bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5942: Mechanism and role of neutrophil recruitment in head and neck squamous cell carcinoma
Authors: Ghias Kulsoom , Shakeel Hina , Sheikh Alisalman , Abidi Syed Hani , Masood Kiran I. , Fatima Saira , Bilwani Fareena
Published: April 2023

The immune system plays a pivotal role in identification and clearance of tumor cells, but can also be counterproductive and enhance tumor progression. Presence of neutrophils in solid tumors, including the sixth most common cancer globally - head and neck squamous cell carcinomas (HNSCC), is associated with poor prognosis. Pro-inflammatory cytokines regulated by NF-κB promote extravasation of neutrophils into tissue; neutrophils are polarized to anti-tumor (N1) or pro-tumor (N2) phenotype through the action of various pathways, including TGF-β. The mechanism and role of neutrophil recruitment in HNSCC were explored this study.

Neutrophils isolated from adult donors were co-cultured for 5 hours with conditioned media (CM) from HNSCC cells SCC-9 and Cal-27, and stained with TNF-α, ICAM-1, CCL2, CCL5 for analysis of neutrophil phenotype by flow cytometry. TGF-β blocking antibody was used to explore the role of TGF-β in polarization of neutrophils. Canonical and non-canonical TGF-β signaling pathway activation was also investigated in these neutrophils through western blots. Using HNSCC patient samples, qPCR was performed for expression of genes involved in apoptosis, epithelial-to-mesenchymal transition, metastasis, and neutrophil function.

Following co-culture with 24-hour HNSCC CM, neutrophils begin to exhibit cell surface markers indicative of N2 phenotype, which is impacted by TGF-β blocking antibody. Neutrophils co-cultured with HNSCC CM exhibited altered expression of TGF-β mediators Smad2/3, p38 and Akt, which was abrogated with the addition of TGF-β blocking antibody indicating specificity of a TGF-β induced signaling. Gene expression analysis exhibited significantly higher expression of anti-apoptotic BCL2 and neutrophil function marker alpha defensin 1 in HNSCC patient samples with greater neutrophil infiltration, while caspase 3 was expressed at significantly higher levels in HNSCC patient samples with low neutrophil infiltration.

HNSCC cells recruit neutrophils; evidence suggests that TGF-β pathway, which typically promotes N2 phenotype in neutrophils, is involved via canonical and non-canonical mediators including p38 and Akt. Further, HNSCC patient samples have varying levels of neutrophil infiltration and differentially express genes involved in apoptosis, EMT, metastasis and invasion, inflammation.

Citation Format: Kulsoom Ghias, Hina Shakeel, Alisalman Sheikh, Syed Hani Abidi, Kiran I. Masood, Saira Fatima, Fareena Bilwani. Mechanism and role of neutrophil recruitment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5942.
Abstract 5948: MAGE-A3 expression with MMR-MSH6 screening suggests MAGE-A3 as a potential target for microsatellite-stable colorectal cancer
Authors: Yom Jina , Gonzalez Rachel , Zewdu Eden , Su Aubrey , Yin Patrick , Gilmore Bailey , Kong Dehe , Han Andy , Guo Zhaoying , Qu Tianli , Zhao Zoe , Christenson Eric , Ma Yan , Guo Hailey , Hu Xiaomin , Ren Qi , Wu Zhaohui , Liu Xuan , Fu Wei
Published: April 2023

Colorectal Cancer (CRC) is the second leading cause of death worldwide. Recent developments in cancer immunotherapy have obtained encouraging clinical responses, including PD-1/PD-L1 blockade therapy, although response rates vary depending on tumor type. For example, despite the lack of response by microsatellite-stable CRC, a subset of CRC with deficient mismatch repair (dMMR) and high microsatellite instability (MSI-H) responds especially well to the PD-1/PD-L1 blockade therapy. For other types of CRC which do not respond to PD-L1 therapy, new targets are being investigated. One family of targets is the cancer testis antigen MAGE-A whose expression is restricted to germline cells in normal tissue but is overexpressed in many cancer cells. In this study, immunohistochemical analysis of MAGE-A3 expression in colon cancer along with IHC screening of mismatch protein MSH6 was performed. Many of the cases in this study observed a co-expression in the tissue. In the tumors, MAGE-A3 was observed to have weak cytoplasmic expression while MSH6 had strong nuclear expression in the epithelial cells. The immune cells adjacent to the epithelial cells were also positive for MAGE-A3. This result suggests that MAGE-A3 may be a promising target for microsatellite-stable colorectal cancer immunotherapy.

Citation Format: Jina Yom, Rachel Gonzalez, Eden Zewdu, Aubrey Su, Patrick Yin, Bailey Gilmore, Dehe Kong, Andy Han, Zhaoying Guo, Tianli Qu, Zoe Zhao, Eric Christenson, Yan Ma, Hailey Guo, Xiaomin Hu, Qi Ren, Zhaohui Wu, Xuan Liu, Wei Fu. MAGE-A3 expression with MMR-MSH6 screening suggests MAGE-A3 as a potential target for microsatellite-stable colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5948.
Abstract 596: Development and assessment of a novel tumor microenvironment activable linker (TMALIN) ADC platform for solid tumor treatments
Authors: Cai Jiaqiang , Song Shuai , Zong Qing , Liu Qigang , Xu Jian
Published: April 2023

Antibody-drug conjugates (ADC) are one of the fastest growing anticancer drugs, which bring cytotoxic drugs into tumors while avoiding high systemic toxicities. To date, all commercial ADCs use intracellular lysosomal cleavage mechanisms for payload release to kill tumor cells which is often limited by the low tumor antigen expression. To this end, a novel systemically stable and extracellular tumor microenvironment activable linker-payload technology platform has been developed.

Preclinical assessments demonstrated that the highly hydrophilic linker-payload, which showed limited impact on the hydrophilicity of antibody, prolonged the retention time in vivo and eventually enhanced the in vivo potency of ADC. The linker-payload is stable with less than 2% payload drop-off after 28-day incubation in plasma under physiological conditions in vitro. The high stability was also illustrated by GLP cynomolgus monkey studies and preliminary clinical trial data, with the overlap of ADC and TAb PK profile. Furthermore, a battery in vitro and in vivo cell line-derived xenograft (CDX) mouse model studies showed the linker-payload is stable in normal tissues but efficiently releases the payload in tumor tissues. The ADCs using TMALIN platform elucidated significant anti-tumor activity advantages when compared with those marketed ADCs targeting the same antigen, besides showing an acceptable safety profile in pivotal NHP studies without lung/liver/kidney toxicities.

In summary, the TMALIN platform addresses the unmet need of ADCs by releasing the payload extracellularly in tumors and tumor microenvironments on top of the known intracellular lysosomal cleavage mechanisms. Continued clinical validation work is underway for several pipeline candidates as well as investigation into other target areas.

Citation Format: Jiaqiang Cai, Shuai Song, Qing Zong, Qigang Liu, Jian Xu. Development and assessment of a novel tumor microenvironment activable linker (TMALIN) ADC platform for solid tumor treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 596.
Abstract 6088: Charting new cancer dependencies with patient derived organoids
Authors: Neiswender James V. , Brenan Lisa , Bertea Tate , Guo Jimmy , Anderson Ashley , Wong Megan , Posner Zoe , Kapner Kevin , Hennessey Connor , Wie Sarah , Boyle Isabella , De Kegel Barbara , Dempster Joshua , Tseng Yuen-Yi (Moony) , Root David , Aguirre Andrew , Vazquez Francisca
Published: April 2023

The number of patients benefiting from precision cancer medicine (PCM) is very limited. The Cancer Dependency Map Initiative aims to dramatically accelerate PCM by systematically identifying the landscape of cancer vulnerabilities across all tumors. Genome-wide knockout screens have been performed in over 1000 cancer cell lines, but these have been sourced from historically derived models, leaving many cancers underrepresented. Advances have been made in model derivation by growing cells in 3D formats, such as organoids or spheroids. These models present an opportunity to screen new cancer types, but come with challenges that require a new pipeline. Here, we present a set of screens using a genome wide CAS12 library in organoid tumor models representing novel subtypes of pancreatic, esophageal, and gastric cancer, along with treatment-resistant breast, ovarian, and prostate cancer organoids. We show that genome-wide screening of these models is feasible and can be achieved with quality comparable to 2D cell lines. Our work uncovered a set of novel dependencies associated with cell-ECM interactions that have not previously been identified in 2D models. Our methods provide a framework for screening future patient derived 3D culture models and discovering vulnerabilities in new tumor types.

Citation Format: James V. Neiswender, Lisa Brenan, Tate Bertea, Jimmy Guo, Ashley Anderson, Megan Wong, Zoe Posner, Kevin Kapner, Connor Hennessey, Sarah Wie, Isabella Boyle, Barbara De Kegel, Joshua Dempster, Yuen-Yi (Moony) Tseng, David Root, Andrew Aguirre, Francisca Vazquez. Charting new cancer dependencies with patient derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6088.
Abstract 6089: Identification of novel YAP/TAZ pathway regulators in the triple-negative breast cancer cell line MDA-MB231 using single-cell CRISPR screening
Authors: Berlak Mareike , Makowska Zuzanna , Klironomos Filippos , Kuehnlenz Julia , Kamburov Atanas , Steffen Andreas , Lange Martin , Nicke Barbara , Lesche Ralf , Staller Peter , Kopitz Charlotte , Naujoks Jan
Published: April 2023

The Hippo signaling cascade is a major pathway that integrates a broad spectrum of mechanosensory signals at the plasma membrane and regulates response via control of cell proliferation, self-renewal, differentiation, and apoptosis. Dysregulation of this pathway has been observed across a range of cancer types and results in an altered activity of its primary downstream effectors, the oncogenic transcription factors YAP/TAZ. For example, both germline and somatic loss-of-function mutations in the tumor suppressor gene NF2, a component of Hippo, induce hyperactivation of YAP/TAZ, transcriptional changes and ultimately result in tumor growth. The Hippo signaling pathway is an attractive target for drug discovery efforts, however, it is highly complex and still incompletely understood. Hence it is indispensable to get a deeper insight into the Hippo - YAP/TAZ signaling axis. To this end, we performed a genome-wide CRISPR knockout screen in the triple-negative breast cancer (TNBC) cell line MDA-MB231 (NF2LOF) expressing a YAP/TAZ reporter construct. We identified both negative and positive regulators of YAP/TAZ in breast cancer cells. In a second step, screening hits were further characterized in a focused single-cell CRISPR screen (Perturb-Seq), aiming at better understanding of the effects on YAP/TAZ activity regulation and downstream effects on gene expression. Here we present the technical details of our screening approaches and the results of perturbing known and novel regulators of YAP/TAZ on single cell level. We discuss the use of Perturb-Seq in the initial validation of hits from genome-wide screens and provide data that may serve as a basis for future drug discovery efforts, seeking for novel and effective treatments for triple-negative breast cancers and other malignancies with Hippo pathway alterations.

Citation Format: Mareike Berlak, Zuzanna Makowska, Filippos Klironomos, Julia Kuehnlenz, Atanas Kamburov, Andreas Steffen, Martin Lange, Barbara Nicke, Ralf Lesche, Peter Staller, Charlotte Kopitz, Jan Naujoks. Identification of novel YAP/TAZ pathway regulators in the triple-negative breast cancer cell line MDA-MB231 using single-cell CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6089.
Abstract 6233: Omacetaxine reduces c-MYC expression and demonstrates antitumor effects in osteosarcoma
Authors: Farrell Kristen B. , Thamm Douglas H.
Published: April 2023

Omacetaxine mepesuccinate (OMX), also known as homoharringtonine, is a protein translation inhibitor approved for use in chronic myeloid leukemia. Recent studies suggest OMX may be effective against other cancer types with high rates of protein translation, and may be successful against protein targets that are difficult to inhibit by reducing their translation rates. Using both canine and human osteosarcoma (OS) cell lines, we have investigated the efficacy of OMX against osteosarcoma and expression of c-MYC. OMX provides significant growth inhibition of OS cells with IC50 values in the low nanomolar range. OMX also reduces migration and invasion capabilities of OS cells. Strikingly, we observed reduction in levels of c-MYC protein in most canine and human OS cell lines after OMX exposure. This reduction was both dose and time dependent. We also investigated the efficacy of OMX against OS in an orthotopic mouse OS model. OMX is a promising candidate for treatment of OS in both dogs and humans, and potentially additional cancers dependent on c-MYC.

Citation Format: Kristen B. Farrell, Douglas H. Thamm. Omacetaxine reduces c-MYC expression and demonstrates antitumor effects in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6233.
Abstract 6237: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia
Authors: Paiboonrungruang Chorlada , Xiong Zhaohui , Li Yahui , Bowman Brittany , Huang Zhi , Chen Vivian , Weissman Bernard , Williams Kevin P. , Major M Ben , Chen Xiaoxin
Published: April 2023

Objective: NRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC.

Design: We performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. Mechanism of action of one compound was explored by biochemical assays.

Results: Using high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity.

Conclusion: We have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic as a radiation and chemotherapy sensitizer for NRF2high ESCC.

Citation Format: Chorlada Paiboonrungruang, Zhaohui Xiong, Yahui Li, Brittany Bowman, Zhi Huang, Vivian Chen, Bernard Weissman, Kevin P. Williams, M Ben Major, Xiaoxin Chen. Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6237.
Abstract 6705: Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma
Authors: Tan Zhen , Ko Josephine Mun-Yee , Yu Valen Zhuoyou , Ng Hoi Yan , Law Simon , Lung Maria Li
Published: April 2023

Esophageal squamous cell carcinoma (ESCC) is a clinically challenging disease that requires a multidisciplinary approach. Unfortunately, the scarcity of ESCC genomic data hinders the understanding of ESCC biology, disease progression and rational therapy design. Circulating tumor cells (CTCs), which are shed from the primary and metastatic tumors and then circulate within the peripheral vasculature, reflect the existing tumor heterogeneity. Here, we utilized the non-biased size-based CTC enrichment strategy in combination with the real-time RT-PCR to molecularly characterize the CTCs with cancer stem cell (CSC) or mesenchymal properties in ESCC. Comprehensive data from ESCC cell lines, mouse ESCC xenograft models and clinical ESCC peripheral blood samples emphasize the importance of TWIST1 (Twist Family BHLH Transcription Factor 1) in ESCC progression. Gain-of-function and loss-of-function analyses demonstrate that TWIST1 promotes cell migration, invasion and colony formation in ESCC cells. Moreover, positive TWIST1 expression is responsible for the chemoresistance of ESCC cells to Cisplatin and is correlated with poor overall survival in ESCC patients. In addition, we show that TWIST1 promotes malignant potential, including tumor growth, invasion and chemoresistance via the TWIST1-TGFBI-ZEB1 signaling pathway in ESCC. Meanwhile, the TWIST1-TGFBI-ZEB1 signaling pathway confers immunosuppressive conditions to the tumor microenvironment, which in turn contributes to EMT to promote tumor progression. Collectively, these findings indicate that TWIST1 has a novel role in ESCC carcinogenesis by regulating tumorigenicity and cancer stem cell properties that may be useful as a prognostic monitoring biomarker and a promising therapeutic target in clinical ESCC treatment.

Citation Format: Zhen Tan, Josephine Mun-Yee Ko, Valen Zhuoyou Yu, Hoi Yan Ng, Simon Law, Maria Li Lung. Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6705.
Abstract 6722: RNAScope in situ hybridization as a novel approach for the spatiotemporal assessment of RD3 mRNA expression in neuroblastoma
Authors: Somasundaram Dinesh Babu , Aravindan Sheeja , Yu Zhongxin , Baker Ashley , Aravindan Natarajan
Published: April 2023

Our recent investigations unveiled the transcriptional and translational loss of Retinal degeneration Protein 3 in neuroblastomas (NB) when compared to the healthy adult and fetal tissues. Further, our preclinical studies with patient derived cellular systems and mouse model of high-risk NB (HR-NB) demonstrated that RD3 loss orchestrates disease progression. More importantly, we defined the therapy pressure driven acquired loss of RD3 in neuroblastomas, its crucial function in the evolution of progressive disease (PD), and further defined the prognostic and predictive significance of RD3 loss in NB. In this study, we investigated the use of RNAScope as a sensitive and specific method for the evaluation of spatiotemporal RD3 transcriptional loss in NB. We investigated the expression of RD3 mRNA in FFPE NB (n = 100) tissues utilizing RNAScope with custom archived RD3 specific probes comparing it with the matched RD3 protein (IHC) expression patterns. With positive (PPIB) and negative (DapB) controls in place, our results displayed definite and specific RD3 mRNA expression in NB. We observed a statistically significant association of RD3 mRNA loss to the disease progression and poor clinical outcomes. Further, we observed significant correlation to the loss of RD3 transcription to the evolution of progressive disease. More importantly, the expression gradients of RD3 mRNA observed with RNAScope assay (compared in terms of no treatment vs progressive disease, primary vs metastatic disease, relapsed vs no relapse, favorable to unfavorable disease) illustrate the concordance with the RD3 IHC outcomes. Our results provide a reference basis for better RD3 mRNA readouts in NB, and more importantly identify the technically sound, adaptable (high throughput/single event, manual/automatic) RNAScope in situ hybridization assay as an alternate approach to detect spatiotemporal expression of RD3 mRNA. Together, the results provide a new, descriptive, and efficient platform, RNAScope for assessing spatiotemporal RD3 mRNA expression that could better inform disease prognosis and therapy response in children with NB.

Funding: This work was partially or in full, funded by Department of Defense DoD-CA210339 and Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-045.

Citation Format: Dinesh Babu Somasundaram, Sheeja Aravindan, Zhongxin Yu, Ashley Baker, Natarajan Aravindan. RNAScope in situ hybridization as a novel approach for the spatiotemporal assessment of RD3 mRNA expression in neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6722.
Abstract 6729: Urea cycle metabolism is disturbed in Fibrolamellar carcinoma
Authors: Shirani Mahsa , Levin Solomon , Tomasini Michael D. , Knox James , Shebl Bassem , Requena David , Clark Jackson , Heissel Søren , Alwaseem Hanan , Surjan Rodrigo , Lahasky Ron , Molina Henrik , Lyons Barbara , Migler Rachael D. , Coffino Philip , Simon Sanford M.
Published: April 2023

In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurrence and occasionally causes death. Using mass spectrometry, we quantitatively analyzed the proteomes of FLC patient’s tumor and adjacent normal, to find pathways that are changed in FLC. These data identified multiple proteins that were altered in the proteome, among these, enzymes involved in metabolism of ammonia. These results were confirmed with immunofluorescence demonstrating that these alterations occur in all tumor cells. These results suggest that FLC cells have defects in the two primary ammonia detoxification pathways in the liver, which are responsible for detoxification of 70% of the ammonia in the body: 1) consumption of ammonia by glutamine synthetase (GLUL), and 2) addition of ammonia by ornithine carbamoyltransferase (OTC) to the urea cycle. Additionally, they also generate extra ammonia because of upregulation of glutaminase (GLS). This was tested with a targeted metabolomics of the reactants and products of these enzymes. The results were consistent with both a loss of the two pathways for consumption of ammonia activation of a pathway for generating ammonia. This production of ammonia is consistent with the observation that surgical resection of fibrolamellar reduces the ammonia in patients. All FLC patients with hyperammonemic encephalopathy and documented urine test results showed increased urinary orotic acid, evidence of blockage of the OTC pathway. This study implies that hyperammonemic encephalopathy in FLC may require alternatives to commonly used therapies.

Citation Format: Mahsa Shirani, Solomon Levin, Michael D. Tomasini, James Knox, Bassem Shebl, David Requena, Jackson Clark, Søren Heissel, Hanan Alwaseem, Rodrigo Surjan, Ron Lahasky, Henrik Molina, Barbara Lyons, Rachael D. Migler, Philip Coffino, Sanford M. Simon. Urea cycle metabolism is disturbed in Fibrolamellar carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6729.
Abstract 673: p38γ MAPK remodels tumor microenvironment facilitating breast cancer progression and brain metastasis
Authors: Yuan Xiangliang , Chen Hao-Nien , Badu-Nkansah Akosua , Duan Yimin , Yu Dihua
Published: April 2023

Major neoplastic diseases have high incidences of brain metastasis (BrM) which is a clinically dreadful consequence of advanced cancers with an abysmal prognosis. Unfortunately, our understanding of the biology and molecular mechanisms underlying BrM remains rudimentary. Improving our understanding of the pathogenesis of BrM will facilitate the rational development and prioritization of new therapeutic strategies. Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine protein kinases that are key players in various intracellular signaling pathways controlling many cellular processes, e.g., growth, differentiation, stress responses, and immune defense which have a key role in cancer progression. However, little is known regarding the role of MAPKs in the pathogenesis of BrM. Here, we report that p38γ mitogen-activated protein kinase (MAPK12) is overexpressed in multiple tumor types and associated with aggressive breast cancer progression and a poor outcome in BrM patients. Interestingly, we found that the lack of p38γ did not impact the proliferation of tumor cells ex vivo, but significantly reduced both the primary tumor growth and BrM in EO771 and other mammary tumor-bearing immunocompetent mouse models. Our preclinical data indicated that p38γ promoted tumor progression and BrM through remodeling tumor microenvironments in vivo. We have mapped the immune landscape of BrM lesions of EO771-bearing mice using high-dimensional single-cell profiling. Clearly, cytometry by time-of-flight (CyTOF) analysis of the BrM immune infiltrates revealed reprogramming of the immunosuppressive brain microenvironment toward a proinflammatory and antitumoral state with tumor-intrinsic p38γ depletion, as indicated by highly enriched dendritic cell (DC) and cytolytic T cell (CTL) populations in BrM. To explore the impact of p38γ depletion in breast cancer cells, we performed RNA sequencing on EO771 cells having p38γ knocked down versus the control cells. We found that p38γ knocked down led to the upregulation of pathways related to immune cell movement, thereby leading to an increased accumulation of antitumor immune cells in BrM. Together, our findings uncover an important role for tumor-intrinsic p38γ in shaping the brain immune microenvironment and underscore the potential of specific p38γ blockade in enhancing antitumor immunity for the treatment of breast cancer BrM.

Citation Format: Xiangliang Yuan, Hao-Nien Chen, Akosua Badu-Nkansah, Yimin Duan, Dihua Yu. p38γ MAPK remodels tumor microenvironment facilitating breast cancer progression and brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 673.
Abstract 6748: Tumor regression grade combined with post-therapy lymph node status: A novel independent prognostic factor for patients treated with neoadjuvant therapy followed by surgery in resectable, locally advanced gastroesophageal junction and gastric carcinoma
Authors: Yin Hongyan , Yao Qian , Jia Keren , Niu Dongfeng , Chong Xiaoyi , Li Yanyan , Feng Xujiao , Li Yilin , Shen Lin , Chen Yang
Published: April 2023

Background: Tumor regression grade (TRG) is a measure of histopathological response to neoadjuvant therapy (NAT). Post-therapy lymph node (ypN) metastasis was reported as a prognostic factor. However, the evaluation of the treatment effectiveness of NAT has not been well studied. Here, we explored whether TRG combined with ypN status could be a prognostic factor for gastroesophageal junction (GEJ) and gastric cancer (GC).

Methods: 376 patients with GEJ or GC receiving NAT in Peking University Cancer Hospital were retrospectively collected from January 1, 2003 to June 30, 2021. According to TRG and ypN status, patients were innovatively categorized into four groups: TRG0N0, TRG1-3N0, TRG0-1N+, and TRG2-3N+. We applied Kaplan-Meier method and log-rank test to examine the differences in disease free survival (DFS) and overall survival (OS) among four groups. Univariate and multivariate analyses were performed to elucidate the relationships between TRG combined with ypN status and prognosis.

Results: We observed significant survival differences among the four groups (P < 0.001, respectively). Median DFS and OS of patients with TRG0N0, TRG1-3N0 and TRG0-1N+ were not reached, whereas median DFS and OS of patients with TRG2-3N+ were 17.37 months (95%CI, 14.14 to 20.60 months) and 39.97 months (95%CI, 27.05 to 52.89 months). TRG combined with ypN status was an independent predictor for both DFS (P < 0.001) and OS (P < 0.001) in multivariate analysis. Additionally, TRG combined with ypN status was significantly associated with different NAT (P < 0.001). The TRG0N0 rate in patients accepting immunotherapy was 31.9%, which was the highest in all regimens.

Conclusion: Our results demonstrate that TRG combined with ypN status is a novel independent predictor of both DFS and OS in resectable, locally advanced GEJ and GC. Neoadjuvant immunotherapy achieved the highest TRG0N0 rate.

Citation Format: Hongyan Yin, Qian Yao, Keren Jia, Dongfeng Niu, Xiaoyi Chong, Yanyan Li, Xujiao Feng, Yilin Li, Lin Shen, Yang Chen. Tumor regression grade combined with post-therapy lymph node status: A novel independent prognostic factor for patients treated with neoadjuvant therapy followed by surgery in resectable, locally advanced gastroesophageal junction and gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6748.
Abstract 675: Understanding SETDB1-mediated tumor immune evasion mechanism in endometrial cancer
Authors: Salari Kiarash , Wells Matthew , Johnson Eleanor R. , Li Tianyue , Hao Jiaqing , Li Bing , Yang Shujie
Published: April 2023

SETDB1 is characterized as an important epigenetic regulator in many different cancers and involved in transcriptional silencing of many different processes and pathways. It is often overexpressed in many different cancers including colon, melanoma, breast and pancreatic cancers and its high expression is correlated with worse patient outcome. Often associated with repressing complexes such as HUSH and KAP1/KRAB, it has been shown to repress many different genes such as p53 and p21 through histone H3K9 methylation. In addition, it has been implicated in regulating transposable elements (TEs), primarily of the LTR types as well as SINEs and LINEs. Activation of TEs is key to induction of anti-viral response genes such as the type I interferon pathway and immune-stimulated genes (ISGs) which in turn promote innate immune response against the tumor. Here, we show that SETDB1 promotes tumor growth in endometrial cancer possibly through establishing an immune evasion from the innate immune system. Mice subcutaneously injected with SETDB1 depleted endometrial cancer cells show prolonged survival time and reduced tumorigenesis. In NSG mice, we demonstrate that subcutaneously injected endometrial cancer cell, ISHIKAWA, shows both increased penetration and recruitment of macrophages in the tumor when SETDB1 is knocked out. Our macrophage-cancer cells co-culture in-vitro assay suggests that macrophages induce greater killing in SETDB1-/- cells when stimulated into M1 phenotype more so than in WT cancer cells. Further, tumor IHC analysis shows greater proliferation capacity for control tumor relative to SETDB1-/- as evident by greater mitotic figures per unit area and Ki67 staining score. We further show that cytokines such as CCL5 and CXCL9 (previously reported for their positive correlation with T-cell infiltration in tumor) are significantly upregulated in SETDB1 knockout tumors relative to non-targeting control tumors (NT) as well as in cell lines. Endometrial cancer TCGA data show significant negative correlation between SETDB1 and markers of T-cell infiltration such as CD8A and IFNG as well as cytokines CCL5 and CXCL9. Overall, these findings show important implication of SETDB1 in macrophage recruitment and macrophage-mediated killing of cancer cells in addition to its role in promoting tumor cells proliferation.

Citation Format: Kiarash Salari, Matthew Wells, Eleanor R. Johnson, Tianyue Li, Jiaqing Hao, Bing Li, Shujie Yang. Understanding SETDB1-mediated tumor immune evasion mechanism in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 675.
Abstract 6756: Ramucirumab plus paclitaxel as second-line treatment in patients with advanced gastric cancer who previously treated with first-line nivolumab plus chemotherapy
Authors: Jeon Youngkyung , Jeong Sun Young , Jang Jaeyeon , Jung Ye Ji , Choi Daeho , Hong Joohyun , Kim Seung Tae , Kang Won ki , Lee Jeeyun
Published: April 2023

Background: For human epidermal growth factor receptor 2 (HER2)-negative advanced gastric cancer (AGC), a combination of fluoropyrimidines and platinum agents was the standard first-line treatment. After the failure of the first-line therapy, ramucirumab plus paclitaxel was the standard second-line treatment. In the CheckMate 649 study, nivolumab plus chemotherapy has been considered a new standard first-line treatment in previously untreated AGC. However, the role of ramucirumab plus paclitaxel as 2nd line after the failure of nivolumab plus chemotherapy is not confirmed.

Methods: We analyzed AGC patients with ramucirumab plus paclitaxel as second-line therapy, who failed the nivolumab plus chemotherapy (capecitabine plus oxaliplatin (XELOX) or 5-fluorouracil plus oxaliplatin (FOLFOX)) as frontline therapy at Samsung Medical Center, South Korea.

Results: Under the routine clinical practice, 23 AGC patients, who progressed after first-line chemotherapy with nivolumab plus chemotherapy, were treated with ramucirumab plus paclitaxel between Dec 2021 to Sep 2022. The median age was 56 (range, 24-76), and 18 (78.3%) patients received nivolumab plus XELOX, while 5 (21.7%) patients received nivolumab plus FOLFOX. The overall response rate (ORR) to ramucirumab plus paclitaxel was 10.0% (2 of PR) and the disease control rate was 55.0%. At the median follow-up of 4.5 months, the median progression-free survival (PFS) from second-line ramucirumab plus paclitaxel commencement was 2.7 months (95% confidence interval (CI), 1.7-3.7); 6.9 months (95% CI, not calculated) in the first nivolumab plus chemotherapy responders(n=7), and 2.3 months (95% CI, 1.6-3.0) in non-responders(n=15) (p=0.232). At the time of data cutoff, 6 patients continued to receive ramucirumab plus paclitaxel and the median OS was 6.3 months (95% CI, 4.9-7.7).

Conclusions: This analysis showed that ramucirumab plus paclitaxel as 2nd line therapy was not sufficient in AGC patients after the failure for nivolumab plus chemotherapy. The new innovative 2nd line therapy might be needed in AGC patients after nivolumab plus chemotherapy.

Citation Format: Youngkyung Jeon, Sun Young Jeong, Jaeyeon Jang, Ye Ji Jung, Daeho Choi, Joohyun Hong, Seung Tae Kim, Won ki Kang, Jeeyun Lee. Ramucirumab plus paclitaxel as second-line treatment in patients with advanced gastric cancer who previously treated with first-line nivolumab plus chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6756.
Abstract 6772: Powerful background reduction in fluorescent tissue stains with an improved proximity-based technology for detection of protein-protein interactions
Authors: Elofsson Hampus , Raykova Doroteya , Wicher Agata Zieba
Published: April 2023

Immunofluorescent staining of tissues via in situ proximity ligation assay (isPLA) is now a well-established tool for highly sensitive detection of protein-protein interactions and their localization. It finds versatile applications in basic research, understanding of cellular processes, visualization of tissue architecture, and identification of biomarkers, among others. However, standard IF and isPLA techniques alike may suffer from high background. Tissues are particularly affected by background problems caused by autofluorescence and tissue infiltration of various cell types (e.g., immune, endothelial) where the fluorescently labelled oligos used for detection in isPLA may bind unspecifically. To address this, we developed a next generation isPLA-based technology for fluorescent detection of protein interactions in FFPE and fresh frozen human and mouse tissues, called NaveniFlex Tissue. In contrast to previously available protocols, it can visualize signal that would otherwise be obscured by background, thereby increasing sensitivity. Here, we compare the ability of NaveniFlex Tissue and similar commercial kits to detect the interaction between low abundance proteins Podocalyxin and Ezrin in the glomeruli of healthy kidney and in breast cancer, where co-expression is a prognostic marker for increased metastatic potential. While other kits generate high background which masks isPLA signal entirely, NaveniFlex Tissue dramatically reduces cell-based background and visualizes the interaction in its specific localization. Furthermore, in a TMA staining for the Mucin 16/Mesothelin interaction, our method successfully demonstrates strong and clear signal in stage III ovarian cancer. Moreover, it sensitively detects even low abundance interaction in a stage Ia tumor. In contrast, a different commercial kit detects significantly less interactions in the stage III tumor, while in the lower grade tumor it does not generate signals above background level. Therefore, NaveniFlex Tissue improves detection in tissues with varying disease progression, thus adding information and prognostic value to the staining. Taken together, our data illustrate that NaveniFlex Tissue outperforms current isPLA-based techniques by efficiently reducing background, which improves visualization of signal and signal-to-noise ratios in various healthy and diseased tissues.

Citation Format: Hampus Elofsson, Doroteya Raykova, Agata Zieba Wicher. Powerful background reduction in fluorescent tissue stains with an improved proximity-based technology for detection of protein-protein interactions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6772.
Abstract 6773: Spatial dynamics of cytotoxic T lymphocyte exhaustion in reactive and tumoral tissue
Authors: De Schepper Maxim , Antoranz Asier , Dubroja Nikolina , Floris Giuseppe , De Smet Frederik , Bosisio Francesca
Published: April 2023

Background The study of cytotoxic T-lymphocytes (Tcy) has shown increased attention from the scientific community in the last years because of their predictive role in anti-cancer therapy with specific interest in immune check point blocking therapy. However, most of these studies have used bulk or dissociation-based single-cell technologies to draw their biological insights. Lately, new spatial -omics technologies are increasing our knowledge on this population by adding the spatial location of these cells in-situ in the native structure of the tissue. In this study, we have compared the activation of Tcy in reactive and cancerous conditions at single cell level and in the micro-anatomical context of the tissue.

Experimental procedures One tissue micro array slide containing: reactive tonsil, inflammatory liver disease, breast cancer, glioblastoma and melanoma was stained using the COMET™ instrument (Lunaphore Technologies SA) for a multiplex panel of 20 markers. The derived multiplexed images were analyzed using the DISSCOVERY software platform developed by the MILAN Unit at KU Leuven (Belgium). 12/20 markers were specifically aimed at identifying the main cell populations in the tissue (CD11b, CD163, CD20, CD3, CD31, CD4, CD56, CD68, CD8, CK, FOXP3, S100) while the remaining 8 markers were aimed at adding functional information on immune cell types of interest (CD69, HLA-DR, Ki67, LAG3, OX40, PD-1, PD-L1, TIM3).

Results The general activation levels of the Tcy in the reactive conditions were higher than in the three cancer subtypes. When looking at Tcy activation as a function of the distance from the vascular structures (identified via CD31+ endothelial cells), we observed an orderly distribution of activated Tcy around CD31+ endothelial cells with a gradient of increasing exhaustion levels with increasing distance from the vessel in the reactive conditions. In the cancer samples, on the contrary, a patchy disorderly organized pattern of exhausted and activated Tcy was observed around the vessels.

Conclusion The spatial location of inflammatory cells plays a critical role to understand their functional behavior and thanks to technological progress it is now possible to start doing this at scale. The study of the dynamics of exhaustion of Tcy in the tissue will help clarify the interplay between cancer cells and immune environment. These results will be crucial to better select patients who may benefit the most of immune check point blockade.

Citation Format: Maxim De Schepper, Asier Antoranz, Nikolina Dubroja, Giuseppe Floris, Frederik De Smet, Francesca Bosisio. Spatial dynamics of cytotoxic T lymphocyte exhaustion in reactive and tumoral tissue. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6773.
Abstract 678: Investigating the mechanisms involved in HMGB1-dependent DNA uptake and STING activation in dendritic cells
Authors: Celias Daiana P. , Hänggi Kay , Ruffell Brian
Published: April 2023

Background: The cGAS-STING pathway is critical for the development of anti-tumor immunity. Activation of host STING can occur by sensing of extracellular DNA by intratumoral dendritic cells (DCs). After chemotherapy and under conditions of chromosomal instability, tumor cells release double-stranded DNA. We have previously shown DCs are able to internalize DNA in a dynamin-dependent manner, suggesting that a membrane receptor might mediate DNA endocytosis. DNA uptake also requires the presence of the high mobility group box protein 1 (HMGB1). HMGB1 has multiple known receptors such as TLR2, TLR4, RAGE and CD24. However, whether these receptors are involved in DNA engulfment by DCs remains unknown. Here, we sought to elucidate the mechanisms facilitating DNA uptake and subsequent STING activation in DCs.

Methods: Bone marrow derived (BM)DCs obtained from wild type (WT), RAGE, TLR2 or TLR4-deficient mice, as well as and WT or CD24-deficient MutuDC1940 cells, were cultured in the presence or absence of Cy5-labeled plasmid DNA, a recombinant Flag-tagged HMGB1 protein (rHMGB1) and/or DMXAA. Cy5-DNA uptake, Flag-HMGB1 binding and IRF3 phosphorylation was analyzed by flow cytometry or immunofluorescence microscopy. MutuDC1940 cells were also treated with plasmid DNA, rHMGB1, tumor debris (HS) and/or L-Leucyl-L-Leucine methyl ester (LLOMe) and galectin-3 clustering was quantified by immunofluorescence. Statistically significant differences were determined by ANOVA test.

Results: DCs take-up Cy5-DNA when rHMGB1 was added, and it was HMGB1 dose-dependent, supporting HMGB1 as a key factor for DNA uptake. We found that TLR2, TLR4, RAGE or CD24 deficient DCs were capable of internalizing Cy5-DNA in the presence of rHMGB1 to the same extent as WT DCs, indicating that none of these receptors are necessary for HMGB1-mediated DNA uptake. DNA-HMGB1 stimulation triggered IRF3 phosphorylation in DCs, suggesting extracellular DNA enters the cytosol and is sensed by cGAS, leading to STING activation. In support of this, DNA-HMGB1 treatment induced an increase in lysosomal membrane permeabilization, as measured by the extent of galectin-3 clusters within cells.

Conclusion: HMGB1 promotes DNA uptake by DCs independently of TLR2, TLR4, RAGE or CD24, suggesting other receptor(s) mediate the process of HMGB1-dependent DNA internalization. Further, DNA-HMGB1 induced lysosomal membrane destabilization which may facilitate DNA release into the cytosol for sensing and trigger activation of the cGAS-STING pathway.

Citation Format: Daiana P. Celias, Kay Hänggi, Brian Ruffell. Investigating the mechanisms involved in HMGB1-dependent DNA uptake and STING activation in dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 678.