bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5027: Targeting ARAF p.S214C mutation with sorafenib in non-small cell lung cancer
Authors: Lee Carol , Chan Sze Man , Yeung Sai Fung , Tsui Stephen Kwok-Wing
Published: April 2023

Sorafenib is an FDA-approved multi-kinase inhibitor for the treatment of several cancer types, but is still under investigation for non-small cell lung cancer (NSCLC). Although sorafenib has not shown significant survival benefit in clinical trials for NSCLC patients, one study identified a patient with advanced lung adenocarcinoma harboring ARAF p.S214C mutation as an exceptional sorafenib responder. As NSCLC lacks clinically validated predictive biomarkers for drug responses, we aim to evaluate the potential of ARAF p.S214C mutation as a biomarker for sorafenib sensitivity and its mechanism of action. Constructs with ARAF p.S214C mutation, ARAF-wild type (WT) and EGFR-vector control were ectopically expressed in a lung adenocarcinoma cell line, H2023, by retroviral transduction. Western blot confirmed stable overexpression of ARAF proteins over time. MTT assay showed that sorafenib inhibited growth of cells expressing ARAF p.S214C more pronouncedly than cells expressing ARAF-WT or vector control. In addition, spheroids formed by cells expressing ARAF p.S214C were more sensitive to sorafenib inhibition, indicating sorafenib sensitivity of ARAF p.S214C in NSCLC in vitro. To evaluate whether such sorafenib sensitivity is dependent on the kinase activity of ARAF, phospho-MEK and MAPK levels in mutant cells will be determined. As ARAF belongs to the Raf protein family which also includes BRAF and RAF1, knockdown studies and co-immunoprecipitation will be performed to determine whether BRAF and RAF1 are essential for ARAF p.S214C activity. The in vivo sorafenib sensitivity of ARAF p.S214C will be validated in tumor xenografts established from cells expressing the above constructs upon sorafenib treatment. Tumor volume will be assessed and expression of the tumor marker cytokeratin will be evaluated by immunohistochemistry. This study may reveal ARAF p.S214C as a potential biomarker for sorafenib sensitivity and a new target for precision medicine in NSCLC.

Citation Format: Carol Lee, Sze Man Chan, Sai Fung Yeung, Stephen Kwok-Wing Tsui. Targeting ARAF p.S214C mutation with sorafenib in non-small cell lung 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 5027.
Abstract 5028: The regulation of inflammatory signaling in cancer via A20 phosphorylation
Authors: Palacios Tania Lopez , Chan Tsz-Yin , Egbert Christina , Truman Jacob , Ashworth Spencer , Vaughan Alec , Andersen Joshua L.
Published: April 2023

A20 is an anti-inflammatory protein with a dual ubiquitin-editing activity. Through its N-terminal ovarian tumor (OTU) domain, A20 negatively regulates NF-ĸB by removing the K63-linked ubiquitin chains on RIP1 that act as platforms for NF-ĸB activation downstream of tumor necrosis factor alpha (TNFα). On the other hand, A20 also catalyzes the addition of K48-linked ubiquitin chains on RIP1 via its C-terminal zinc finger domain 4 (ZnF4) to promote its proteasomal degradation. Through both mechanisms, A20 is a means of negative feedback on TNFα-induced NF-ĸB activity. Importantly, inactivation of A20 is frequently found in B-cell lymphomas, where loss-of-function A20 truncations promote cell proliferation and upregulation of NF-ĸB signaling. Among the post-translational modifications (PTMs) reported in A20, there are several phosphorylation events in its C-terminus, including within the ZnF4 domain. Besides its known E3 ubiquitin ligase activity, previous reports show that the C-terminal region of A20 is important for ubiquitin binding, having preference for K63-linked poly-ubiquitin. However, a little is known about the effects of phosphorylation at this region on A20 activity as well as its consequences in downstream signaling. Our current work suggests that phosphorylation in the C-terminus of A20 inhibits its E3 ubiquitin ligase activity and disrupts its interaction with ubiquitin, which may also affect its deubiquitinase activity by preventing its recruitment to ubiquitinated proteins in the TNFα receptor (TNFR) complex. We also propose that the C-terminal region is important for the formation of the A20 ubiquitin-editing complex, necessary to downregulate NF-ĸB-activated inflammatory signals. Based on these observations, we hypothesize that not only gene-inactivating mutations, but also post-translational mechanisms, such as phosphorylation, inhibit A20 to promote cancer development. Our research is now focused on understanding the upstream signaling that regulates A20 phosphorylation.

Citation Format: Tania Lopez Palacios, Tsz-Yin Chan, Christina Egbert, Jacob Truman, Spencer Ashworth, Alec Vaughan, Joshua L. Andersen. The regulation of inflammatory signaling in cancer via A20 phosphorylation. [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 5028.
Abstract 5053: A critical role for copper in enhanced radioirdide uptake in thyroid cancer cells
Authors: Brookes Katie , Zha Ling , Kim Jana , Kannappans Vinodh , Wang Weiguang , Sunassee Kavitha , Blower Philip J. , Smith Vicki E. , Read Martin L. , McCabe Christopher J.
Published: April 2023

New drug approaches are urgently required to improve radioiodide (RAI) uptake for efficient ablation of thyroid cancer cells in RAI-refractory disease. Employing high-throughput screening of FDA-approved compounds we recently identified drugs capable of robust induction of sodium iodide symporter (NIS) activity to promote RAI uptake1. In particular, a leading drug candidate - the well-established anti-alcoholism drug disulfiram (DSF) - had not been previously implicated in regulating NIS. Here, we demonstrate that the ability of DSF to increase RAI-uptake in thyroid TPC-1 (3.1-fold; p<0.01) and 8505C (4.9-fold; p<0.001) cells can be significantly potentiated by combination with Cu2+ to 5.1-fold and 18.9-fold increases respectively. Despite promising data, DSF is known to have poor bioavailability in vivo due to its rapid metabolism to diethyldithiocarbamate (DDC) within the stomach and circulation with subsequent methylation in the liver. Whilst methylated-DDC did not have any effect on NIS function, DDC chelated to divalent copper ions [Cu(DDC)2] was highly effective at increasing RAI uptake (up to 8-fold; P<0.001) in multiple thyroid cell types and induced significant NIS protein expression (up to 36.2-fold; 250nM; P<0.001). Importantly, Cu(DDC)2 retained the ability to enhance NIS function in thyroid cells ablated for expression of either VCP or its co-factor NPL4, indicating its effect on NIS was via VCP-independent pathways. Instead, a transcriptional effect of Cu(DDC)2 was revealed by significant induction of NIS mRNA levels in TPC-1 (8.5-fold; P<0.001) and 8505C (104.8-fold; P<0.001) cells. In wild-type Balb/c mice the intraperitoneal administration of albumin nano-encapsulated Cu(DDC)2 significantly induced thyroidal uptake of technetium-99m (99mTc) after 30 min (~40% increase; n=11 per group; 3 mg/kg dose; P<0.001). Thus, our study demonstrates a promising drug strategy utilizing copper to significantly enhance NIS function with clinical potential to improve treatment effectiveness in RAI-refractory thyroid cancer patients.

1Read ML, Brookes K, Thornton CEM, Fletcher A, Nieto HR, Alshahrani M, Khan R, Borges de Souza P, Zha L, Webster JRM, Alderwick LJ, Campbell MJ, Boelaert K, Smith VE, McCabe CJ (2022) Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter. Cell Chem Biol. 29(3):502-516.e7.

Citation Format: Katie Brookes, Ling Zha, Jana Kim, Vinodh Kannappans, Weiguang Wang, Kavitha Sunassee, Philip J. Blower, Vicki E. Smith, Martin L. Read, Christopher J. McCabe. A critical role for copper in enhanced radioirdide uptake in thyroid cancer 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 5053.
Abstract 5199: B7-H3 & PD-L1 double-humanized BALB/c mouse: a novel animal model for preclinical studies of human B7-H3 antibodies or bispecific antibodies
Authors: Guo Shiying , Song Lingyu , Qin Xin , Xu Jianming , Sun Hongyan , Ju Cunxiang , Wang Hongyu , Chen Santi Suryani , Li Zhiying , Moore Mark Wade , Zhao Jing , Gao Xiang
Published: April 2023

Immune checkpoint molecule B7-H3, also known as CD276, is a member of the B7-CD28 family of immunomodulatory proteins. It is a type I membrane protein with sequence similarities to the extracellular domain of programmed death-ligand 1 (PD-L1).

B7-H3 is highly expressed in most human cancers, but has limited distribution in normal tissues, remaining elusive of its receptor. Due to its promising safety as a dominant tumor target, various strategies have been developed to modulate the effect of B7-H3 via monoclonal antibodies, bispecific antibodies, ADC, or CAR-T. To study the effect of these therapies in an immunocompetent mouse model, we established a double humanized B7-H3 and PD-L1 mouse model on BALB/c background (BALB/c-hB7-H3/hPD-L1). In this model, the extracellular domain of murine fragments was replaced by the human counterparts while the trans-membrane and cytoplasmic domain were kept intact. When engrafted with CT26 colon cancer cells, which stably overexpress human B7-H3 and PD-L1 while endogenous murine counterparts were knocked out, the tumor growth was inhibited certain degree by anti-B7-H3 antibody (8H9 Biosimilar, 20mpk, TGI=18.56%) treatment while inhibited significantly after the monotherapy of anti-PD-L1 (Tecentriq, 3 mpk, TGI=55.89%, p<0.001). The same dosage combination of anti-B7-H3 and anti-PD-L1 had a significant inhibition on tumor growth (TGI=76.85%, p<0.001), and had a synergistic effect (CDI<0.7) compared with the monotherapy. Analysis of tumor-infiltrating lymphocytes (TILs) at the end of the efficacy study showed that the proportion of CD45+ immune cells was significantly increased in all of the treated groups. The NK cells were significantly increased and the Treg cells were significantly decreased especially in the combined treatment group.

Based on the above, the B7-H3 and PD-L1 double humanized mouse model is suitable for the pre-clinical evaluation of mono or combined immune checkpoint blockade with anti-human B7-H3 and PD-L1 therapy.

Citation Format: Shiying Guo, Lingyu Song, Xin Qin, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. B7-H3 & PD-L1 double-humanized BALB/c mouse: a novel animal model for preclinical studies of human B7-H3 antibodies or bispecific antibodies. [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 5199.
Abstract 5201: TNFR2 humanized mice: an ideal model for studying anti-TNFR2 antibodies
Authors: Wang Huiyi , Li Ying , Zhang Mingkun , Su Xu , Chen Santi Suryani , Li Zhiying , Moore Mark Wade , Zhao Jing , Gao Xiang , Ju Cunxiang
Published: April 2023

Tumor necrosis factor (TNF) receptor 2 (TNFR2) is mainly expressed on the surface of regulatory T cells (Tregs) and CD8+ T cells, which can stimulate the proliferation of these cells through NF-kB activation. TNFR2 is also highly expressed on the surface of various human tumors, such as colorectal cancer and lung cancer. Therefore, a TNFR2 antagonist blocking the binding of TNFR2 to its ligand that targets TNFR2+ tumor-infiltrating Tregs can directly enhance the killing ability of effector T cells (Teff) against TNFR2-expressing tumors. Many pharmaceutical and biotech companies have lined up TNFR2-targeting drugs, and most TNFR2 antibodies related research is in the early preclinical/clinical stage.

We established a humanized TNFR2 (hTNFR2) genetically engineered mouse model on the BALB/c background, in which the extracellular domain of the mouse TNFR2 was replaced with its human counterpart, and the murine transmembrane and cytoplasmic domain kept intact. The hTNFR2 chimeric protein is mainly expressed on Teff and Treg cells in BALB/c-hTNFR2 and BALB/c-hPD1/hPDL1/hTNFR2 mice, which is similar to mouse TNFR2 protein expression profiles in wild-type mice. Our data showed that anti-TNFR2 antibodies could significantly inhibit tumor growth in BALB/c-hTNFR2 mice bearing CT26 tumor cells or BALB/c-hTNFR2 mice bearing CT26-hTNFR2 tumors. Furthermore, within the tumor-infiltrating lymphocytes, there was a reduction in Tregs cells and an increase in cytotoxic CD8 T cells, which accurately mimics the mechanism of action of anti-TNFR2 antibodies.

In conclusion, BALB/c-hTNFR2 and BALB/c-hPD1/hPDL1/hTNFR2 mice are ideal models for studying the efficacy and pharmacodynamics of anti-TNFR2 antibodies as a single agent or in combination with anti-PD-1/PD-L1 therapies.

Citation Format: Huiyi Wang, Ying Li, Mingkun Zhang, Xu Su, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao, Cunxiang Ju. TNFR2 humanized mice: an ideal model for studying anti-TNFR2 antibodies. [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 5201.
Abstract 521: Discovery of novel TNKS inhibitors through structure-based virtual screening
Authors: Ryu Hwani , Seo Hye-Ran , Kim Hyo Jeong , Ahn Jiyeon
Published: April 2023

More than 80% of colorectal cancers (CRCs) have somatic mutations in the adenomatous polyposis coli (APC) gene, which affects abnormal WNT/β-catenin signaling. Since inhibition of tankyrases (TNKS1 and TNKS2) belonging to the poly(ADP-ribose) polymerase family stabilizes AXINs and induces degradation of β-catenin, drug development as a therapeutic target for APC-mutated CRCs is required. To identify selective small molecule inhibitors of TNKS, we performed pharmacophore modeling and structural docking studies targeting nicotinamide, adenosine, and both binding subsites of TNKS. Among the 8 million compounds, 149 of the nicotinamide site binders, 23 of the adenosine site binders, and 15 of the dual-site binders of TNKS were selected through computer-based virtual screening. Using in vitro TNKS1 and TNKS2 enzyme assays, we identified that TI2-N17 and TI3-D11 significantly inhibited TNKS1 and TNKS2 enzyme activities at low nanomolar levels. TI2-N17 and TI3-D11 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM, APC-mutated CRC cells. Both compounds exhibited anticancer effects on COLO320DM and SW403 cells. In addition, the combination treatment of 5-Fluorouracil (5-FU), an anticancer agent for CRC patients, and these compounds synergistically inhibited the proliferation of CRC cells compared to single compound treatment. Our findings demonstrated that TI2-N17 and TI3-D11 are therapeutic candidates targeting TNKS for APC-mutated CRCs treatment and suggested that the combined treatment with the compounds and 5-FU chemotherapy may be an effective therapeutic strategy for APC-mutated CRCs. Based on these compounds, further study will develop potent and selective TNKS inhibitors by designing and synthesizing novel derivative compounds.

Citation Format: Hwani Ryu, Hye-Ran Seo, Hyo Jeong Kim, Jiyeon Ahn. Discovery of novel TNKS inhibitors through structure-based virtual 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 521.
Abstract 5697: Predicting response to PD-L1 inhibition in NSCLC using a quantitative systems pharmacology model guided by immunogenomic data
Authors: Wang Hanwen , Arulraj Theinmozhi , Popel Aleksander S.
Published: April 2023

Introduction: Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer with an overall 5-year survival rate of 26%, which varies by the disease stage. Novel drug combinations involving immune checkpoint inhibitors are being investigated in clinical trials in NSCLC. In this study, we generated about 450 virtual patients that resembled actual patients with NSCLC and simulated tumor growth during PD-L1 inhibition using a quantitative systems pharmacology (QSP) model.

Methods: We recalibrated a previously developed QSP model of the cancer-immunity cycle (1) to NSCLC with parameters estimated by published experimental data. Proportions of CD8, CD4, and regulatory T cells (Tregs), M1 and M2 macrophages in lung adenocarcinoma and lung squamous cell carcinoma were downloaded from iAtlas database and used to calculate the ratios of CD8/CD4, Tregs/CD8, and M1/M2. The three immune subset ratios (observed data) were used to guide the virtual patient generation. We first randomly generated deviations around the baseline model parameters to account for inter-patient variability and then selected virtual patients with model-predicted immune subset ratios statistically matching the observed data. Finally, we simulated tumor growth during PD-L1 inhibition and predicted clinical response in the virtual patient cohort.

Results: Our model predicted an overall response rate of 16.3% with a 95% bootstrap confidence interval of (11.8, 22.4)% for the overall virtual patient cohort. It also showed that CD8 T cells, PD-L1 expression, CD8/CD4, CD8/Tregs, M1/M2 ratios, and the number of NSCLC-specific T cell clones were significantly higher in responders to PD-L1 inhibition. Furthermore, Tregs/CD8 and CD8/CD4 ratios were predicted to be the best predictive biomarkers according to ROC analysis with areas under ROC curve of 0.71 and 0.66.

Conclusion: Immunogenomic analysis of RNA-seq data on iAtlas allowed generation of realistic virtual patients using QSP models in immuno-oncology. With a virtual patient cohort guided by immunogenomic data, our model provided reliable efficacy prediction for immunotherapy and a tool to identify predictive biomarkers in NSCLC. 1. Wang H, Zhao C, Santa-Maria CA, Emens LA, Popel AS. Dynamics of tumor-associated macrophages in a quantitative systems pharmacology model of immunotherapy in triple-negative breast cancer. iScience. 2022;25:104702.

Citation Format: Hanwen Wang, Theinmozhi Arulraj, Aleksander S. Popel. Predicting response to PD-L1 inhibition in NSCLC using a quantitative systems pharmacology model guided by immunogenomic data. [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 5697.
Abstract 5982: A potent and selective CDK8 inhibitor ABM-3249 with excellent efficacies in multiple in vivo cancer models
Authors: Chen Chen , Huang Charles , Xu Min , Chen Xiuquan , Lv Xiaobing , Yang Chen , Zhao Lanjiao , Chen YouQin , Li Jizhi , Zhao Li
Published: April 2023

CDK8 is a member of the CDK family in which belongs to the serine/threonine protein kinase. It binds with cyclin C to regulate the transcription. By modulation of various gene expression programs, CDK8 associated with the mediator complex to sustain proliferation and viability of cancer cells. CDK8 has been identified as an important factor in cancer occurrence and development. It also involves in the regulation of cancer cell stress response to radiotherapy and chemotherapy, assists tumor cell invasion, metastasis and drug resistance. Therefore inhibition of CDK8 is regarded as a promising target for cancer therapy. ABM-3249 was discovered as a potent and selective CDK8 inhibitor with a novel chemical structure. It inhibited CDK8 kinase activity in biochemical assay and cellular binding assay, with IC50 values of 1.4 nM and 19nM respectively. ABM-3249 displayed good kinase selectivity: <50% inhibition on all other CDK family members at 1uM. Consistently, ABM-3249 reduced STAT1-S727 phosphorylation with an IC50 of less than 100nM. ABM-3249 showed good ADME properties with high oral bioavailability (~90%) in rodents. In efficacy studies, ABM-3249 exhibited excellent anti-tumor activities in multiple in vivo models. In an AML MV4-11 xenograft model at 25mpk oral dosing ABM-3249 achieved complete remission in all mice within 17 days and no tumor recurrence within one month after drug withdrawal. ABM-3249 showed good efficacies as a single agent, as well as a synergetic effect with anti-PD1 combination in a murine colon cancer MC38 model. It also showed an efficacy in a murine colon cancer B16F10 metastasis model. In addition, ABM-3249 displayed a good safety margin in a preliminary tox screen in rodents.

Citation Format: Chen Chen, Charles Huang, Min Xu, Xiuquan Chen, Xiaobing Lv, Chen Yang, Lanjiao Zhao, YouQin Chen, Jizhi Li, Li Zhao. A potent and selective CDK8 inhibitor ABM-3249 with excellent efficacies in multiple in vivo cancer models. [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 5982.
Abstract 600: Tumour and local lymphoid tissue interaction determines prognosis in high grade serous ovarian cancer
Authors: Lu Haonan , Wengert Georg , Lou Hantao , Paudel Reema , Patel Naina , Desai Saral , Crum Bill , Linton-Reid Kristofer , Chen Mitchell , Li Dongyang , Ip Jacey , Mauri Francesco , Pinato David J. , Rockall Andrea , Copley Susan J. , Ghaem-Maghami Sadaf , Aboagye Eric O.
Published: April 2023

Tertiary lymphoid structure (TLS) is an ectopic lymphoid organ found in some tumors. The presence of TLS is recently proposed as a predictive biomarker for immune checkpoint inhibitors (ICI) in mutation-driven tumors. It is also associated with prognosis in copy number-driven tumors, including high grade serous ovarian cancer (HGSOC), although the function of TLS and its interaction with copy-number alterations in HGSOC is not fully understood. In the current study, we comprehensively analyze immune profile, copy number profile and quantitative imaging profile from 242 HGSOC patients (HH cohort), and combine these with two public single cell RNA-sequencing datasets (Lambrechts cohorts; n=9, 29,999 cells). TLS-high HGSOC patients show significantly better progression free survival in both HH and TCGA cohorts. We show that presence of TLS in HGSOC tumors is associated with B-cell maturation, resulting in higher and more specific immunoglobulin production; TLS also leads to more cytotoxic tumor-specific T-cells activation and proliferation. Through integrated analysis of single cell RNA-sequencing and whole exome sequencing data, we reveal that the copy number loss of IL15 and CXCL10 may limit TLS formation in HGSOC; we also propose a list of genes that may dysregulate TLS function. A candidate gene, DCAF15, is found to confer resistance to NK-mediated cytotoxicity in HGSOC cell lines. Lastly, we develop a radiomics-based signature to predict presence of TLS, which independently predicts PFS in both HGSOC patients and ICI-treated NSCLC patients. Overall, we reveal that TLS coordinates intratumoral B-cell and T-cell response against HGSOC tumor, while cancer genome evolves to counteract TLS formation and function.

Citation Format: Haonan Lu, Georg Wengert, Hantao Lou, Reema Paudel, Naina Patel, Saral Desai, Bill Crum, Kristofer Linton-Reid, Mitchell Chen, Dongyang Li, Jacey Ip, Francesco Mauri, David J. Pinato, Andrea Rockall, Susan J. Copley, Sadaf Ghaem-Maghami, Eric O. Aboagye. Tumour and local lymphoid tissue interaction determines prognosis in high grade serous ovarian 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 600.
Abstract 6031: KYNU upregulation is a prominent feature of NRF2-activated cancers and is associated with tumor immunosuppression and poor prognosis
Authors: Letelier Ricardo A. Leon , Sater Ali H. Abdel , Chen Yihui , Wu Ranran , Dennison Jennifer B. , Park Soyoung , Irajizad Ehsan , Katayama Hiroyuki , Vykoukal Jody , Hanash Samir , Ostrin Edwin J. , Fahrmann Johannes F.
Published: April 2023

Activation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, either through gain-of-function mutations in the NRF2 encoding gene NFE2L2 or through loss-of-function of its suppressor, Kelch-like ECH-associated protein 1 (KEAP1), is a frequent manifestation in various malignancies. NRF2 activation exerts pro-tumoral effects in part by altering cancer cell metabolism. We previously reported a novel mechanism of NRF2 tumoral immune suppression through selective upregulation of the tryptophan metabolizing enzyme kynureninase (KYNU) in lung adenocarcinoma. In the current study, we explored the Pan-Cancer relevance of NRF2-mediated KYNU upregulation. We analyzed the gene expression dataset for 9,801 tumors representing 32 cancer types in The Cancer Genome Atlas (TCGA). Elevated KYNU expression levels paralleled increased gene-based signatures of NRF2-activation and was strongly associated with an immunosuppressive tumor microenvironment, marked by high expression of gene-based signatures of Tregs as well as immune checkpoint blockade-related genes CD274 (PDL-1), PDCD1 (PD-1), and CTLA4, regardless of cancer type. Cox proportional hazard models further revealed that increased tumoral KYNU gene expression was prognostic for poor overall survival in several cancer types, including thymoma, acute myeloid leukemia, low grade glioma, kidney renal papillary cell carcinoma, gastric adenocarcinoma, and pancreatic ductal adenocarcinoma (PDAC). Using PDAC as a model system, we confirmed that siRNA-mediated knockdown of NRF2 reduces KYNU mRNA expression, whereas activation of NFE2L2 (the encoding gene for NRF2) through either small molecule agonists or siRNA-mediated knockdown of KEAP1 upregulated KYNU. Metabolomic analyses of conditioned media from PDAC cell lines revealed elevated levels of KYNU-derived anthranilate, confirming KYNU as enzymatically functional. Collectively, our findings highlight the multi-cancer relevance of the NRF2-KYNU axis and of tumoral KYNU as a prognostic marker of poor overall survival associated with immunosuppression.

Citation Format: Ricardo A. Leon Letelier, Ali H. Abdel Sater, Yihui Chen, Ranran Wu, Jennifer B. Dennison, Soyoung Park, Ehsan Irajizad, Hiroyuki Katayama, Jody Vykoukal, Samir Hanash, Edwin J. Ostrin, Johannes F. Fahrmann. KYNU upregulation is a prominent feature of NRF2-activated cancers and is associated with tumor immunosuppression and poor prognosis [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 6031.
Abstract 6045: Role of PP2A methylation on methionine dependence of cancer
Authors: Andronicos Anna , Lin Da-Wei , Su Yi-Chen , Gabra Mari Ishak , Kong Mei , Kaiser Peter
Published: April 2023

Dependence on exogenous methionine is a well-known, but poorly understood cancer-specific metabolic requirement. Although most cancer cells can convert homocysteine to methionine, they are unable to grow when methionine depleted media is supplemented with homocysteine. In contrast, non-tumorigenic cells proliferate well in homocysteine media. Previous work from our laboratory has shown that cancer cells cultured in homocysteine medium increase metabolic flux towards the transsulfuration pathway, away from S-adenosylmethionine (SAM) synthesis resulting in overall reduced methylation potential. We have shown that methionine sensing occurs independently of the mTOR pathway, which is known to link amino acid abundance and cell growth. Instead, our results suggest that Protein Phosphatase 2A (PP2A) methylation is sensitive to changes in the SAM/SAH ratio. Reduced PP2A methylation correlates with reduced cell proliferation in homocysteine medium. Furthermore, overexpression of the PP2A demethylase Pme1 forcibly reduces PP2A methylation and induces methionine dependence in cells that normally proliferate in homocysteine medium. These findings suggest a model that links methionine metabolism to cell cycle progression via regulation of PP2A methylation. Here we use quantitative proteomics to define changes in B-subunit binding to PP2A during methionine starvation to investigate the molecular mechanism of this methionine dependence seen in cancer.

Citation Format: Anna Andronicos, Da-Wei Lin, Yi-Chen Su, Mari Ishak Gabra, Mei Kong, Peter Kaiser. Role of PP2A methylation on methionine dependence of 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 6045.
Abstract 6046: Enhanced Acod1 expression in cancer cells promotes immune evasion
Authors: Schofield James H. , Longo Joseph , Murphy Sean , Sheldon Ryan , Hawk Mark A. , Albano Emma , Jones Russell G. , Lu Xin , Schafer Zachary T.
Published: April 2023

The monoclonal antibody αPD-1 used in immune checkpoint blockade (ICB) therapy has made a substantial impact in clinical outcomes for melanoma, lung, and breast cancer patients. Despite the success of ICB in these contexts, such strategies are not universally effective due to unresponsiveness or development of resistance by the malignant cells. Poor understanding of the molecular mechanisms leading to ICB resistance is a crucial barrier to more effective ICB. Recent studies have demonstrated that the metabolic milieu of the tumor microenvironment (TME) can modulate the anti-tumor immune response. However, the specific mechanisms by which the secretion of cancer cell metabolites into the TME impacts the efficacy of ICB in unresponsive tumors remains poorly understood. To better characterize the factors that influence ICB, we utilized a murine model (Smad4−/−/Pten−/−/Trp53−/−) of castration-resistant prostate cancer (CRPC) that is responsive to αPD-1. Serial culture and passage of residual tumor cells following exposure to αPD-1 treatment into new recipient mice allowed us to develop an ICB-insensitive CRPC cell line. Unbiased metabolic analysis revealed that the αPD-1 resistant cells produced elevated levels of itaconate, a metabolite whose biological functions in epithelial cells remain poorly characterized. Consistent with this finding, the resistant cells display increased expression of aconitate decarboxylase 1 (Acod1), the enzyme responsible for itaconate production. Additionally, we have identified established human epithelial cancer cell lines which express high levels of ACOD1. Interestingly, conditioned media (CM) from the Acod1-high cell lines, but not the Acod1-low lines, restricts the proliferation and activation of naïve CD8+ T cells in vitro. CRISPR-mediated Acod1 reduction in the αPD-1 insensitive cells (Acod1KO) produces CM which promotes increased naïve CD8+ T proliferation and activation, suggesting a link between Acod1 abundance in cancer cells and the activity of cytotoxic T cells. Furthermore, Acod1KO tumors have increased CD8+ tumor infiltrating lymphocytes (TILs) and increased sensitivity towards αPD-1 ICB in vivo. Taken together, these data suggest that cancer cell intrinsic ACOD1 abundance may be predictive of ICB efficacy.

Citation Format: James H. Schofield, Joseph Longo, Sean Murphy, Ryan Sheldon, Mark A. Hawk, Emma Albano, Russell G. Jones, Xin Lu, Zachary T. Schafer. Enhanced Acod1 expression in cancer cells promotes immune evasion [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 6046.
Abstract 614: CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects
Authors: Glez-Vaz Javier , Azpilikueta Arantza , Ochoa Maria Carmen , Olivera Irene , Gomis Gabriel , Cirella Asunta , Luri-Rey Carlos , Álvarez Maite , Ciordia Sergio , Berraondo Pedro , Teijeira Álvaro , Corrales Fernando , Zapata Juan M , Melero Ignacio
Published: April 2023

CD137 (4-1BB) is a member of the TNFR family, whose expression is induced on antigen-primed T cells and that mediates potent costimulatory signals upon ligation by CD137L or agonist monoclonal antibodies. CD137 agonists attain immunotherapeutic antitumor effects in cancer mouse models and multiple agents of this kind are undergoing clinical trials. We show that cIAP1 and cIAP2 are physically associated with the CD137 signaling polyprotein complex. Moreover, cIAPs are required for CD137 signaling towards the NF-κB and MAPK pathways and for costimulation of human and mouse T lymphocytes. Functional evidence was substantiated with SMAC-mimetics that trigger proteasomal degradation of cIAPs and transfecting dominant-negative variants of cIAPs. Importantly, antitumor effects of agonist anti-CD137 mAbs are critically dependent on the integrity of cIAPs in cancer mouse models.

Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Maria Carmen Ochoa, Irene Olivera, Gabriel Gomis, Asunta Cirella, Carlos Luri-Rey, Maite Álvarez, Sergio Ciordia, Pedro Berraondo, Álvaro Teijeira, Fernando Corrales, Juan M Zapata, Ignacio Melero. CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects [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 614.
Abstract 6195: Assessing replication stress as an actionable therapeutic opportunity in chordoma
Authors: Punturi Nindo , Dolat Lee , Leung Wendy , Levy Joan , Zou Lee , Freed Daniel M. , Cote Gregory M.
Published: April 2023

Chordoma is a rare bone cancer with a high rate of disease recurrence and no approved systemic therapies. Tumor profiling studies indicate a significant number of chordomas are characterized by alterations in SWI/SNF chromatin remodeling and DNA damage repair genes, which have been associated with replication stress in other cancer types. We demonstrate here that preclinical chordoma models exhibit a replication stress phenotype and dependence on ATR activity. To assess the effect of ATR inhibition on cell viability, a panel of chordoma cell lines were treated with the ATR inhibitor elimusertib (BAY 1895344). In a subset of cell lines, elimusertib inhibited viability with low-nanomolar IC50 values. Upon treatment with the ATR inhibitor ceralasertib (AZD6738), DNA fiber assays revealed a mild decrease in replication fork speed and a significant increase in fork asymmetry in cells sensitive to ATR inhibitor, suggesting possible fork stalling or collapse in response to ATR inhibition. To assess genomic instability as a consequence of ATR inactivation and fork stalling or collapse, alkaline and neutral comet assays were performed to determine the presence of genome-wide ssDNA and/or dsDNA breaks, respectively. In a panel of chordoma PDX models, striking differential sensitivity to ATR inhibition was observed. In sensitive models, elimusertib (50 mg/kg, po, bidx3) promoted 85-90% tumor growth inhibition, in contrast to resistant models, where elimusertib treatment had no effect on tumor growth. Moreover, PDX models sensitive to ATR inhibition were sensitive to other therapies that exacerbate replication stress; for example, gemcitabine promoted multiple complete responses in a pediatric chordoma PDX model. Coupled with preclinical efficacy data, genomic and transcriptomic sequencing of differentially-sensitive chordoma models provided insight into the molecular factors associated with the replication stress phenotype in chordoma - illuminating potential biomarkers for patient selection. Finally, we explore various combination therapies and show that combining elimusertib with cisplatin or gemcitabine synergistically inhibits the viability of chordoma cell lines. Collectively, our data indicate that a subset of chordomas are characterized by intrinsic replication stress that can be therapeutically exploited by inhibiting ATR.

Citation Format: Nindo Punturi, Lee Dolat, Wendy Leung, Joan Levy, Lee Zou, Daniel M. Freed, Gregory M. Cote. Assessing replication stress as an actionable therapeutic opportunity in chordoma [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 6195.
Abstract 6205: Visualizing dynamics of long non-coding RNA TUG1 in live glioblastoma cells
Authors: Minematsu Ayaka
Published: April 2023

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that are not translated into proteins. lncRNA TUG1 (taurine upregulated gene 1) is highly expressed in various cancers, including glioblastoma. Previously we found that TUG1 is an essential molecule for cancer cells to cope with excessive replication stress in the S phase. Albeit its importance to cell cycle-related function, how it is dynamically regulated during the cell cycle is entirely unknown. Here, we established a system visualizing endogenous TUG1 molecules in live cells. TUG1 was tagged with stem-loop motifs recognized by a specific binding protein fused with GFP. We tracked TUG1 RNA by the GFP signal for 22 hours in T98G glioblastoma cells and found that the expression started from the later G1 phase. When cells were treated with Hydroxy Urea, a chemical that interferes with replication and increases replication stress, the expression of TUG1 was further upregulated in the S phase within 20 mins. We also observed some TUG1 molecules accumulated in several nuclear locations. It may indicate that TUG1 assembles to protect damaged DNA loci efficiently. Our results suggest that the expression of TUG1 is regulated in a cell cycle-dependent manner and is strongly connected with its function of TUG1. The current live-cell imaging system could help further discover the critical roles of TUG1 in cancer cells.

Citation Format: Ayaka Minematsu. Visualizing dynamics of long non-coding RNA TUG1 in live glioblastoma 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 6205.
Abstract 621: Aging dampens lymphoma-provoked phenotypic and epigenomic changes to the adaptive immune system
Authors: Hesterberg Rebecca S. , Cheng Chia-Ho , Yao Jiqiang , Song Xiaofei , Yu Xiaoqing , Cleveland John L.
Published: April 2023

Lymphoma is a disease that principally afflicts older individuals and also impairs the immune system, yet how aging and cancer interact to affect the immune system remains poorly understood. This is particularly the case for malignancies that arise within hematopoietic tissues that closely interact with immune cells. We hypothesized that lymphoma-provoked changes to the adaptive immune system would differ based on age. To explore how the immune system is altered by both B-cell lymphoma and the aging process, we transplanted Eμ-Myc lymphoma B-cells into mice aged 6-12 weeks or 20-24 months. Interestingly, as marked by reduced splenomegaly and lymphoma cell numbers, tumor burden was significantly reduced in aged hosts, suggesting lymphoma cells grow more slowly in the aged tissue microenvironment. Further, while lymphoma progression provoked losses in total numbers of normal T-cells, NK-cells, and B-cells in young individuals, aged lymphocytes were surprisingly resistant to lymphoma. Similarly, profound phenotypic changes in T-cell subpopulations that are provoked by Eμ-Myc lymphoma in young individuals, such as increases in expression of CD44, CD69, PD-1, Foxp3, and Tox, were not manifest in lymphoma-bearing aged mice. In addition, immature NK cell population dynamics were altered by lymphomas differently among aged mice when compared to young mice. Finally, analyses of the chromatin state of enriched CD4+ T cells from young and aged mice by ATAC-seq revealed that the aging process provoked marked alterations in the epigenome, and that lymphoma only provoked significant changes to the epigenome among young CD4+ T cells. We conclude that aging-driven epigenetic alterations manifest in the adaptive immune system renders these cells surprisingly resilient to lymphoma-mediated immune suppression.

Citation Format: Rebecca S. Hesterberg, Chia-Ho Cheng, Jiqiang Yao, Xiaofei Song, Xiaoqing Yu, John L. Cleveland. Aging dampens lymphoma-provoked phenotypic and epigenomic changes to the adaptive immune system [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 621.
Abstract 6213: Disruption of XPF function by HPV promotes alternative end joining and sensitivity to cisplatin in HNSCC
Authors: Liu Qi , Zuo Nan , Ma Lin , Wei Lanlan
Published: April 2023

Human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) usually show a tremendous better clinical outcome compared to their HPV-negative counterparts. Although the HPV associated survival advantage is partially explained by the enhanced cisplatin sensitivity in HNSCC (Clin Ca Res 2018, 24(23): 6001), the underlying mechanisms are still poorly elucidated. Cisplatin causes DNA interstrand crosslinks (ICLs) that requires Fanconi anemia (FA) pathway for DNA repair. Consistently, our study showed that HPV-positive HNSCC cells exhibited a FA defective cellular phenotype. In clonogenic cell survival assay, 1h treatment with 5μM cisplatin eliminated approximately 56.7% more HPV-positive HNSCC cells than HPV-negative ones. Furthermore, HPV-positive cells treated with cisplatin showed prolonged G2/M cell cycle arrest and more 53BP1 foci, indicating profound deficiency in repairing ICLs. Consistent with these findings, increased aberrant chromosome formation was observed in HPV-positive cells following Mitomycin C treatment. In order to reveal the mechanism, we further interrogated HPV-labeled HNSCC samples in TCGA database, and identified XPF, an endonuclease protein in FA pathway, was downregulated in HPV-positive HNSCC. Further analysis of cellular and clinical samples confirmed that both mRNA and protein expressions of XPF were significantly lower in HPV-positive HNSCC (P<0.001). To test the importance of XPF in HPV-induced cisplatin sensitivity, we performed a cell viability assay, and found that knock-down of XPF increased the effects of cisplatin on HPV-negative HNSCC by 39.54% (± 4.42%), while no significant change was observed in HPV-positive HNSCC (P>0.05). Notably, inhibition of XPF function by a small molecule inhibitor recapitulated the effect of HPV, resulted in enhanced error-prone DNA repair with alternative end-joining (alt-EJ). In a probe-based ddPCR assay, XPF inhibition increased 32.02% (± 5.80%, P<0.001) alt-EJ events in HPV-negative HNSCC cells, while little difference was detected in HPV-positive cells. Thus, we speculated that combined inhibition of XPF and alt-EJ may improve clinical outcome in the difficult-to-treat, HPV-negative HNSCC, which worth further research efforts.

Citation Format: Qi Liu, Nan Zuo, Lin Ma, Lanlan Wei. Disruption of XPF function by HPV promotes alternative end joining and sensitivity to cisplatin in HNSCC. [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 6213.
Abstract 6520: Combination of serum human satellite RNA and miR-21 levels as a biomarker for diagnosis of pancreatic cancer
Authors: Seimiya Takahiro , Kishikawa Takahiro , Otsuka Mtoyuki , Fujishiro Mitsuhiro
Published: April 2023

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis due to the difficulty of its diagnosis. Because human satellite II (HSATII) RNA, a satellite repeat RNA, is highly and specifically expressed in human PDAC, the serum HSATII RNA level may be a biomarker for diagnosis of PDAC. To measure the serum HSATII RNA level with high sensitivity and reproducibility, we previously developed a convenient method, tandem repeat amplification by nuclease protection (TRAP) combined with droplet digital PCR (ddPCR). Here, we refined the original method by simultaneously measuring the serum miR-21 level to enhance the detection of PDAC. A total of 135 patients were enrolled in this study, including 65 patients with pancreatic cancer and 70 healthy controls. We developed the PDAC-Index as a combination of the HSATII RNA and miR-21 levels to discriminate PDAC from healthy controls. The PDAC-Index demonstrated good diagnostic performance (AUC: 0.92 (95% CI: 0.86-0.99); sensitivity: 0.94; specificity: 0.85) which was significantly better than HSATII RNA alone (AUC: 0.92 vs 0.82, p=0.004). Importantly, the PDAC-index was elevated in an early-stage PDAC case that was negative for CA19-9. The PDAC-index also discriminated PDAC from chronic pancreatitis and intraductal papillary mucinous neoplasm more accurately than CA19-9. These results suggest that the PDAC-Index may be a promising biomarker for diagnosis of PDAC.

Citation Format: Takahiro Seimiya, Takahiro Kishikawa, Mtoyuki Otsuka, Mitsuhiro Fujishiro. Combination of serum human satellite RNA and miR-21 levels as a biomarker for diagnosis of pancreatic 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 6520.