we set up a novel preclinical mouse model based on expanded melanoma DTC. For this, we established a human immune system in immunodeficient mice, which were then transplanted with melanoma DTC-derived cells. Interestingly, the presence of a human immune system significantly induced tumor formation, as well as dissemination of melanoma cells and improved the response to a targeted therapy compared to mice without a humanized immune system. The development of DTC based pre-clinical in vitro/in vivo models enables mechanistic studies on DTCs and drug tests against the target cells of adjuvant therapies in the presence of human immune cells. These models may help to identify candidate adjuvant therapies targeting DTCs and to understand mechanisms of drug resistance of currently applied targeted therapies.

Citation Format: Christian Werno, Kathrin Weidele, Steffi Treitschke, Catherine Botteron, Sebastian Scheitler, Bernhard Polzer, Melanie Werner-Klein, Christoph Andreas Klein. Characterization of in vitro and in vivo models generated from disseminated tumor cells of patients without manifest metastasis [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 LB-363.
Abstract LB-377: Validation and application of a comprehensive next generation sequencing system for molecular characterization of solid tumor tissues
Authors: Dehghani Mehdi , Li Lei , Brobey Reynolds , Wang Yue , Rosenblatt Kevin P. , Amato Robert J.
Published: August 2018

Identification of somatic molecular alterations in solid tumors and metastases can provide critical information regarding tumor biology and tumor heterogeneity. These alterations can also reveal activated pathways in a patient's disease, enabling personalized treatment assignment. However, the optimal methods and panels remain unknown. The present study was conducted to assess the use of the Oncomine Comprehensive Cancer Panel in identifying molecular alterations in fresh and preserved cancer tissues from a solid tumor registry at The University of Texas Health Science Center at Houston. Results were compared with pathologic findings. The assay was found to be highly specific (>99%) and sensitive (>99%), with low false-positive and false-negative rates for single-nucleotide variants, indels, copy number alterations, and gene fusions. This tumor-agnostic molecular assessment assay holds potential as a straightforward and reliable method to determine molecular alterations in solid tumor samples. Overall, 23 (88%) of these 26 patients had at least one loss-of-function or gain-of-function molecular alteration (80/180 total high-priority alterations), of which 68% (66/80) led to selection of targeted therapy. This next-generation sequencing assay not only detected at least one clinically actionable alteration, but also it can be a great translational research tool. For instance, using this panel we detected EGFRvIII in a significant number of renal cell cancer core biopsies (43%). The biological importance of this alteration in renal cancer growth and progression and its clinical importance as a therapeutic or prognostic marker needs to be investigated further.

Citation Format: Mehdi Dehghani, Lei Li, Reynolds Brobey, Yue Wang, Kevin P. Rosenblatt, Robert J. Amato. Validation and application of a comprehensive next generation sequencing system for molecular characterization of solid tumor tissues [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 LB-377.
Abstract LB-378: Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing
Authors: Cortés-Ciriano Isidro , Lee June-Koo , Xi Ruibin , Jain Dhawal , Jung Youngsook L. , Yang Lixing , Gordenin Dmitry , Klimczak Leszek J. , Zhang Cheng-Zhong , Pellman David S. , Park Peter J.
Published: August 2018

Chromothripsis is a newly discovered mutational phenomenon involving massive, clustered genomic rearrangements that occurs in cancer and other diseases. Recent studies in cancer suggest that chromothripsis may be far more common than initially inferred from low resolution DNA copy number data. Here, we analyze the patterns of chromothripsis across 2,658 tumors spanning 39 cancer types using whole-genome sequencing data. We find that chromothripsis events are pervasive across cancers, with a frequency of >50% in several cancer types. Whereas canonical chromothripsis profiles display oscillations between two copy number states, a considerable fraction of the events involves multiple chromosomes as well as additional structural alterations. In addition to non-homologous end-joining, we detect signatures of replicative processes and templated insertions. Chromothripsis contributes to oncogene amplification as well as to inactivation of genes such as mismatch-repair related genes. These findings show that chromothripsis is a major process driving genome evolution in human cancer.

Citation Format: Isidro Cortés-Ciriano, June-Koo Lee, Ruibin Xi, Dhawal Jain, Youngsook L. Jung, Lixing Yang, Dmitry Gordenin, Leszek J. Klimczak, Cheng-Zhong Zhang, David S. Pellman, Peter J. Park. Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing [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 LB-378.