bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 688: Targeting tumor associated host cells by modifiers of integrated endoplasmic reticulum stress response
Authors: Upcin Berin , Szi-Marton Daniel , Henke Erik , Ergün Süleyman , Aktas Bertal Huseyin
Published: September 2020

Solid tumors are composed of cancer cells, tumor associated host cells (TAHCs) and extracellular matrix components. TAHCs form tumor vasculature, secrete extracellular matrix components, provide protection from host immune-defense, and contribute to therapy resistance. Agents that can abrogate the survival and/or proliferation of both cancer cells and TAHCs would be more potent and less vulnerable to acquired resistance.

Solid tumors are under stress due to host immune surveillance, lagging vascularization, and an unstable nascent vasculature that results in nutrients and oxygen restriction. Tumors utilize adaptive pathways including hypoxia activated signaling, integrated endoplasmic reticulum (ER) stress response (IERSR) signaling, and immune evasion for survival and progression. The IERSR is a double-edged sword that is pro-survival if activated transiently but cause cell death if activated in a sustained manner. In particular, sustained activation by eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation (eIF2α-P) arm of the IERSR causes cell death. eIF2α-P reduces overall protein synthesis while inducing expression of some pro-apoptotic and tumor suppressor genes. Because TAHCs actively synthesize proteins in supporting tumor cells, we hypothesized that specific activators of eIF2α kinases will abrogate tumor survival and/or progression by dual effect on TACHs and cancer cells. We tested this hypothesis by evaluating chemical activators of eIF2α kinase heme regulated inhibitor (HRI) in ex-vivo aortic ring angiogenesis assay and against tumor organoids formed by co-culture of macrophages, fibroblasts, endothelial cells, and human breast or melanoma cancer cells. All HRI activators inhibited angiogenesis in the aortic ring assay (ARA) and had profound effects on tumor angiogenesis and macrophages. At sublethal concentration, HRI activators disrupted tumor vasculature and significantly reduced macrophages in the tumor organoids. These agents also significantly inhibited cell migration as measured by wound healing assay and by cell-migration assay of xCELLigence® RTCA DP instrument. These data indicate that HRI activators can target both tumor and TAHCs in a dose dependent manner and support further development of these agents for cancer therapy.

Citation Format: Berin Upcin, Daniel Szi-Marton, Erik Henke, Süleyman Ergün, Bertal Huseyin Aktas. Targeting tumor associated host cells by modifiers of integrated endoplasmic reticulum stress response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 688.
Abstract 748: The small leucine-rich proteoglycan fibromodulin is a potential, novel serum and urinary biomarker for the detection of prostate cancer
Authors: Reyes Niradiz , Bettin Alfonso , Geliebter Jan
Published: September 2020

Background: Prostate cancer (PCa) is the most common malignant tumor diagnosed in males and the fifth leading cause of cancer deaths worldwide. The impact of this cancer in the population is due, in part, to the lack of satisfactory methods for early diagnosis and accurate prognosis, which results in delayed clinical treatment of patients. Effective therapy exists for early stages, but it is practically incurable in advanced stages. Therefore, new and better molecular biomarkers are urgently needed for the opportune diagnosis and adequate prediction of the clinical course of the disease. We have previously identified the small leucine-rich proteoglycan, fibromodulin, as a novel, potential tissue biomarker for prostate cancer. Fibromodulin is a secreted proteoglycan which can be detected in different body fluids, such as blood, urine, or prostatic secretions. The aim of the present study was to determine whether fibromodulin concentration in serum and/or urine is able to differentiate PCa patients from patients with benign disease.

Methods: Serum and urine samples were collected from a cohort of patients newly diagnosed with PCa (n=44) or with other prostatic disorders (n=46). Samples were also obtained from a group of control healthy individuals within the same age range. The concentration of fibromodulin in the blood and urine samples was measured with a commercial ELISA kit. Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic performance of fibromodulin in serum and urine. This work was approved by the Institutional Review Board of the University of Cartagena.

Results: Both serum and urinary levels of fibromodulin were significantly higher in PCa patients compared to patients with benign disorders and healthy controls (p<0.05). Overall, urinary levels were higher than serum levels for each of the three groups.

Conclusion: Our results support the potential of fibromodulin as a novel serum and urinary biomarker for PCa. Validation of these results requires prospective studies using a larger cohort of patients, as well as multicenter clinical trials, to define the real value of this protein as a biomarker for PCa.

Keywords: Prostate cancer, fibromodulin, biomarkers, proteoglycan.

Citation Format: Niradiz Reyes, Alfonso Bettin, Jan Geliebter. The small leucine-rich proteoglycan fibromodulin is a potential, novel serum and urinary biomarker for the detection of prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 748.
Abstract 810: Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples
Authors: Erdmann Gerrit , Dudys Przemylaw , Arndt Anja , Regenbrecht Christian , Templin Markus , Sachse Christoph
Published: September 2020

Precision medicine's goal of achieving a better response rate by avoiding ineffective therapies has sparked new approaches, including the testing of patient-derived 3d (PD3Ds) tumor cultures for modeling individual patient response, and the use of various molecular pathology techniques for advanced tumor profiling. An intrinsic limitation of well-established genomics methodologies is, however, that they cannot directly assess cell-signaling activity within the tumor cells defined by the phosphorylation status of cellular signaling pathway networks.

Here we present the development of a robust protein profiling strategy utilizing the DigiWest immuno-assay platform, to obtain data on the activation status of key cellular signaling networks implicated in cancer, and on proteins targeted by FDA-approved drugs. This includes targeted cancer therapies for e.g. EGFR, HER2, PI3K, mTOR, ALK and AKT.

Accordingly, we compiled a list of relevant pathway nodes and their phosphorylation sites that yield activity information on RAS/RAF/ERK, PI3K/AKT and mTOR signaling pathways. Based on this, we validated 242 total and phospho antibodies in a pre-set oncoproteomic DigiWest panel that yields information on the activity of all major signaling pathways from the receptor level down to transcription factors, apoptosis and proliferation. This oncoproteomic panel can be utilized for elucidating drug response in pre-clinical cell models, in PD3D organoid models or in clinical tumor samples. Exemplary, we show differential activation states in cancer cell lines representing the most prevalent cancer types and the impact of pathway activation in different models. Finally, we tested this panel in PD3D organoids that were subjected to screening against common targeted therapies.

While the initial results are promising, further work on evaluating how such an oncoproteomic panel profiling might contribute to the rationale for personalized therapy decisions is required.

Disclosure Statement: NMI TT Pharmaservices offers DigiWest protein profiling as a service. CELLphenomics offers PD3D cell cultures and drug testing as a service.

Citation Format: Gerrit Erdmann, Przemylaw Dudys, Anja Arndt, Christian Regenbrecht, Markus Templin, Christoph Sachse. Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 810.
Abstract 823: Gene expression analysis reveals a pitfall in the molecular research of prostate tumors relevant to Gleason score
Authors: Zhang Wensheng , Dong Yan , Zhang Kun
Published: September 2020

Gleason score (GS) is a powerful prognostic factor in prostate cancer (PCa). A GS-7 tumor typically has the primary architectural pattern and secondary prevalent one being graded as 3 and 4 or 4 and 3, respectively. Due to the well-known multifocal occurrence of different patterns, a biological sample from a GS-7 tumor used in a molecular experiment will be uncertain regarding the actually represented pattern if no special attention is given to specimen preparation. In this study, by an integrative analysis of public gene expression datasets for PCa, we demonstrate that such an uncertainty is not uncommon. First, based on a unique dataset (Sowalsky, et.al, Clin. Cancer Res., 2017) of paired Gleason pattern 3 (GP-3) and Gleason pattern 4 (GP- 4) specimens of 13 GS-7 tumors, we identify 303 differentially expressed genes with respect to those two architectural patterns. Then, using the TCGA data, we show that the profile of these genes is a strong signature for distinguishing the GS-6 (3+3) tumors from the GS-8 (4+4) ones (AUC = 0.96, p-value = 9*10E-17), implying that the differences of transcription program between the GS-8 (4+4) and GS-6 (3+3) samples resemble the differences between the GP-3 and GP-4 specimens. We further demonstrate that the co-expression pattern of the signature genes in the subset of the GS-7 samples resembles that present in the aggregate of GS-6 and GS-8 samples. Finally, we verify the findings using the GSE21032 data. Our results suggest that the GS-7 specimens used to generate two frequently-cited genome-wide expression datasets largely are individual GP-3 or GP-4 specimens rather than the “intermediate” specimens of GP-3 and GP-4. This provides molecular evidence for the aforementioned uncertainty of GS-7 tumor samples, indicating a potential pitfall in the existing molecular and translational research of prostate tumors relevant to GS.

Citation Format: Wensheng Zhang, Yan Dong, Kun Zhang. Gene expression analysis reveals a pitfall in the molecular research of prostate tumors relevant to Gleason score [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 823.
Abstract 828: gDR suite: an integrative solution for handling cell line drug response data
Authors: Hafner Marc , Gladki Arkadiusz , Li Jane , Lin Eva , Lun Aaron , Martin Scott , Potocka Natalia , Scigocki Dariusz , Vartanian Steffan , Vogel Jan , Vuong Allison
Published: September 2020

Assessing the response of cancer cell lines to drugs and treatments that affect their growth is the cornerstone of drug discovery and development. On one hand, large screens are performed across many lines and drugs in a semi-automated manner. On the other hand, small-scale studies, for example focused on factors that contribute to sensitivity and resistance, are generally performed in labs with limited automation. Data from these complementary approaches are rarely handled in the same manner: commercial software are available for the former case, whereas experimentalists in the latter case often handle files manually and process data in spreadsheets. Here we propose a suite of computational tools that enable the processing, archiving, and visualization of drug response data from any experiment, thus ensuring reproducibility and implementation of the FAIR principles.

The core of the gDR suite is a database designed to host drug response data from large screens, single-agent treatments, combination experiments, as well as data from complex experimental designs including ligand co-treatments or shRNA. Experimentalists can upload metadata files along with unprocessed output files from plate readers in an interactive web application built with R Shiny. Data normalization and quality control are performed by the software before the data is stored in the database. Data from commercial software or other databases can also be easily pushed into the database through a REST API. To fetch the processed data, we developed an R package as well as another R shiny-based web application. Both tools allow users to efficiently search the database, easily plot the data from the selected experiments, and perform basic analyses. Overall, the gDR suite is a modular software that provides an end-to-end solution for managing drug response data.

At the conference, we will describe our implementation and demonstrate how to use the gDR suite. We hope that this tool will facilitate the handling of drug response data and thus contribute to the quality of published data. In addition, we hope that the community will contribute to the gDR suite by adding new functionalities for either importing, plotting, or analyzing the data.

Citation Format: Marc Hafner, Arkadiusz Gladki, Jane Li, Eva Lin, Aaron Lun, Scott Martin, Natalia Potocka, Dariusz Scigocki, Steffan Vartanian, Jan Vogel, Allison Vuong. gDR suite: an integrative solution for handling cell line drug response data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 828.
Abstract 84: E2F8 tumor suppressive role in Non-Alcoholic Steatohepatitis
Authors: Ochoa-Rios Shaaron L. , Kent Lindsey N. , Leone Gustavo
Published: September 2020

Non-Alcoholic Steatohepatitis (NASH) is the aggressive form of Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD is the accumulation of extra fat in liver cells, most commonly known as fatty liver. Some of the main conditions that distinguish NAFLD from NASH are severe liver inflammation, increased lipid accumulation and fibrosis. NASH is caused by an excess of fat in the liver which is linked to obesity and diet. Diets high in fats and sugars, typical of a Western diet, have been shown to induce NASH. Approximately 25% of NASH cases progress to Hepatocellular Carcinoma (HCC). Currently, there is no approved therapy for NASH and complications due to NASH is estimated to be the main reason for liver transplant. E2F8, a member of a family of transcription factors that are well known for regulating the cell cycle, has been shown to promote hepatic steatosis in a zebrafish model. Previous research from our lab has demonstrated that E2F8 has a tumor-suppressive role and loss of E2F8 leads to HCC in a carcinogen-induced model. Here, we use a mouse model to conditionally delete E2f8 in hepatocytes to sensitize to liver cancer and evaluate the effects of E2F8 on NASH development. Mice were fed with a low-fat diet (LFD), high-fat diet (HFD), choline-deficient high-fat diet (CDHFD) or Western diet (WD). Our results show that control mice in a LFD maintained a normal liver and E2F8 had a tumor-suppressive role for all diets including LF. However, a HFD induced NAFLD while a CDHFD and WD caused NASH by inflammation and fibrosis, respectively. We demonstrate that E2f8 ablation influences collagen deposition, lipid accumulation, and E-cadherin expression. Additionally, experimental mice fed a HFD had an incidence of less than 40% of spontaneous HCC, while experimental mice fed a WD had an incidence of more than 50% of spontaneous HCC; demonstrating that E2F8 tumor-suppressive role is essential to prevent progression to HCC in a NASH model. We hypothesize, that E2F8 has a role in the regulation of liver metabolism. Collectively, our study demonstrates that a WD and E2F8 hepatocyte deletion leads to an increase in lipid and collagen accumulation and HCC occurrence.

Citation Format: Shaaron L. Ochoa-Rios, Lindsey N. Kent, Gustavo Leone. E2F8 tumor suppressive role in Non-Alcoholic Steatohepatitis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 84.
Abstract 896: Enhanced sensitivity detection of replication competent lentivirus by qPCR
Authors: Burke David , Drafahl Kristine , Fjeld Clark , Galderisi Chad , Spittle Cindy
Published: September 2020

Introduction: Chimeric antigen receptor T (CAR T) cells are an important emerging therapy for hematologic malignancies and are currently being investigated for other cancer types as well. CAR-T cells require a retrovirus or lentivirus to introduce the chimeric antigen receptor gene into the activated T cells. Safety measures are put in place by design to eliminate any chance to create replication competent virus. Multiple plasmids carrying specific components of the virus are used to reduce the chances of creating a replication competent lentivirus. Despite these measures, safety concerns have been posed by the FDA regarding use of a lentivirus for this treatment due to the potential for replication competent lentiviruses (RCL) to arise. FDA requires monitoring of both CAR T products as well as patients undergoing therapy. It has proven to be difficult to achieve acceptable detection sensitivity of the VSV-G sequence in a background of gDNA due to the high level of homology to many areas in gDNA.

Methods: We have developed a new rapid and sensitive qPCR assay targeting the vesicular stomatitis virus G glycoprotein (VSV-G), an envelope protein on lentiviral vectors. Incorporation of the envelope gene would be required to generate a replication competent virus. The intended use of the validated assay is to detect and monitor for RCL presence in human blood samples collected during clinical monitoring. A validated reference assay targeting RPPH1 will be used to quantify the amount of amplifiable genomic DNA in each extracted DNA sample. The size of the amplicons are 66 and 87 base pairs for VSV-G and RPPH1 respectively. The VSV-G assay test includes one positive and one negative control per assay run. The VSV-G positive control is composed of plasmid containing the VSV-G target sequence spiked into a background of VSV-G negative human gDNA. The negative control is composed of negative gDNA without VSV-G plasmid.

Results: Improved sensitivity without non-specific amplification was achieved by optimized primer and probe selection. Additionally sensitivity and specificity were enhanced by employing a step-down PCR protocol whereby the annealing and extension temperature is lowered in stepwise fashion over the first five cycles of PCR. Genomic DNA inputs up to 200ng per reaction showed high efficiency of amplification from 10 to 1e6 copies target sequence per reaction.

Conclusion: This assay will provide a rapid and sensitive method for safety monitoring of patients undergoing CAR-T therapy with therapeutic cells generated using lentiviral vectors.

Citation Format: David Burke, Kristine Drafahl, Clark Fjeld, Chad Galderisi, Cindy Spittle. Enhanced sensitivity detection of replication competent lentivirus by qPCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 896.
Abstract 909: The role of checkpoints in Rituximab-mediated NK cell activation
Authors: Wang Zhaoming , Weiner George J.
Published: September 2020

Purpose: Despite the remarkable clinical success of anti-CD20 monoclonal antibody (mAb) therapy, the mechanisms of action by which such mAb mediate their anti-tumor effects are not completely understood. This includes a need to better understand the role of various NK cell subtypes and checkpoint molecules in anti-CD20 mAb therapy.

Experimental procedures:

Raji B cell lymphoma cells and peripheral blood mononuclear cells (PBMCs) from normal donors (n=21) were co-cultured with rituximab (RTX) or trastuzumab as a control mAb for up to 15 days. Analysis included assessment of proliferation, remaining viable cells and phenotype of various cell types.

Results:

As expected, RTX was superior to trastuzumab in eliminating Raji cells. RTX also induced proliferation of NK cells and a shift of NK cells from a CD56dim to CD56bright population. Both NK cell proliferation and the increase in the CD56bright population occurred at day 5-7 when elimination of Raji cells was largely complete. Addback of additional Raji cells at day 5 delayed proliferation of NK cells and the shift from a CD56dim to CD56bright population suggesting this shift occurs once activation of the NK cell via Fcγ;;R is complete. Evaluation of checkpoint molecule expression by NK cells demonstrated RTX, in the presence of Raji target cells, upregulates NK cell expression of TIGIT and TIM3, but not PD1, CTLA-4, or LAG-3. This upregulation was maintained for the full duration of incubation (up to 15 days). The level of expression of TIGIT and TIM3 on NK cells induced by RTX was dose dependent and peaked at 1-5 ug/ml. NK cells induced by RTX to express TIGIT or TIM3 degranulated (CD107a), produced cytokines (IFNγ, TNFα) and expressed activation markers (CD69 and CD25) to a greater degree than did TIGIT or TIM3 negative NK cells. Anti- TIGIT mAb enhanced CD107a expression and TNFα production by RTX-activated NK cells.

Conclusion:

NK cells proliferate and shift from CD56dim to CD56bright population after being activated and eliminating RTX-coated target cells.

TIGIT and TIM3 are biomarkers for RTX-mediated NK cell activation.

Blocking TIGIT can enhance RTX-mediated NK cell degranulation and cytokine production.

Citation Format: Zhaoming Wang, George J. Weiner. The role of checkpoints in Rituximab-mediated NK cell activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 909.
Abstract 923: Pulsatile MEK inhibition improves anti-tumor immunity and T cell function in Kras mutant lung cancer
Authors: Choi Hyejin , Deng Jiehui , Li Shuai , Silk Tarik , Dong Lauren , Brea Elliott J. , Houghton Sean , Redmond David , Zhong Hong , Boiarsky Jonathan , Akbay Esra A. , Smith Paul D. , Merghoub Taha , Wong Kwok-Kin , Wolchok Jedd D.
Published: September 2020

KRAS is one of the most commonly identified driver oncogenes in non-small cell lung cancer (NSCLC) and is frequently associated with disease refractory to currently available modalities of treatment. Targeted therapy to inhibit MEK signaling has shown promising tumor growth control in preclinical models, but is followed by quick rebound in tumor growth. Recently, immune checkpoint blockade has demonstrated clinical activity by dis-inhibiting T cells suppressed in the tumor microenvironment. Thus, we sought to identify the most effective therapy for the treatment of KRAS mutant NSCLC patients by targeting cancer cells and activating immune infiltrating cell populations concurrently. To achieve this, we focused on studying the impact of pulsatile MEK inhibition on the immune microenvironment utilizing Kras mutant lung cancer cell lines and Kras mutant transgenic lung cancer mouse models. We found that pulsatile MEK inhibition maintained T cell activation better than continuous treatment ex vivo and in vivo. Treatment with two different MEK inhibitors (selumetinib and trametinib) each resulted in higher CTLA-4 and PD-1 expression in T cells using pulsatile treatment, compared to continuous treatment ex vivo. In addition, the pulsatile schedule showed superior anti-tumor effects and delayed drug resistance compared to continuous administration in vivo. Further, a combination of pulsatile MEK inhibitor treatment and CTLA-4 blockade resulted in the most prolonged survival of Kras tumor-bearing mice. Similar experiments in immune-deficient mice confirmed that this prolonged survival is conferred by adaptive immunity. Our findings set the foundation for a combinatorial therapeutic strategy using pulsatile targeted therapy together with immune checkpoint blockade in patients to optimally enhance tumor apoptosis and promote long-term immune response simultaneously. We are now continuing our studies using a KRAS G12C inhibitor to define its immune modulating properties and will then rationally combine with immune based therapy.

Citation Format: Hyejin Choi, Jiehui Deng, Shuai Li, Tarik Silk, Lauren Dong, Elliott J. Brea, Sean Houghton, David Redmond, Hong Zhong, Jonathan Boiarsky, Esra A. Akbay, Paul D. Smith, Taha Merghoub, Kwok-Kin Wong, Jedd D. Wolchok. Pulsatile MEK inhibition improves anti-tumor immunity and T cell function in Kras mutant lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 923.
Abstract 93: AMPK alpha1 and AMPK alpha 2 control TNBC proliferation through cell cycle regulation
Authors: Johnson Jeremy , Richahou Piotr , Evers B. Mark
Published: September 2020

Introduction. Triple negative breast cancer (TNBC) is very aggressive, has a higher metastatic rate than other breast cancer subtypes, and lacks traditional targetable factors. AMPK-activated protein kinase (AMPK) is an important energy regulator within the cell that also impacts cancer progression. AMPK's catalytic subunit is composed of α1 and α2 isoforms, but little is known about their individual roles in TNBC. The purpose of this study was to establish the specific function of each isoform in TNBC with respect to expression, cell proliferation, cell cycle progression, and metabolism.

Methods. (i) Immunohistochemistry, immunofluorescence, and immunoblotting were used to determine the expression and localization of AMPKα1 and AMPKα2 in TNBC PDX samples or cell lines. (ii) To study the role of each isoform individually, TNBC cells were transfected with siRNA to NTC, AMPKα1, or AMPKα2 (50 nM) and were either harvested for immunoblotting or subseeded for cell cycle analysis, proliferation assays, or Seahorse analysis.

Results. (i) AMPKα1 expression was localized in the cytoplasm of all 9 TNBC PDX cell lines, whereas AMPKα2 was expressed predominantly in the nucleus in 6 of the 9 of TNBC samples. IF analysis of MDA-MB-231 cells confirmed expression findings in the PDX cell lines with AMPKα1 expression noted in the cytoplasm and AMPKα2 expression in both the cytoplasm and nucleus. (ii) siRNA knockdown of AMPKα1 or AMPKα2 induced G1 cell cycle arrest and decreased proliferation of MDA-MB-231 cells. Moreover, knockdown of AMPKα1 decreased levels of cyclin D1 in MDA-MB-231, MDA-MB-468, and BT-20 cells. siRNA knockdown of AMPKα1, but not AMPKα2, led to decreased basal and compensatory glycolysis in MDA-MB-231 cells.

Conclusions. Our findings indicate that both AMPKα1 and AMPKα2 can impact cell cycle progression and proliferation in TNBC; both isoforms promote progression through the cell cycle, thus supporting the proliferative capacity of TNBC. Importantly, AMPKα1 may also affect TNBC proliferation by increasing energy availability through glycolysis.

Citation Format: Jeremy Johnson, Piotr Richahou, B. Mark Evers. AMPK alpha1 and AMPK alpha 2 control TNBC proliferation through cell cycle regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 93.
Abstract 931: Assessment of tumor mutational burden using a multiplex amplicon-based NGS target enrichment approach
Authors: LI Chenyu
Published: September 2020

Tumor mutational burden (TMB) is gaining significant importance in cancer therapeutics with its correlation to patient response to checkpoint inhibitor-base immunotherapy. A whole exome sequencing approach is traditionally used to assess TMB, but this approach is expensive and time consuming, requiring high sample inputs and complex workflows. Alternatively, a targeted enrichment strategy using multiplexed PCR can provide better coverage of regions of interest and reduce costs. But increasing the multiplexity of PCR, as required for tumor profiling, can lead to problems with non-specific interactions, poor uniformity, and high bias. In this study, we present a multiplex PCR-based strategy for targeting hundreds of genes for tumor profiling applications and assessment of TMB.

Paragon Genomics designed a 500-gene TMB panel which uses about 27,000 pairs of primers for target enrichment via multiplex PCR. The amplicons were designed to target short segments of about 113 bp on average to allow for target amplification from challenging fragmented samples such as FFPE DNA and cfDNA. The 27,000 pairs of PCR primers cover a total of 1.6 Mb (1.2 Mb of coding sequence). Paragon Genomics' CleanPlex® technology enables a streamlined library preparation workflow which includes a multiplex PCR step with the targeted primers, a subsequent cleanup step to remove by-products, and a final PCR step to add sequencer flow cell adapters and sample indexes.

The 500-gene TMB panel exhibits on-target rates of ~96% and uniformity at 0.2X mean of ~94%. Little to no GC bias or amplicon length bias is observed despite the highly multiplexed nature of this panel. At an average read depth of 3000 paired-end reads, 97% of the expected mutations with allele frequencies ≥ 5% were detected. Lastly, Paragon Genomics utilized an internally developed variant-calling algorithm to calculate the TMB values from sequenced libraries prepared from tumor cell line DNA. The advanced design of this panel along with the CleanPlex technology enables the rapid interrogation of these genes and of the TMB value in a robust assay.

Citation Format: Chenyu LI. Assessment of tumor mutational burden using a multiplex amplicon-based NGS target enrichment approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 931.
Abstract 933: Pre-clinical characterization of TIM-3 antibody NB002
Authors: Dong Xin , Dong Jannie , Ma Jun , Zhao Lianqi , Wang Haojie , Wang Binbin , Gao Qian , Pan Hui , Wang Dong , Shao Liegang
Published: September 2020

Targeting immune checkpoint molecules PD1 and CTLA-4 by activating the immune system have improved the lives of many cancer patients. However, this type of immunotherapy is not effective for all patients, suggesting that there are additional mechanisms of immune evasion. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) has been reported to be expressed on exhausted T and NK cells within the tumor microenvironment. The data in many preclinical models suggest that targeting TIM-3 pathway might provide an additional immune modulation of this anti-tumor axis in cancer patients. We have developed a novel monoclonal antibody NB002 against TIM-3,which forms a high-affinity interaction with a unique epitope. NB002 can enhance IFN-gamma secretion and proliferation of these exhausted T cells that has increased TIM-3 expression. Moreover, TIM-3 is constitutively expressed on NK cells. TIM-3 blockade could enhance in vitro cytotoxic activity of NK cells. Altogether, our data shows that NB002 is capable of targeting exhausted T cells and NK cells resulting activation of antitumor immunity. Further preclinical assessment in vivo and in vitro for solid tumors will support clinical development of NB002.

Citation Format: Xin Dong, Jannie Dong, Jun Ma, Lianqi Zhao, Haojie Wang, Binbin Wang, Qian Gao, Hui Pan, Dong Wang, Liegang Shao. Pre-clinical characterization of TIM-3 antibody NB002 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 933.
Abstract 935: Optimization of checkpoint inhibitor-TKI combinations in renal cell carcinoma using an ex vivo 3D tumor organoid model of fresh patient tissue with intact TME
Authors: Ehrhart Jared C. , Pabón Mibel M. , Agrawal Vijayendra , Pastoor Tina , Kreahling Jenny M. , Altiok Soner
Published: September 2020

Introduction: Renal Cell Carcinoma (RCC) is highly resistant to systemic chemotherapy. Different tyrosine kinase inhibitors (TKIs), such as sunitinib, sorafenib and axitinib have been introduced in the treatment of RCC with improved outcomes in the subsets of RCC patients. Tumor microenvironment (TME) plays a critical role in response to TKIs as well as to immuno-oncology drugs including the PD1 inhibitor nivolumab. Given the heterogeneity of the TME in each patient's tumor, optimization of combination treatments is highly desirable. Here, we employed an ex vivo 3D-tumor organoid model with unaltered TME to identify the most effective TKI-nivolumab combinations in individual RCC tumors and further explored the impact on both tumor cell viability and immune cell activation.

Materials and Methods: All RCC tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D organoids measuring 150 micron in size were treated with sunitinib, sorafenib, axitinib alone or in combination with nivolumab ex vivo. Treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages, cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Multiplex cytokine assays were performed to assess drug-induced changes in cytokine release. High content confocal analysis was used to quantify tumor cell killing.

Results: We showed that TKI and nivolumab treatments alone had limited immune cell activation and tumor cell killing efficacy in RCC while their combination showed significant variations in T-cell activation by flow cytometry analysis and tumor cell killing by high content confocal imaging among different patient samples. We correlated tumor response to ex vivo treatments with tumor characteristics and further characterized the impact of treatment on TME in each tumor sample in an attempt to identify markers associated with drug responsiveness.

Conclusion: In this comprehensive study we used a physiologically relevant 3D tumor organoid model to demonstrate the impact of the heterogeneity of TME on TKI/nivolumab treatments in renal cell carcinoma. This approach can be used to identify the most effective drug and drug combinations in RCC and may improve personalized immunotherapy for individual patients in clinical studies.

Citation Format: Jared C. Ehrhart, Mibel M. Pabón, Vijayendra Agrawal, Tina Pastoor, Jenny M. Kreahling, Soner Altiok. Optimization of checkpoint inhibitor-TKI combinations in renal cell carcinoma using an ex vivo 3D tumor organoid model of fresh patient tissue with intact TME [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 935.
Abstract 939: Evaluating anti-PD-1/4-1BB combination therapy: In vivo efficacy with humanized double knock-in mice
Authors: Gan Tian , Zhou Xiaofei , Hu Yuting , Shang Chengzhang , Ng Gary
Published: September 2020

The transmembrane protein receptor 4-1BB is a member of the tumor necrosis factor (TNF) receptor super family and is involved in the co-stimulation of immune cells. 4-1BB is expressed in activated CD4 helper T cells, B cells, natural killer cells, natural killer T cells, dendritic cells, and activated endothelium. Most regulatory T cells (Tregs) also express 4-1BB, but it remains unclear that whether agonistic antibody treatment exerts pro- or anti-suppressive effect on these cells. 4-1BB enhances tumor rejection because it is upregulated on T cells following activation as its engagement increases T cell proliferation and pro-inflammatory cytokine production. Despite of the toxicity of 4-1BB-targeting drugs associated with overt immune activation, new therapeutic modalities using 4-1BB targeting aptamers, as well as combination therapies with other immuno-modulatory and traditional anti-cancer treatments, have revived excitement for the use of 4-1BB agonists in the clinic.

Biocytogen has developed a double humanized B-hPD-1/h4-1BB mouse model to unlock the tremendous potential of combination therapy involving 4-1BB agonists and anti-PD-1 antibodies. In such model, mouse PD-1 gene exon 2, and 4-1BB gene exons 2-7 were replaced with their human counterparts. Murine colon cancer MC38 cells, humanized PD-L1 in MC38 cells, humanized PD-L1 in murine melanoma B16F10 cells, and murine lymphoma EL4 cells were subcutaneously implanted into homozygous B-hPD-1/h4-1BB mice. Combination treatment of Urelumab (anti-4-1BB) and Pembrolizumab (anti-PD-1) has shown enhanced inhibitory effects compared to single antibody treatments using the B-hPD-1/h4-1BB mice models. Based on these findings, it could be concluded that the B-hPD-1/h4-1BB double-humanized mouse model is a validated tool for in vivo evaluation of hPD-1 and h4-1BB antibody combination therapy.

Citation Format: Tian Gan, Xiaofei Zhou, Yuting Hu, Chengzhang Shang, Gary Ng. Evaluating anti-PD-1/4-1BB combination therapy: In vivo efficacy with humanized double knock-in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 939.
Abstract 954: Adverse immunomodulatory effects of chemotherapy on myeloid-derived suppressor cell compromise immune-checkpoint blockade efficacy
Authors: Kwong Tsz Tung , Wong Chi Hang , Zhou Jing Ying , Cheng Alfred Sze Lok , Chan Anthony Wing Hung , Chan Stephen Lam
Published: September 2020

Introduction: Intrinsic immunologic composition in liver tumor microenvironment (TME) may play a role in the heterogenous response towards immune-checkpoint blockade in hepatocellular carcinoma (HCC). In some cancers, it has been shown that cytotoxic chemotherapy is synergistic with checkpoint inhibitors. In this study, we evaluated the efficacy and immunomodulation of 5-FU and anti-PD-L1 in orthotopic liver cancer mouse model.

Method: The orthotopic mouse model was established with the murine HCC cell line RIL-175 through intrahepatic injection. The tumor-bearing mice was then treated with 5-FU (i.p.) 3 times per week at 20mg/kg. Anti-PD-L1(10mg/kg) was delivered (i.p.) every 5 days. Tumor growth was reflected by the luciferase intensity via in vivo imaging. Upon sacrifice, the tumor tissue, liver, spleen and blood sample were harvested, followed by subsequent immune profiling analysis using flow cytometry.

Results: Based on the tumor growth rate and endpoint tumor weight, anti-PD-L1 monotherapy induced a significant tumor reduction whereas 5-FU, either alone or combined with anti-PD-L1, did not have therapeutic effect as compared to vehicle control. Proportions of immunosurveillance cells including natural killer (NK) cells, CD8+ T cells and CD4+ T cells were drastically increased in the tumor site after anti-PD-L1 single treatment, but reduced in 5-FU-treated tumor. To depict the underlying regulation by chemotherapy, immunosuppressive components were also examined. Accumulation in myeloid cells, particularly CD11b+Gr-1+Ly6G+Ly6Cint polymorphonuclear myeloid-derived suppressor cells (P-MDSCs) were found to be a contributing factor to lymphocytes depletion when treated with 5-FU, hence counteracting the anti-tumor activity by PD-L1 blockade.

Conclusion: In summary, our data indicate that chemotherapeutics may impose immunosuppressive modulation in HCC TME and latently alter the therapeutic outcome against immune-checkpoint blockade. This study suggests that combination of chemotherapy with checkpoint inhibitors may not be a feasible approach for HCC. Acknowledgement: The study is supported by the Terry Fox Foundation.

Key Words: Tumor microenvironment (TME), Hepatocellular carcinoma, Chemotherapy, myeloid-derived suppressor cell (MDSC), anti-PD-L1, immunotherapy

Citation Format: Tsz Tung Kwong, Chi Hang Wong, Jing Ying Zhou, Alfred Sze Lok Cheng, Anthony Wing Hung Chan, Stephen Lam Chan. Adverse immunomodulatory effects of chemotherapy on myeloid-derived suppressor cell compromise immune-checkpoint blockade efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 954.
Abstract 963: Metabolic signatures of chemotherapy damage in mature T cells that lead to poor proliferation
Authors: Das Rajat K. , Barrett David M.
Published: September 2020

We have previously described progressive T cell dysfunction (characterized by poor response to ex vivo stimulation) with cumulative chemotherapy exposure in pediatric patients. Chemotherapy appears to sensitize surviving T cells to activation induced cell death (AICD) with hallmarks of disordered metabolism, though the mechanism for each agent and the correct antidote varies. We used an in vitro model of chemotherapy exposure to identify signatures of damage in surviving T cells. T cells from healthy donors were exposed in vitro to doxorubicin, cytarabine and 4-hydroperoxycyclophosphamide (4-HPCP) and allowed to recover to assess the surviving cells as a model of the kinds of cells that are collected from pediatric patients for future adoptive cell therapies. T cells were assessed via the Agilent Seahorse platform for metabolic parameters including oxygen consumption (OCR), spare respiratory capacity (SRC) and glycolysis via extracellular acidification rate (ECAR). All classes of chemotherapy reduce OCR, whereas cytarabine reduces SRC. Doxorubicin increases ECAR and basal glycolysis. We analyzed the T cells via metabolites detected by mass spectrometry, which identified disordered glycolysis in doxorubicin treated cells, evidence of reactive oxygen species/glutathione in 4-HPCP and disordered polyamine synthesis with cytarabine. All classes inhibited T cell proliferation in response to CD3/28 stimulation, primarily due to AICD. We targeted each metabolic deficit with agents designed to support the metabolic pathways identified (2-deoxyglucose for doxorubicin, n-acetylcysteine for 4-HPCP and polyamine supplementation for cytarabine). Each agent was able to improve the proliferative response specific to the deficit, but there was no rescue across classes. We manufactured chimeric antigen receptor T cells (CART) from chemotherapy exposed T cells with and without rescue, and demonstrated improved cell recovery but not improved in vitro killing. Autologous T cells for adoptive cell therapy are currently collected after chemotherapy cycles in children, which may impact their suitability for CAR manufacture which requires ex vivo stimulation. Here we demonstrate each class of chemotherapy increases the susceptibility to AICD with a distinct metabolic hallmark in surviving cells. These metabolic pathways may be specifically targeted to reduce AICD during CAR manufacture, and thus potentially increase the yield from these patients.

Citation Format: Rajat K. Das, David M. Barrett. Metabolic signatures of chemotherapy damage in mature T cells that lead to poor proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 963.
Abstract 974: Immune modulation following combination of TAK-164 with checkpoint inhibitors
Authors: Appleman Victoria A. , Smith Michael D. , Ganno Michelle , Hatten Tiquella , Borland Maria , Abu-Yousif Adnan O.
Published: September 2020

TAK-164 is an antibody drug conjugate comprised of a full length, fully human IgG1 monoclonal antibody (mAb) directed towards the human extra-cellular domain of guanylyl cyclase C (GCC). The mAb is conjugated using ImmunoGen's peptide-linked indolinobenzodiazepine DNA alkylator DGN549. Previously presented preclinical studies using TAK-164 have demonstrated single agent activity of TAK-164 in a panel of patient derived xenograft models of colorectal cancer grown in immunodeficient mice. With emerging evidence demonstrating the immunostimulatory activity of cytotoxic antibody drug conjugates we sought to explore the potential of TAK-164 in immune competent mice. A CT26 model was engineered to express human GCC, allowing us to test the ability of TAK-164 to modulate the tumor microenvironment, thereby potentiating the activity of checkpoint inhibitors. huGCC-CT26 tumor cells were implanted in the right flank of 6-8 week old BALB/c female mice and randomized when tumors were between 50-150mm3 and treated with a single intravenous administration of TAK-164 alone or in combination with checkpoint inhibition administered Intraperitoneally biweekly for 2 weeks and monitored for tumor growth. To understand if responses observed were driven by a response capable of inducing immunological memory we separated mice with complete responses and rechallenged with CT26 tumor cells on the opposite flank. While memory was observed in the mice that achieved complete response in the single agent groups, both frequency of complete response and tumor rejection was increased in groups treated with a combination of TAK-164 and immune checkpoint inhibition. We performed immunophenotyping studies which revealed broad immunomodulation of lymphoid and myeloid cells by TAK-164 and combination regimens. Collectively these data support further interrogation of immune checkpoint inhibition in combination with TAK-164.

Citation Format: Victoria A. Appleman, Michael D. Smith, Michelle Ganno, Tiquella Hatten, Maria Borland, Adnan O. Abu-Yousif. Immune modulation following combination of TAK-164 with checkpoint inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 974.