bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4690: ARD1-mediated NRF2 acetylation promotes human colon cancer cell proliferation
Authors: Fang Xizhu , Lee Yeon-Hwa , Zheng Jie , Kim Seong Hoon , Jang Jeong-Hoon , Kim Do-Hee , Surh Young-Joon
Published: September 2020

The transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2) is regarded as one of the main orchestrators of the cellular antioxidant and carcinogen-detoxifying response. NRF2 plays a pivotal role in protecting normal cells from carcinogenic insults, but it may also accelerate the proliferation and progression of cancer cells. Abnormally elevated expression or activation of NRF2 has been attributed to mutation, especially in the sequences involved in interaction with its negative regulator KEAP1. However, other mechanisms responsible for NRF2 overactivation/overexpression are less well understood. Arrest defective (ARD1), an N-terminal acetyltransferase that catalyzes N-terminal acetylation of target proteins, has lysine acetyltransferase activity and is involved in mediating various (patho)physiological processes, such as proliferation, apoptosis, autophagy, and differentiation. Aberrant overexpression of ARD1 is correlated with metastasis and poor prognosis in several types of cancer, suggesting that it may act as a tumor promoter. In the present study, we found that NRF2, acteyl-NRF2, and ARD1 were highly expressed in some human colon cancer cell lines (HCT-116, HCT-15, DLD1) and tissues obtained from colorectal cancer patients. Furthermore, overexpression of NRF2 and ARD1 was also verified by quantitative immunofluorescence analysis of colorectal tissue microarray. We noticed that silencing of NRF2 and ARD1 individually in human colon cancer cells (HCT-116) led to the decreases in viability, anchorage-independent growth, and migration capacity of HCT-116 cells. While there was no change in the protein level of ARD1 when NRF2 expression was knock down by siRNA, protein expression of Nrf2 and acteyl-NRF2 was markedly reduced by silencing of the ARD1 gene. This prompted us to explore the association between ARD1 and NRF2 in the human colon cancer cell proliferation and progression. Co-immunoprecipitation and immunocytochemical analyses showed that two proteins colocalized in the cytosol, and ARD1 directly bound to NRF2. Taken together, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer cells by acetylating and stabilizing this transcription factor. This study was supported by the Global Core Research Center (GCRC) Grant (No. 2011-003-0001) from the National Research Foundation (NRF) of Republic of Korea.

Citation Format: Xizhu Fang, Yeon-Hwa Lee, Jie Zheng, Seong Hoon Kim, Jeong-Hoon Jang, Do-Hee Kim, Young-Joon Surh. ARD1-mediated NRF2 acetylation promotes human colon cancer cell 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 4690.
Abstract 4709: Loss of GPSM2 promotes the metastasis of non-small cell lung cancer by inducing the expression of Snail
Authors: Liu Bofang , Deng Mingming , Zhang Jingdong , Li Feifei , Han Weidong , Pan Hongming
Published: September 2020

Background/Aims: Non-small cell lung cancer (NSCLC) is the leading global cause of cancer-related death. Due to the lack of reliable diagnostic or prognostic biomarkers, the prognosis of NSCLC remains poor. Consequently, there is an urgent need to explore the mechanisms underlying this condition in order to identify effective biomarkers. G protein signaling modulator 2 (GPSM2) is widely recognized as a determinant of mitotic spindle orientation. However, its role in cancer, especially NSCLC, remains uncertain.

Methods: Tissue microarray (TMA) were used to compare differences in GPSM2 expression, and its relationship with clinical characteristics and prognosis. Wound healing assay, transwell assay and lung metastatic mice model were used to explore the biological function of GPSM2 on NSCLC cells in vitro and in vivo. Western blot and immunohistochemistry were conducted to investigate the potential pathways of GPSM2.

Results: In this study, we found that GPSM2 was downregulated in NSCLC tissues and was correlated with a poor prognosis,and the expression status of GPSM2 was a favorable independent prognostic factor(HR = 0.222; 95% CI = 0.052 - 0.959; P = 0.044). Furthermore, the knockdown of GPSM2 promoted NSCLC cell metastasis in vitro and in vivo and accelerated the process of epithelial-to-mesenchymal transition (EMT). Mechanistically, we demonstrated that silencing GPSM2 induced cell metastasis and EMT via the ERK/GSK-3β/Snail pathway.

Conclusion: These results confirm that GPSM2 plays an important role in NSCLC. Moreover, GPSM2, as an independent prognostic factor, may be a potential prognostic biomarker and drug target for NSCLC.

Citation Format: Bofang Liu, Mingming Deng, Jingdong Zhang, Feifei Li, Weidong Han, Hongming Pan. Loss of GPSM2 promotes the metastasis of non-small cell lung cancer by inducing the expression of Snail [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 4709.
Abstract 4849: YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood cancer
Authors: Yoo Jakyung , Lee Jun Hee , Lee Mi Jung , Lee Kang Woo , Min Ji Eun , Kim Jinhwan , Min Ki-Nam , Jeon Da-Hye , Lim Dae Seong , Lee Kwang-Ok
Published: September 2020

Background: Cyclin dependent kinase 7 (CDK7) has been recognized as one of potential therapeutic targets in Myc-driven cancers. Previously, we synthesized selective irreversible CDK7 inhibitor and we demonstrated a significant inhibition of the proliferation of TNBC and HCC cells by down-regulation of c-Myc and MCL-1. We further report YPN-005 of the preclinical data with biological characterization.

Methods: Antiproliferative activity of YPN-005 was measured in solid tumor cell lines with Myc amplification and in hematologic cancer cells with MCL-1overexpression. To investigate the mechanism of action of YPN-005, the expression levels of phospho-RNA polymerase II, phospho-CDK2 and -CDK4, BClXL, cleaved-PARP, c-Myc and MCL-1 were analyzed by Western Blot analyses. CDK7 occupancy was determined by using a biotinylated small molecule probe following incubating of TNBC, HCC, AML cells and hPBMC with YPN-005. The therapeutic efficacy of YPN005 was evaluated in TNBC and HCC xenograft mouse model.

Results: YPN-005 treatment induced apoptosis and cell cycle arrest. Selectivity of YPN-005 on CDK7 was confirmed in the 468 kinases panel assay. YPN-005 significantly inhibited the proliferation of TNBC, ER+ breast cancer, HCC, ovarian cancer, small cell lung cancer (SCLC), pancreas, prostate and various types of blood cancer cells and IC50's were determined in 1-20 nM range. Inhibition of cell proliferation was accompanied by down-regulation of RNA PolII phosphorylation, c-Myc and MCL-1 expression. Washout assay by Western Blot demonstrated that YPN-005 irreversibly blocked RNA PolII phosphorylation. In addition, YPN-005 downregulated the adaptive immune checkpoint PD-L1 by the depletion of c-Myc in TNBCs. CDK7 occupancy over 90% was observed in TNBC, HCC, and AML cells with 100nM of YPN-005. In vivo xenograft models of TNBC and HCC showed that daily oral administration of YPN-005 for 21 days (once, and 10~20mg/kg) efficiently inhibited the growth of tumor. All the mice survived and there was no significant changes in their hematologic profiles.

Conclusion: Our findings suggest that YPN-005 is an orally available, potent, and selective CDK7 inhibitor which can be used for the treatment of Myc and MCL-1 overexpressing cancer patients. These data support the rationale for advancing YPN-005 towards IND-enabling studies.

Citation Format: Jakyung Yoo, Jun Hee Lee, Mi Jung Lee, Kang Woo Lee, Ji Eun Min, Jinhwan Kim, Ki-Nam Min, Da-Hye Jeon, Dae Seong Lim, Kwang-Ok Lee. YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood 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 4849.
Abstract 4855: Development of highly selective CDK7 inhibitor Q901 for solid tumors
Authors: Yu Donghoon , Jeon Yeejin , Park Dongsik , Seo Mooyoung , Ahn Wongyun , Kim Jaeseung , Park Choa , Jeong Euna , Yoon Sukjoon , Nam Kiyean
Published: September 2020

Cyclin-dependent kinase 7 (CDK7) a member of the cyclin-dependent kinase family, forms a functional CDK-activating kinase (CAK) with cyclin H and MAT1, which regulates the cell cycle by phosphorylating other CDKs. CDK7 is also a component of transcription factor II H (TFIIH) that regulates transcriptional initiation and elongation through the phosphorylation of the RNA polymerase II C-terminal domain (CTD). Inhibition of CDK7 has been reported as a potential cancer therapeutic by addressing aberrant cell cycle regulation and transcription, as these abnormalities are considered main factors for tumor progression in specific types of cancer. Q901 is a novel highly selective CDK7 inhibitor in IND enabling development stage. Q901 shows extreme selectivity by singular inhibition of CDK7 among tested kinome panel. Q901 triggers G1 cell cycle arrest in specific cancer models in vitro and in vivo. Good correlation has been demonstrated between Q901 tumor growth inhibition and target engagement of a pharmacodynamics marker in treated models, suggesting CDK7 is a main driver of tumor progression in the selected models. The Q901 data supports the continued preclinical evaluation and potential of the compound as investigational treatment for patients with multiple solid tumors.

Citation Format: Donghoon Yu, Yeejin Jeon, Dongsik Park, Mooyoung Seo, Wongyun Ahn, Jaeseung Kim, Choa Park, Euna Jeong, Sukjoon Yoon, Kiyean Nam. Development of highly selective CDK7 inhibitor Q901 for solid tumors [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 4855.
Abstract 4887: Direct mutational consequences of CRISPR-cas9 gene-edited DNA repair genes
Authors: Zou Xueqing , Koh Gene , Nanda Scott , Degasperi Andrea , Urgo Katie , Bushell Wendy , Agu Chukwuma , Perez-Alonso Vanesa , Rueda Daniel , Foreman Julia , Harris Rebecca , Jiricny Josef , Skarnes Bill , Nik-Zainal Serena
Published: September 2020

Cancer whole-genome sequencing has revealed characteristic mutational signatures associated with defective DNA repair that underpin human genetic diseases. To define the direct mutagenic effects of DNA repair deficiency at the genome-wide level, we investigate mutational signatures generated by CRISPR-Cas9-based knockouts of 42 genes involved in DNA repair/replication using a human-induced pluripotent stem cell line. Knockouts (Δ) of nine DNA repair genes reveal substitution/indel mutational signatures. Notably, dissection of signatures of defective mismatch repair (MMR) uncovers gene-specific characteristics including distinguishing features of ΔMLH1, ΔMSH2, and ΔMSH6 from ΔPMS2. This gene-specificity is also exhibited by hIPSCs derived from patients with autosomal recessive Constitutional Mismatch Repair Deficiency (CMMRD) that carry biallelic germline mutations of MMR genes. Furthermore, gene-specificity manifests in whole genome sequenced primary human cancers. Additionally, detailed analyses reveal putative sources of endogenous DNA damage that contribute to MMR signatures, including guanine oxidation, errors of DNA polymerases and reversed template slippage or double slippage. Finally, we find that using all mutational signatures of MMR-deficiency as identified in this study results in improved sensitivity and specificity in classifying MMR-deficient tumors, critical for accurate patient stratification for therapeutic intervention.

Citation Format: Xueqing Zou, Gene Koh, Scott Nanda, Andrea Degasperi, Katie Urgo, Wendy Bushell, Chukwuma Agu, Vanesa Perez-Alonso, Daniel Rueda, Julia Foreman, Rebecca Harris, Josef Jiricny, Bill Skarnes, Serena Nik-Zainal. Direct mutational consequences of CRISPR-cas9 gene-edited DNA repair genes [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 4887.
Abstract 491: Encapsulation of the stem cell inhibitor Salinomycin in novel QUATRAMER sustained injectable suspension (HSB-1216) for the treatment of small cell lung cancer
Authors: Kharbanda Surender M. , Mohammad Anees , Appajosyula Sireesh , Rosenberg Mark , Hill James , Laumas Sandeep , Kufe Donald W. , Singh Harpal
Published: September 2020

Small cell lung cancer (SCLC) represents approximately 15% of all cases of lung cancer. SCLC patients have a poor prognosis and limited treatment options. Conventional chemotherapy can reduce the bulk of SCLC lesions, but cancer stem cells (CSCs) survive, reconstitute the tumor and contribute to metastases. Thus, targeting CSCs, which are resistant to many current treatments, may significantly improve outcomes of SCLC patients. Salinomycin (SAL), a potassium ionophore, preferentially targets CSCs. However, SAL is extensively metabolized in animal models and is associated with substantial toxicity at higher doses, limiting its potential for anti-cancer activity in patients In this study, we report a novel QUATRAMER polymeric nanoparticle (NP)-based formulation, that potentially overcomes toxicity of SAL. SAL was encapsulated in polymeric NPs comprised of a polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG-modified PLA-tetra-block copolymer (SAL-NPs; HSB-1216). SAL release from the NPs was controlled by both PEG/PEG-PPG-PEG chain length and the PLA molecular weight, permitting time-dependent release over sustained periods. Treatment of the human SCLC cell line NCI-H69 with HSB-1216 demonstrated an IC50 of 0.5 uM. Once weekly treatment of BALB/c nu/nu mice harboring NCI-H69 xenografts with HSB-1216 at a dose of 5 mg/kg resulted in substantial tumor growth inhibition The tumor growth inhibition was without any effect on the body weights of the mice. These results demonstrate that a novel formulation for sustained delivery of SAL in a sustained injectable suspension represents an approach for targeting CSCs in SCLCs and potentially other cancer types.

Citation Format: Surender M. Kharbanda, Anees Mohammad, Sireesh Appajosyula, Mark Rosenberg, James Hill, Sandeep Laumas, Donald W. Kufe, Harpal Singh. Encapsulation of the stem cell inhibitor Salinomycin in novel QUATRAMER sustained injectable suspension (HSB-1216) for the treatment of small cell 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 491.
Abstract 4931: Yap1 regulates the transcription of cofilin to facilitate the metastatic movement of colorectal cancer cells
Authors: Islam S M Anisul , Acevedo-Duncan Mildred
Published: September 2020

Colorectal cancer (CRC) is one of the most leading death-causing cancers in the United States which accounts for 23,380 estimated death cases in 2019 as per American Cancer Society. Surgical removal of cancerous tissue coupled with chemotherapeutic intervention is the primary treatment of metastatic CRC and the only hope of enhanced survival. Dysregulation of kinases is pivotal in the various pathological process, including cancer. In addition, the metastatic events in a cancer cell are usually brought by the alteration of different key molecules. One such key molecule is called cofilin that acts on the actin cytoskeleton to induce metastatic phenotype in cancerous cells. In this study, we used two atypical Protein Kinase C (PKC) inhibitors, namely ICA-I (a PKC-ι inhibitor) and ζ-Stat (a PKC-ζ inhibitor), to investigate the role of atypical PKCs in regulating the expression of cofilin in metastatic colorectal cells. Our findings suggested that the atypical PKC inhibitors reduced the expression and translocation of Yap1 from cytosol to nucleus which ultimately leads to the reduced expression of cofilin in CRC cells. This mechanistic study provides an important insight into the metastatic behavior of the cancerous colorectal cells that can be used to target metastasis regulating molecules to improve the survival rate in patients with CRC.

Citation Format: S M Anisul Islam, Mildred Acevedo-Duncan. Yap1 regulates the transcription of cofilin to facilitate the metastatic movement of colorectal cancer cells [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 4931.
Abstract 4988: Multiplex immunofluorescence detection of resident memory T cells in solid tumors
Authors: Baranova Elena , Finan-Marchi Amanda , Motte Manon , Tliba Maroua , Iglesias Sabine , Belda Carole , Mace Alexandra , Hunt Sylvie , Lazzaro Domenico , Coton Jean-Philippe , Burrer Renaud
Published: September 2020

Tissue-resident memory T cells (TRM) represent a lineage of T cells localized in peripheral tissues that do not reenter the circulation. Their establishment in the tissue makes them the initial T cell response in local infections and inflammatory conditions. TRM are characterized by the expression of CD103, CD69, and CD49a. TRM play a role in the efficacy of cancer vaccines and have been correlated with improved prognosis and/or survival in a number of cancers. Comparisons of TRM and cytotoxic CD8 T cells in the tumor microenvironment are limited. Due to this, we have developed a Histoprofile TRM multiplex immunohistochemistry panel, validated by in-depth image analysis, for TRM and CD8 T cell profiling. Here we present our in depth multiplex immunofluorescence validation assay including tests of repeatability and reproducibility. Human tissue microarrays of tumors in a range of tissues were investigated with the TRM multiplex panel. After multi-spectral acquisition, the TRM and CD8 T cells could be differentiated in all of the tissue types examined. After delineation by a pathologist, we demonstrate the capability to quantify TRM and CD8 cells within the tumor and stroma compartments. The presented approach has the power to expand the clinical implications of TRM in cancer vaccine and immunotherapy.

Citation Format: Elena Baranova, Amanda Finan-Marchi, Manon Motte, Maroua Tliba, Sabine Iglesias, Carole Belda, Alexandra Mace, Sylvie Hunt, Domenico Lazzaro, Jean-Philippe Coton, Renaud Burrer. Multiplex immunofluorescence detection of resident memory T cells in solid tumors [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 4988.
Abstract 5049: Development of huPBMC-NCG mice to study xenogenic graft versus host disease (GVHD)
Authors: Ju Cunxiang , Sun Hongyan , Wang Dingyu , Guo Shiying , Zhang Mingkun , Wu Hainan , Li Shuai , Ju Chao , Zhao Jing , Gao Xiang
Published: September 2020

Graft versus host disease (GVHD) is a severe complication of allogeneic stem cell transplantation for leukemia treatment. NCG mice lacking functional murine T, B, NK cells as well as decreasing functional macrophage and dendritic cells could be used as recipients of human peripheral blood mononuclear cells (huPBMC) for the studies of human GVHD. In this study, huPBMC-NCG mice were developed by the engraftment of huPBMC into unconditioned or irradiated NCG mice. The engraftment kinetics, irradiation effect, and preclinical treatment were explored in these mice. After huPBMC engraftment, NCG mice developed GVHD symptoms, including body weight loss, fur loss, reduced activity, hunched posture and severe ruffling, and eventually led to mortality. We also observed human T cell trafficking to target organs including gut, heart, liver, skin, and lung by flow cytometry and immunohistochemistry. Interestingly, irradiation could significantly accelerate the onset of GVHD following huPBMC engraftment. Meanwhile, irradiated huPBMC-NCG mice presented less variability in the kinetics of GVHD and the frequency of this disease in comparison with unconditioned counterparts, thus providing a more stable model for human GVHD studies. For preclinical studies, we found immunomodulatory drugs including Abatacept (CTLA4-Ig) targeting APC cells or anti-Ly6G antibody targeting neutrophils could alleviate GVHD-associated symptoms along with improving overall survival of huPBMC-NCG mice. Taken together, this study established stable huPBMC-NCG mice to mimic the onset and development of human GVHD, which was helpful for the development of therapeutic intervention.

Citation Format: Cunxiang Ju, Hongyan Sun, Dingyu Wang, Shiying Guo, Mingkun Zhang, Hainan Wu, Shuai Li, Chao Ju, Jing Zhao, Xiang Gao. Development of huPBMC-NCG mice to study xenogenic graft versus host disease (GVHD) [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 5049.
Abstract 5080: Prognostic immune subtypes in glioblastoma
Authors: Joy Anna , Dong Xishuang , Kim Seungchan
Published: September 2020

Variations in the tumor immune microenvironment between Glioblastoma (GBM) patients complicates development and testing of immunotherapies. We used publicly available single cell RNA sequencing data of GBM patients to extract pan immune-specific genes in GBM. Interrogating bulk GBM sequencing or microarray data with pan immune-specific genes should allow examination of the immune component. We used these genes to investigate subtypes of GBM immune microenvironments. Consensus clustering using these genes on GBM TCGA RNAseq data gave 4 distinct subtypes while 12% of tumors were not assigned to any of those subtypes. Immune subtypes had distinct clinical outcomes. Differences in median survival (16.6 vs 5.8 months; longest vs. shortest survival; p < 1e-05 Logrank, Wald and Likelihood ratio tests) suggests the immune microenvironment significantly impacts clinical course. Cox univariate analysis indicates age had no contribution to risk (HR = 1.034; 95% confidence interval 1.017-1.052). We propose that immune-subtyping of GBM with pan immune-specific genes will lead to biomarkers that predict immunotherapeutic response. It will also help to identify molecular pathways and targets that suppress the immune anti-tumor response.

Citation Format: Anna Joy, Xishuang Dong, Seungchan Kim. Prognostic immune subtypes in glioblastoma [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 5080.
Abstract 5148: ADGRG1 promotes tumorigenesis, invasion/migration, and cell-cell adhesion in triple-negative breast cancer cells
Authors: Bhat Raksha , Abdulkareem Noor M. , Yasser Hala , Thangavel Hariprasad , Trivedi Meghana V.
Published: September 2020

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with the highest risk of metastatic recurrence and shortest survival in patients. Chemotherapy, the current standard of treatment for systemic therapy in TNBC, has suboptimal efficacy but high toxicity. Therefore, drugs directed against new targets exhibiting superior efficacy and excellent safety profiles are urgently needed in the treatment of metastasis in TNBC. Recently, adhesion G protein-coupled receptors (GPCRs) have shown an important role in various cancer processes. Traditionally, GPCRs are considered excellent drug targets. In this study, we aimed to evaluate the role of ADGRG1, an adhesion GPCR, in TNBC. ADGRG1 was highly expressed in basal/TNBC compared to luminal subtypes in the Tumor Cancer Genome Atlas (TCGA) Provisional datasets. Higher expression of ADGRG1 predicted poor recurrence-free survival in TNBC patients as determined using the Kaplan-Meier plotter. ADGRG1 gene and protein expression was higher in LM2 derivative with higher lung metastatic potential compared to parental MDA-MB-231 cells. ADGRG1 knockdown using 2 independent target-specific validated siRNAs reduced cell growth by MTT assay in both MDA-MB-231 parental and LM2 cells. ADGRG1 knockdown decreased mammosphere formation in MDA-MB-231 Parental cells but not in LM2 cells. ADGRG1 knockdown significantly inhibited invasion and migration of both Parental MDA-MB-231 cells and their LM2 derivatives in Trans-well assay. In addition, ADGRG1 knockdown significantly decreased cell-cell interaction in the VybrantTM Cell Adhesion assay in both cell types. ADGRG1 was significantly overexpressed in circulating tumor cell (CTC) clusters compared to single CTCs from the LM2 xenograft models. Overall, our results indicate pro-tumorigenic and pro-metastatic role of ADGRG1 which needs further validation in animal studies. Understanding the role of ADGRG1 in cell-cell adhesion as well as its signaling in TNBC are crucial in discovering its potential as a novel and druggable target in TNBC.

Citation Format: Raksha Bhat, Noor M. Abdulkareem, Hala Yasser, Hariprasad Thangavel, Meghana V. Trivedi. ADGRG1 promotes tumorigenesis, invasion/migration, and cell-cell adhesion in triple-negative breast cancer cells [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 5148.
Abstract 5160: A GPCR candidate in pancreatic ductal adenocarcinoma: A potential repurposing opportunity
Authors: Salmerón Cristina , Sriram Krishna , Baird Alyssa , Insel Paul A.
Published: September 2020

The 5-year survival of patients with pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer (90%), is very low (~9% all stages). PDAC thus requires new, effective and safe therapies. By comparing transcriptomic data for PDAC (in The Cancer Genome Atlas) with that of normal pancreas (in the GTEx database), we discovered that many G protein-coupled receptors (GPCRs) are increased in expression in PDAC. One such GPCR is the histamine receptor H1 (HRH1). GPCRs regulate metabolism, growth/death and functional activities of normal and cancer cells, including features of the malignant phenotype. GPCRs are the largest family of targets of approved drugs but have been largely ignored in cancer therapy. We found that HRH1 is prominently (~32-fold) overexpressed in PDAC tumors compared to normal pancreatic tissue and that high HRH1 expression is an unfavorable prognostic marker for PDAC patients. HRH1 mRNA is also highly expressed in multiple human PDAC cell lines. Studies with such cell lines revealed that histamine, acting via HRH1, increases intracellular Ca2+, cell proliferation and migration, effects blocked by multiple FDA-approved HRH1 antihistamines. PDAC tumors and cancer cells isolated from KPC mice also have high HRH1 expression compared to normal mouse pancreas. Similar to human PDAC cells, the KPC cells showed histamine/HRH1-promoted increase in intracellular Ca2+. Altogether, our findings identify HRH1 as a prominently overexpressed, functional GPCR in human and mouse PDAC tumors and cells. HRH1 may thus be a novel therapeutic target; its blockade by FDA-approved antihistamines represents a novel potential repurposing approach for the treatment of PDAC.

Citation Format: Cristina Salmerón, Krishna Sriram, Alyssa Baird, Paul A. Insel. A GPCR candidate in pancreatic ductal adenocarcinoma: A potential repurposing opportunity [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 5160.
Abstract 5169: Tissue analysis of NAD(P)H: quinone oxidoreductase (NQO1) and catalase expression in patients with germ cell tumors (GCT)
Authors: Sadraei Neda Hashemi , Adra Nabil , Temm Constance , Kesler Kenneth A. , Idrees Muhammad T. , Boothman David A. , Einhorn Lawrence
Published: September 2020

Background: Testicular germ cell tumor (GCT) is the most common cancer in young men between 15 and 35 years of age. Cisplatin-based combination chemotherapy will cure 70% of patients with metastatic GCT. Patients who relapse can still be cured with salvage standard-dose or high-dose chemotherapy. However, about 15% of patients with relapsed/refractory GCT are incurable. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron oxidoreductase elevated in several tumor types compared to normal tissue. NQO1 detoxifies quinones and reduces oxidative stress. NQO1 bioactivatable drugs induce DNA damage in cells overexpressing NQO1, but not in cells lacking it, hence delivering tumor-selective damage and cell death. Alterations in catalase expression can cause cytoprotection. The ratio of NQO1:catalase activities is presumed to be a predictive marker for therapeutic activity of NQO1 bioactivatable drugs. There is no existing data on NQO1 or catalase expression in GCTs.

Methods: Patients with testicular or extra-gonadal GCT who had surgery between January 2016 and December 2018 were identified from the Indiana University Melvin and Bren Simon Cancer Center Tissue Bank database. Patients with non-seminomatous GCT (NSGCT) of testis or primary mediastinal NSGCT were selected and specimens obtained. Immunohistochemistry staining was performed on tumor tissue to determine the quantity and intensity of NQO1 and catalase expression.

Results: NQO1 and catalase expression were determined in 16 patients. Thirteen specimens stained moderately to strongly positive for NQO1. Only 1 specimen stained negative. Conversely, no specimen stained strongly positive for catalase, and only 2 stained moderately positive. Fourteen specimens were catalase deficient or only mildly positive for catalase. The details of the immunostaining is summarized in the table below.

Immunohistochemistry staining for NQO1 and catalase in GCTImmunostainNegative(0)Mildly positive (<10%)Moderately positive (10-50%)Strongly positive (>50%)NQO1Quantity01114Intensity0547Combined score1267CatalaseQuantity3472Intensity31120Combined score7720

Conclusions: NSGCTs appear to have high levels of NQO1 and deficiency of catalase. NQO1 bioactivatable agents could serve as a potential therapeutic approach in patients with refractory testicular GCTs.

Citation Format: Neda Hashemi Sadraei, Nabil Adra, Constance Temm, Kenneth A. Kesler, Muhammad T. Idrees, David A. Boothman, Lawrence Einhorn. Tissue analysis of NAD(P)H: quinone oxidoreductase (NQO1) and catalase expression in patients with germ cell tumors (GCT) [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 5169.
Abstract 5237: High-throughput slide preparation for spatially-resolved, multiplexed quantification of protein or mRNA in tumor tissues with automation of GeoMx™ DSP Assays on Leica Biosystems BOND RX
Authors: Zollinger Daniel R. , Ketting Sarah , Sorg Kristina , Appelbe Oliver , Aaker Danielle , Cockfield Damian , Merritt Chris , Beechem Joseph M.
Published: September 2020

Characterization of the spatial distribution and abundance of proteins and mRNAs with morphological context within tissues enables a better understanding of biological systems in many research areas, including immunology and oncology. The GeoMxTM Digital Spatial Profiler (DSP) enables the highly-multiplexed detection of mRNA or protein targets within distinct regions of tissues, including FFPE. This assay utilizes in situ hybridization (ISH) probes or antibodies linked to indexing oligo barcodes via a photo-cleavable linker. The GeoMx DSP enables profiling of RNA or protein targets by releasing and collecting these barcodes from user-defined regions-of-interest.

Manual preparation of FFPE tissue uses conventional immuno-fluorescence (IF) and ISH reagents, however it requires greater than two hours of hands-on-time over 2 days. Protocols have been developed to enable the automation of these tissue preparation protocols using the Leica Biosystems BOND RX and BOND RXm Fully Automated Research Stainers. Here we describe protocols for the semi-automated preparation of GeoMx RNA and protein assays as well as the fully-automated preparation of GeoMx RNA assays. These protocols reduce the hands-on time of performing the assay to 72 minutes and 35 minutes for the semi- and fully-automated protocols, respectively. Signal-to-noise ratios (SNR) are highly concordant between serial sections processed manually and processed on the Leica BOND RXm using the GeoMx Immune Cell Profiling Panel for protein (58-plex) and GeoMx Immune Pathways Panel for RNA (84-plex). Furthermore, automated assays show high concordance between the BOND RX and BOND RXm instruments. Lastly, we demonstrate the use of the fully automated slide preparation protocol with the GeoMx Cancer Transcriptome Atlas, a panel that targets >1,600 key cancer mRNAs that are read out by next-generation sequencing.

GeoMx Assays are for RESEARCH USE ONLY. Not for use in diagnostic procedures.

Citation Format: Daniel R. Zollinger, Sarah Ketting, Kristina Sorg, Oliver Appelbe, Danielle Aaker, Damian Cockfield, Chris Merritt, Joseph M. Beechem. High-throughput slide preparation for spatially-resolved, multiplexed quantification of protein or mRNA in tumor tissues with automation of GeoMx™ DSP Assays on Leica Biosystems BOND RX [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 5237.
Abstract 5247: Interleukin-11 could be a novel therapeutic target for lung adenocarcinoma patients
Authors: Ojeda Laura , Cirauqui Cristina , Molina-Pinelo Sonia , Garrido-Martín Eva M , Ramos-Paradas Javier , Yagüe Patricia , Santos Alba , Carrizo Nuria , Enguita Ana B. , Muñoz Maria Teresa , Solorzano Jose Luis , Paz-Ares Luis , Ferrer Irene
Published: September 2020

Interleukin-11 (IL11) has been associated with tumorigenesis in a wide variety of tumors, including lung cancer, and it has been proposed as a diagnostic biomarker for lung adenocarcinoma. However, it is still not clear how IL11 affects the tumorigenesis. It is possible that, as other cytokines, it has a dual role in the tumor cell and tumoral microenvironment. Thus, the inhibition of IL11 could be an interesting therapeutic option to test in these patients. First, we confirmed the pro-tumorigenic effect of IL11 in patients and mouse models of lung adenocarcinoma (cancer cell lines xenografts, patient derived xenografts; PDXs and genetically engineered mouse models; GEMMs). Later, we knocked-down the expression of IL11 or its specific receptor IL11RA in adenocarcinoma cell lines in order to analyze their tumorigenic properties in vitro and in vivo. We confirmed that fibroblasts are a relevant source of IL11, so we knocked-down IL11 expression in order to analyze how it affects the fibroblasts´ properties, including the secretion of other pro-tumorigenic cytokines and growth factors. We reported that an increased expression of IL11 correlates with a poorer survival in lung adenocarcinoma patients and that IL11 stimulation increases the proliferation rates in xenografts, PDXs and GEMMs. On the contrary, IL11 or IL11RA knockdown in adenocarcinoma cell lines reduces their pro-tumorigenic properties in vitro and in vivo. Finally, the silencing of IL11 in fibroblasts reduces their proliferation, migration and secretion of pro-tumorigenic cytokines and growth factors. In conclusion, we propose that IL11 plays a direct pro-oncogenic role in lung adenocarcinoma tumoral cell and an indirect role in tumoral microenvironment. The genetic ablation of IL11 has an anti-tumoral effect, so it could be interesting to develop a pharmacological tool which neutralizes the IL11-IL11RA signaling to test as a therapeutic strategy in preclinical models. Thus, IL11 could represent a potential therapeutic target that deserves to be more exhaustively studied in the clinical settings.

Citation Format: Laura Ojeda, Cristina Cirauqui, Sonia Molina-Pinelo, Eva M Garrido-Martín, Javier Ramos-Paradas, Patricia Yagüe, Alba Santos, Nuria Carrizo, Ana B. Enguita, Maria Teresa Muñoz, Jose Luis Solorzano, Luis Paz-Ares, Irene Ferrer. Interleukin-11 could be a novel therapeutic target for lung adenocarcinoma patients [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 5247.
Abstract 5276: Secretory pathway calcium ATPase 2 (SPCA2) promotes cell survival and chemoresistance in receptor positive breast cancer cells
Authors: Makena Monish Ram , Dang Donna , Mekile Allatah , Buckhaults Phillip , Rao Rajini
Published: September 2020

Introduction: Dysregulation of the Ca2+ toolkit has profound consequences for tumor growth and metastasis, raising hopes for novel avenues of therapeutic intervention. The Secretory Pathway Ca2+-ATPase Isoform 2 (SPCA2) has a dual function in sequestering Ca2+ and Mn2+ into secretory stores as well as eliciting their entry into cells by activating plasma membrane ion channels. We have previously showed that SPCA2 is implicated in breast cancer progression, microcalcifications, epithelial-mesenchymal transition, and metastasis.

Methods and Results: High SPCA2 expression was associated with poor overall survival in receptor positive breast cancer patients (n=548, P =8.7e-0.6; KM Plotter). Depletion of SPCA2 (SPCA2 KD) in MCF-7 cells significantly reduced cell proliferation (P < 0.05). Analysis of the cell cycle (PI staining, Flow cytometry) revealed a significant increase (P < 0.05) in the number of cells in the G0/G1 phase, and significant increase in apoptosis (via TUNEL assay) (P < 0.05) in SPCA2 KD cells compared to control. Microarray analysis showed the p53 pathway is the most significantly upregulated pathway (P < 0.05) in SPCA2 KD cells compared to control. P53 knockout abrogated SPCA2 mediated cell cycle arrest and apoptosis. Depletion of SPCA2 increased reactive oxygen species (ROS, measured by DCFDA dye and MitoSox Red using flow cytometry) leading to DNA damage (confocal microscopy), and activation of ATM/ATR pathway (Immunoblotting, P < 0.05). As a consequence, SPCA2 KD cells showed specific hypersensitivity to DNA damaging agents used in breast cancer therapy (carboplatin, radiation, and doxorubicin) (determined by MTT assay) (P < 0.05). Thus SPCA2 is a novel molecular target in breast cancer therapy. Curcumin is currently being evaluated in clinical trials of breast cancer. We show that curcumin causes dose-dependence inhibition of SPCA2 expression (P < 0.05) in multiple receptor-positive cell lines, and that forced overexpression of SPCA2 decreased curcumin cytotoxicity. Interestingly, we did not observe these results with SPCA1, the housekeeping SPCA isoform, revealing isoform-specific function of Golgi calcium pumps in breast cancer subtypes.

Conclusion: These novel findings point to a causal link between SPCA2, breast cancer progression, and chemoresistance to DNA damaging agents. Decrease of SPCA2 expression by curcumin may have therapeutic potential in treating receptor positive breast cancer.

Citation Format: Monish Ram Makena, Donna Dang, Allatah Mekile, Phillip Buckhaults, Rajini Rao. Secretory pathway calcium ATPase 2 (SPCA2) promotes cell survival and chemoresistance in receptor positive breast cancer cells [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 5276.
Abstract 5328: mTOR inhibitor enhances anti-tumor effect of BET bromodomain inhibitor in small cell lung cancer
Authors: KUMARI ANJU , Gesumaria Lisa , Fetsch Patricia , Miettinen Markku , Hughitt Keith , Mock Beverly , Thomas Craig , Schrump David S. , Chen Haobin
Published: September 2020

Introduction: Small cell lung cancer (SCLC) is one of the two recalcitrant cancers designated by NCI, and its 2-year survival rate has not improved over the past two decades. Novel therapy is urgently needed. BET bromodomain inhibitor (BETi), which targets transcriptional coactivator BET bromodomain proteins, has been reported to be effective in SCLC. However, given that SCLC is notorious for rapid development of drug resistance, it is necessary to identify drugs that can synergize with BETi to form a combinatorial drug regimen.

Methods: A high-throughput drug combination matrix screen was performed to identify potential agents that can synergize with BETi. Synergy between the identified candidate drugs and BETi was further validated in various SCLC cell lines by using the CellTiter-Glo assay as well as in several SCLC PDX models by examining tumor growth suppression and survival of tumor-bearing mice. Cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP) was analyzed by IHC and western blot in SCLC PDX tumors and in vitro cell lines to assess apoptosis after drug treatment, respectively.

Results: The high-throughput screen identified multiple inhibitors of the PI-3K/AKT/mTOR pathway to synergize with BETi by causing a significantly higher rate of apoptosis and cell growth inhibition. A comparison among the inhibitors that specifically target PI-3K, AKT or mTOR revealed that the combination of BETi and mTOR inhibitor (mTORi) have the highest level of synergy. The BETi/mTORi combo also showed synergy in several SCLC cell lines that are initially resistant to single agent BETi. The antitumor effect of the BETi/mTORi combinatorial treatment was further confirmed in several SCLC PDX mouse models. When compared to either single agent or vehicle, the BETi/mTORi combo treatment led to a greater degree of tumor growth suppression and a more prolonged survival of the tumor-bearing mice. This enhanced antitumor effect is connected with a significantly more apoptosis (cleaved caspase 3 or PARP) found in SCLC tumor tissues and cell lines treated with the BETi/mTORi combo.

Conclusion: mTORi can enhance the antitumor effect of BETi in SCLC by causing more apoptosis.

Citation Format: ANJU KUMARI, Lisa Gesumaria, Patricia Fetsch, Markku Miettinen, Keith Hughitt, Beverly Mock, Craig Thomas, David S. Schrump, Haobin Chen. mTOR inhibitor enhances anti-tumor effect of BET bromodomain inhibitor in small cell 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 5328.
Abstract 5547: Synergistic action of sitagliptin and checkpoint inhibition for ovarian cancer therapy
Authors: Moffitt Laura R. , Wilson Amy L. , Basri Bashirah , Wilson Kirsty L. , Plebanski Magdalena , Stephens Andrew N.
Published: September 2020

Ovarian cancer is the most lethal gynecological malignancy and treatment options have not improved in decades. Whilst ovarian tumors have an immune-mediated component, emerging immunotherapies (e.g. anti-PD-1, anti-PD-L1) have failed to achieve robust clinical response for ovarian cancer patients. There is an urgent need to develop new therapeutic strategies to improve survival rates in women, particularly those with recurrent, chemoresistant disease. In an ongoing study we are investigating the repurposing of sitagliptin, an anti-diabetes drug, as a novel immunomodulatory therapy in ovarian cancer. Ovarian tumors were generated in C57BL/6 wild-type mice by intrabursal implantation of ID8 murine ovarian cancer cells. Following the formation of primary tumors (~2 weeks), mice received a daily oral dose of sitagliptin (50mg/kg) until ethical endpoint. Anti-tumor immune responses were examined by flow cytometry and immunohistochemical staining according to tumor stage. Mice were assessed for overall survival compared to paclitaxel chemotherapy as standard-of-care. Daily sitagliptin treatment increased circulating Teff:Treg cell ratios in the blood, peritoneal fluid and tumor tissue at an early stage, and this was sustained throughout disease. Sitagliptin also enhanced T effector cell activation and proliferation, indicating that increased Teff:Treg ratios were a positive indicator of the anti-tumor immune response. Treatment with sitagliptin alone increased median survival time by 27% compared to both untreated controls and paclitaxel as standard-of-care, suggesting a beneficial effect mediated by anti-tumor immune responses. Of particular interest, sitagliptin treatment specifically increased the percentage of CXCR3+ T effector cells in the peritoneal cavity. Recent data has suggested that CXCR3+ T cells are functionally required for immune checkpoint inhibitors to exert efficacy. Thus, we hypothesize that sitagliptin may enhance the efficacy of immune checkpoint inhibitor therapies in ovarian cancers. Our ongoing studies are investigating the combination of sitagliptin with anti-PD-1 and anti-PD-L1 antibodies, as a rational approach to improving their efficacy in ovarian cancers. These studies will identify a clinically relevant and directly translatable approach to improve prognosis for ovarian cancer patients.

Citation Format: Laura R. Moffitt, Amy L. Wilson, Bashirah Basri, Kirsty L. Wilson, Magdalena Plebanski, Andrew N. Stephens. Synergistic action of sitagliptin and checkpoint inhibition for ovarian cancer therapy [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 5547.