bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3101: Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in cancer cells
Authors: Mahmoud Mentalla , Chen Jianyu , Cai Jianfeng , Xie Hao
Published: June 2023

Introduction: Autophagy pathway activation and upregulation is an important mechanism for cancer cell survival upon exposure to chemotherapeutic agents. LC3, a ubiquitin-like protein, plays a key role in the regulation of cargo selection and autophagosome formation. Given small molecule inhibitors have not been well developed to target LC3, we aim to design and synthesize an LC3 binder and evaluate its activity in the inhibition of autophagy pathway in cancer cells.

Methods: The crystal structure of LC3 and p62 complex (PDB: 2ZJD) was used to assist our design of helical sulfonyl-γ-AApeptides to the LC3 interacting region of p62. The peptidomimetics were synthesized on the solid phase and purified by HPLC. We used fluorescent polarization assay to assess the binding affinity of the peptidomimetics to LC3. We then utilized an LC3 HiBiT reporter cell line (U2OS) from Promega, which was engineered to have a fused human LC3-HiBiT with a HaloTag in between to study the effect of the lead LC3 binder on autophagic flux. In addition, confocal microscopy enabled direction visualization of the colocalization of FITC-labeled LC3 binder with LC3 and lysosome, respectively. Cytotoxicity of LC3 binder was evaluated in colorectal cancer cell line HCT116.

Results: We designed and synthesized 6 helical sulfonyl-γ-AApeptides (JC1-6) to target the binding interface of LC3 and p62. These peptidomimetic compounds were designed to retain essential LC3 and p62 interactions at the W and L regions and yet have different functional groups on the helical scaffold. Compounds JC1 and JC6 have binding affinity of Kd 280 nM and 150 nM with LC3, respectively. FITC-labelled JC6 was able to penetrate U2OS and HCT116 cells. It had intracytoplasmic colocalization with not only lysosomes but also LC3 aggregates. Direct visualization of intracytoplasmic LC3 aggregates showed that JC6 at 50 µM significantly inhibited the autophagic degradation of LC3 aggregates. This effect was augmented by the presence of 2 µM autophagy activator PP242. Lastly, JC6 demonstrated cytotoxicity with IC50 approximately 50 µM in HCT116 cells bearing baseline autophagy activation compared to lack of cytotoxicity in U2OS cells with no baseline autophagy activity.

Conclusion: We designed and synthesized novel helical sulfonyl-γ-AApeptide LC3 binders. With the ability of cellular penetration, they demonstrated inhibition of autophagic flux and preliminary cytotoxicity in cancer cells. Future studies may focus on structural activity relationship and further evaluation of its mechanisms of action in cancer cells.

Citation Format: Mentalla Mahmoud, Jianyu Chen, Jianfeng Cai, Hao Xie. Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in 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 3101.
Abstract 312: The ATR inhibitor ART0380 shows preclinical efficacy in monotherapy or in combination with gemcitabine, aPD1, PARP inhibitors and topoisomerase 1 poisons
Authors: Majithiya Jayesh Bhanabhai , Luzarraga Marina Roy , Newman Richard , Costales Paula , Jones Philip , Francesco Maria Emilia Di , Giuliani Virginia , Caroll Chris , Do Mary Geck , Heffernan Tim , Feng Ningpeng , Ma XiaoYan , lively Sarah , Johnson Michael G. , Geo Lerin , Piscitello Desiree , Rajendra Eeson , Grinkevich Vera , Ranzani Marco , Stockley Martin , Langford Gillian , Heald Robert , Robinson Helen M. , Smith Graeme C.
Published: June 2023

ART0380 is a potent, selective and orally active inhibitor of ataxia telangiectasia and Rad3-related (ATR) that has been developed for treatment for cancer patients with DNA repair defects. Here we show preclinical data of ART0380 in a range of tumour xenograft models, both as monotherapy and in combination with other DNA-damaging agents. As monotherapy, ART0380 exhibits tumour PD (γH2AX) effects and efficacy comparable to the ATR inhibitor BAY-1895344 but causing less toxicity to the gut. As ATM has been highlighted as synthetic lethal with ATR, ART0380 monotherapy was also evaluated in ATM-deficient models showing strong tumour growth inhibition using both continuous and intermittent scheduling. ART0380 was also shown to have enhanced efficacy when dosed in combination with other anti-cancer agents. Here we show strong synergy when combining ART0380 with gemcitabine, the PARP inhibitor olaparib, the topoisomerase 1 poison irinotecan as well as the immuno-oncology therapeutic aPD1. Overall, these data demonstrate the high potential of ART0380 as an anti-cancer agent given as monotherapy or in combination

Citation Format: Jayesh Bhanabhai Majithiya, Marina Roy Luzarraga, Richard Newman, Paula Costales, Philip Jones, Maria Emilia Di Francesco, Virginia Giuliani, Chris Caroll, Mary Geck Do, Tim Heffernan, Ningpeng Feng, XiaoYan Ma, Sarah lively, Michael G. Johnson, Lerin Geo, Desiree Piscitello, Eeson Rajendra, Vera Grinkevich, Marco Ranzani, Martin Stockley, Gillian Langford, Robert Heald, Helen M. Robinson, Graeme C. Smith. The ATR inhibitor ART0380 shows preclinical efficacy in monotherapy or in combination with gemcitabine, aPD1, PARP inhibitors and topoisomerase 1 poisons [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 312.
Abstract 316: Engineering large chromosomal deletions to advance precision oncology for prostate cancer
Authors: Wu Jinjin , Wu Chen , Lawitts Joel A. , Bock Cheryl B. , Huang Jiaoti , Chen Ming
Published: June 2023

Human prostate cancer is notable for its high number of somatic copy number alterations (CNAs), which include both focal and broad arm-level amplifications and deletions. Up to 90% of prostate tumors harbor aberrant CNAs, and genomic deletions affect about 40% of these tumors. Many chromosomal deletions are highly recurrent and occur in more than 10% of prostate tumors, including 8p (40~50%), 6q14-16, 10q23, 13q14, and 16q22-24 (20~30% each). Most of these deletions are large, often including hundreds of genes. Notably, several large deletions, including 6q14-16 and 16q22-24, are significantly associated with poor patient outcomes, suggesting that these large chromosomal deletions play a functional role in prostate tumorigenesis, and highlighting the critical need for elucidation of the mechanisms underlying large-deletion driven tumorigenesis. However, due to the limited cloning capacity of targeting vectors and the rarity of on-target homologous recombination events in traditional gene-targeting technology, modelling large chromosomal deletions has proven highly challenging, and the role of such lesions in tumorigenesis has therefore been significantly understudied. Here, we developed an innovative approach to engineering the chromosome over large genetic distances through CRISPR/Cas9 technology in both mouse embryonic stem (ES) cells and human prostate cancer cells. Upon completion of the desired genome edits, the resulting mouse ES clones make possible the creation of conditional knockout mice that accurately mimic prostate cancer-associated large deletions, while the engineered human cancer cell clones serve as an indispensable in vitro model to validate the biological relevance of large chromosomal deletions in prostate tumorigenesis. Further characterization of the human prostate cell line bearing 10q23 deletion revealed that 10q23 loss conferred RWPE1 cells growth advantage. The development and characterization of these in vivo and in vitro models will contribute greatly to the implementation of precision oncology research, open new opportunities for modelling human diseases associated with large chromosomal deletions, and lead to novel insights into the biology of prostate cancer-associated large deletions, which will facilitate the identification of deletion-specific vulnerabilities that can be exploited for prostate cancer treatment.

Citation Format: Jinjin Wu, Chen Wu, Joel A. Lawitts, Cheryl B. Bock, Jiaoti Huang, Ming Chen. Engineering large chromosomal deletions to advance precision oncology for prostate 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 316.
Abstract 3165: ImmuneSig: A deep learning framework to develop gene expression signature of immune checkpoint inhibitor therapy
Authors: Liu Yang , Jhaveri Aashna , Bodapati Sudhenshna , Wong Cheryl , Michor Franziska
Published: June 2023

Immune checkpoint inhibitor (ICI) therapy targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death 1 (PD1), and programmed death-ligand 1 (PDL1) induces durable remission in some patients with various cancer types. Given the variable patient outcomes, many studies have been conducted to identify prognostic biomarkers associated with ICI response. However, due to intra- and inter-tumor heterogeneity and the complexity of the immune system, those biomarkers have diverse efficacy for predicting outcomes in clinical trials. Here we propose a deep learning framework with unsupervised self-learning autoencoders that identifies predictive features from patient transcriptomics data. This approach allows us to determine the contribution of each gene to the response classifier and identifies potential gene signatures for predicting immunotherapy response by using the learned weights of trained autoencoders. We applied our framework to 20+ ICI clinical cohorts across different cancer types and compared the prediction performance of our method with other well-known immune response markers evaluated using a suite of machine learning algorithms (random forest, logistic regression, and XGboosting) with cross-validation. We found that in some cohorts, the learned features from autoencoders improve the predictive power of ICI response when evaluated with the area under the operating characteristic curve. Our framework uncovers potential immune gene signatures associated with patient outcomes. Our work will help advance cancer immunology and immuno-oncology research, provide insights into tumor immunity, and lead to the discovery of progressively more immune markers and novel immune targets.

Citation Format: Yang Liu, Aashna Jhaveri, Sudhenshna Bodapati, Cheryl Wong, Franziska Michor. ImmuneSig: A deep learning framework to develop gene expression signature of immune checkpoint inhibitor therapy [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 3165.
Abstract 3327: Proteomic profiling of extracellular vesicles in patients with biliary tract cancers
Authors: Kim Dong Uk , Park Sunghee , Han Sung Yong , Jeon Daye , Lee Hyeji , Lee Jonghyun , Nam Hyeong Seok , Lee Dong Wook , Cho Chang-Min , Jung Min Kyu , Heo Jun , Kang Un-Beom , Park Seong-Jun , Huh Sunghyun , Shin Yourae
Published: June 2023

Background and Aim: Biliary tract cancers (BTCs) have a poor prognosis. One of the reasons for the prognosis is the difficulty of early diagnosis. The absence of a sufficient biomarker to screen for BTCs in high-risk populations makes the patient to be recognized in advanced status. Therefore, it is urgent to discover a biomarker for early detection of BTCs. This study analyzed extracellular vesicle (EV) peptides to find a new serum biomarker of BTCs.

Methods: Plasma samples were obtained from 5 healthy people as control, 10 from patients with benign biliary diseases such as choledocholithiasis, sclerosing cholangitis, and gallbladder polyp, and 29 from BTCS patients. The EV peptides have been obtained from 2.5ml of patient plasma. The EV peptide samples were analyzed by liquid chromatography-data-dependent acquisition-tandem mass spectrometry (LC-DDA-MS/MS). The acquired peptides and proteins were investigated by searching in Fragpipe-MSFragger. Characterization was performed using the EVs database of previous studies. After that, Differential Expression Protein (DEP) analysis and Pathway/GO analysis are performed to know the biological process.

Results: Total 25,382 peptides and 3,313 proteins were defined (PSM, peptide-level FDR 1%). Most of the gene ontology biological processes were related to vesicle-mediated transport. On DEP analysis, total 72 proteins were up-regulated, and 29 proteins were down-regulated. Compared to cancer samples, 9 proteins were up-regulated to both healthy and benign samples and 2 were down-regulated. According to biological process/pathway analysis, up-regulated 9 proteins were mostly related to cytoskeleton/ECM and Hemostasis. Down-regulated 2 proteins were related to endocytosis and immunity.

Conclusions: By analyzing EV peptides, a total of 11 proteins have been identified. With validation experiments, we expect to finalize biomarker candidates.

Citation Format: Dong Uk Kim, Sunghee Park, Sung Yong Han, Daye Jeon, Hyeji Lee, Jonghyun Lee, Hyeong Seok Nam, Dong Wook Lee, Chang-Min Cho, Min Kyu Jung, Jun Heo, Un-Beom Kang, Seong-Jun Park, Sunghyun Huh, Yourae Shin. Proteomic profiling of extracellular vesicles in patients with biliary tract cancers [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 3327.
Abstract 3343: Liquid biopsy screening tool for the earlier detection of brain tumors
Authors: Theakstone Ashton G. , Brennan Paul M. , Baker Matthew J.
Published: June 2023

Background: There is a need for a rapid, cost-effective and non-invasive screening tool for the earlier detection and diagnosis of brain tumors. Serum based ATR-FTIR combined with machine learning algorithms can reliably predict which patients with symptoms will actually have a tumor on brain imaging. This technique gives a broad overall serum composition of the patient. However, an approach combining spectroscopy and specific cytokines within the serum could potentially provide a more accurate detection and permit prediction of tumor subtypes before histological diagnosis.

Method: Patient serum samples (300uL) were collected prospectively from patients with a new brain tumor diagnosis, and patients without a brain tumor as non-cancer controls. Each patient was analysed using both ATR-FTIR and an electrochemical approach targeting specific cytokine markers. MMP-3, MMP-9 and osteopontin were selected from an unsupervised screening of 64 cytokines. The patients were discriminated using machine learning techniques in three approaches; spectral data alone, biomarker data alone and the combination of both spectral and biomarker data. Receiver operator characteristic (ROC) curve analysis was completed to display the discriminatory ability of each approach.

Results: The spectral and biomarker data was collected for 49 patient samples (25 non-cancer, 24 GBM). All three approaches gave excellent discriminatory ability between cancer and non-cancer control patients. The spectral profiles alone had an area under the curve (AUC) of 0.958, the biomarker data alone had an AUC of 0.937 and the combination of spectral and biomarker data for each patient had an AUC of 0.975. The clinical datatset is being expanded to include more patients and this paper will discuss further patient analysis as well.

Conclusion: The combination of using patient data from both the spectroscopic and electrochemical approaches improved the discrimination between cancer and non-cancer patients. The addition of specific marker information could support prioritization for brain imaging, and prediction of tumor subtype to guide pre-operative management decision-making.

Citation Format: Ashton G. Theakstone, Paul M. Brennan, Matthew J. Baker. Liquid biopsy screening tool for the earlier detection of brain tumors [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 3343.
Abstract 3345: Tissue of origin (TOO) localization using targeted methylation biomarkers discovered by whole-genome bisulfite sequencing
Authors: Wei Qiang , Deng Wanglong , Xu Zhihui , Han Yihui , Yang Yuan , Zhang Chao , Wang Xiaoqiang , Tang Xuan , Song Chao , Xu Qing , Ren Yong
Published: June 2023

DNA methylation is a promising biomarker for tissue of origin (TOO) localization, which is important for classification of cancer of unknown primary site (CUP) in multiple cancer early detection. The current most of CpG sites of TOO focused on CpG islands based on The Cancer Genome Atlas (TCGA) data sets, but remains largely under-explored across the whole genome. In this study, we conducted the whole-genome bisulfite sequencing (WGBS) in 481 tumor samples and 361 matched adjacent normal tissue samples for 9 common types of cancers, including lung, breast, colon, liver, stomach, lymph, pancreas, esophageal and ovarian. After adjusted the tumor cells’ DNA methylation rates, 3,000 CpG sites, 60% of which were not on CpG islands, were selected through Random Forest classifier (multi-class) to identify cancer type. To evaluate these TOO methylation biomarkers, we used Random Forest Model to predict TOO of the methylation data sets in The Cancer Genome Atlas (TCGA), including 1,704 tumor samples. The results showed that accuracy of TOO could achieve 98.5% for these tumor samples of TCGA. Among these samples, the accuracy of TOO of breast, liver, lung, colorectal and pancreas were 99.6%, 99.9%, 97.7%, 99.2% and 99.3% respectively. Several samples from Esophagus and Stomach were classified to each other, and their accuracy of TOO was relatively low, 97.6% and 96.7%. It demonstrated that these methylation biomarkers could detect multiple cancers, and the multi-class model had good performance for TOO localization.

Citation Format: Qiang Wei, Wanglong Deng, Zhihui Xu, Yihui Han, Yuan Yang, Chao Zhang, Xiaoqiang Wang, Xuan Tang, Chao Song, Qing Xu, Yong Ren. Tissue of origin (TOO) localization using targeted methylation biomarkers discovered by whole-genome bisulfite sequencing [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 3345.
Abstract 3383: Liquid biopsy for PDL-1 status by analysis of cfRNA: Clinical data from a large community practice
Authors: Hart Lowell L. , Bergh Magali Van den
Published: June 2023

PD-L1 testing via immunochemistry has become a widely used tool to predict response to immune checkpoint inhibitor (ICI) therapy in many tumor types. However this marker can be expressed heterogeneously, and can vary with time, treatment effects, and local factors including cytokine expression. Gene expression profiles of immunologically active genes are currently being studied to more accurately predict response to ICI treatment. To evaluate a potentially more straightforward way to accomplish this, we utilized an assay of cfRNA in plasma for PD-L1, utilizing a real-time PCR based assay (Circulogene, Inc, Birmingham, AL). The demonstrated limit of detection for PD-L1 RNA was 1 copy/ul. Results were reported as either not detected, or low at over 1%, or high at over 50%. We used the PCR 30th percentile Ct value corresponding to a tissue IHC PD-L1 of>50%, and the 66th percentile Ct value corresponding to a tissue PD-L1 expression of >1%. We then collected data on over 400 patients over a two year period at a large community oncology practice and at a university clinic (Florida Cancer Specialists and Atrium Health/Wake Forest-Baptist Comprehensive Cancer Center) who had a clinical necessity for molecular testing in a commercially available setting with a CLIA certified lab. In some cases the corresponding tissue PD-L1 testing was also available, and may have been either positive or negative.To date 41 patients have been found to have high levels of cfRNA for PD-L1. Their clinical course has been analyzed using the electronic medical record. Some patients have received standard of care therapy, and some have been given CPI therapy with or without chemotherapy, if the attending oncologist felt this to be the best palliative option. These include tumors such as CRPC, metastatic low grade neuroendocrine carcinoma and HR+ metastatic breast carcinoma, that have rarely been treated successfully with CPI therapy. Tumor types - all are stage IV solid tumor patients: 19 NSCLC, 7 breast carcinoma, 3 colorectal cancer, 3 castrate resistant prostate carcinoma, 9 other tumor types.Data collection for tumor response or clinical benefit on the 41 patients is ongoing and will be presented at the meeting, and compared to published CPI response data using standard tissue IHC testing.

Citation Format: Lowell L. Hart, Magali Van den Bergh. Liquid biopsy for PDL-1 status by analysis of cfRNA: Clinical data from a large community practice [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 3383.
Abstract 3387: ctDNA dynamics in early stage node negative lung cancers
Authors: Feng Jamie , Waddell Tom , Yasufuku Kazuhiro , Pierre Andrew , Keshavjee Shaf , Yeung Jonathan , Cypel Marcelo , Donahoe Laura , Wakeam Elliot , de Perrot Marc , Law Jennifer , Salvarrey Alexandra , Le Lisa W. , Lister Jennifer , Cabanero Michael , Tsao Ming , Pipinikas Christodoulos , Howarth Karen , Leighl Natasha
Published: June 2023

Introduction: Patients with early-stage lung cancers have a high risk of relapse and death even after curative surgery. Detection of circulating tumour DNA (ctDNA) in plasma perioperatively is associated with shorter recurrence free survival (RFS). ctDNA-Lung-Detect is an investigator-initiated prospective study of ctDNA detection and association with RFS in patients with early stage non-small cell lung cancer (NSCLC).

Methods: Patients with clinically staged T<4cm N0 NSCLC planned for surgical resection at the Princess Margaret/University Health Network underwent ctDNA assessment before and after surgery (~1 month and 1 year). ctDNA minimal residual disease (MRD) was detected using the highly sensitive and specific tumor-informed Residual Disease and Recurrence (RaDaR®, Inivata, Cambridge, UK) assay, which can track up to 48 tumor-specific variants in plasma.

Results: Since August 2021, 64 eligible patients were enrolled (Table 1). One had isolated metastasis from a remote non-lung primary and subsequently excluded; another had their preoperative sample missed but was included. Preoperative ctDNA was detected in 15/62 (23.8%) patients and 1/63 (1.6%) post-operatively at the 1-month landmark timepoint (occult N2 disease found at surgery). All but one (93.3%) had ctDNA clearance with surgical resection. Two patients have recurred radiographically and in plasma at 1 year post-operatively, 1/15 with preoperative ctDNA detected (6.7%) and 1/47 without preoperative ctDNA detected (2.1%). Patients with higher T stage, tumor size, squamous histology, and without actionable driver alterations were more likely to have ctDNA detected at any timepoint.

Conclusions: In patients with early stage NSCLC (small node negative tumors), 24% have detectable ctDNA pre-operatively. Surgical resection led to ctDNA clearance in 93% of cases. Our results support ongoing testing beyond the initial MRD landmark. Assessment of the impact on RFS post resection is ongoing.

Citation Format: Jamie Feng, Tom Waddell, Kazuhiro Yasufuku, Andrew Pierre, Shaf Keshavjee, Jonathan Yeung, Marcelo Cypel, Laura Donahoe, Elliot Wakeam, Marc de Perrot, Jennifer Law, Alexandra Salvarrey, Lisa W. Le, Jennifer Lister, Michael Cabanero, Ming Tsao, Christodoulos Pipinikas, Karen Howarth, Natasha Leighl. ctDNA dynamics in early stage node negative lung cancers [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 3387.
Abstract 3591: Does the metastatic immunoenvironment determine poor immunotherapy responses
Authors: Barth Nicole D. , van Dalen Floris J. , Duran Camille , Karmakar Utsa , Mendive-Tapia Lorena , Kitamura Takanori , Verdoes Martjin , Condeelis John S. , Oktay Maja , Vendrell Marc , Brunton Valerie , Entenberg David
Published: June 2023

Despite new treatments, cancer remains the second leading cause of death worldwide with breast cancer being the most common type. Of these deaths, the majority are due to the formation of distant metastases. While new immunotherapies have shown some success in primary tumors, their efficacy in combating metastases has been lacking. Thus we aim to investigate the hypothesis that immune responses at the metastatic site contribute to limitations of current therapies.Within the last decade, new microscopy technologies now enable real-time visualization of immune responses at primary and secondary sites. Here, we present use of these microscope technologies, along with the development of novel probes to detect apoptotic cancer cells, to investigate the success of checkpoint inhibitors in the primary and secondary sites. We validate, in multiple in vitro and in vivo assays (including intravital imaging), that fluorescent cyclic amphipathic peptide probes specifically label cells undergoing therapy-induced cancer death. In addition, we introduce a newly developed fluorescent, enzyme-activatable, chemokine-conjugate capable of targeting defined macrophage subsets in live tumors. This chemokine-conjugate exploits the high expression of chemokine receptors (e.g. CCR2) and the activity of cysteine cathepsins in tumor associated macrophages to selectively target and eradicate them without affecting other macrophages or immune cells (e.g., neutrophils, T cells, B cells). This research will help to elucidate the role of combination therapy on the metastatic foci and will provide the first insights into the benefit and limitations of combination therapy on secondary site immune responses.

Citation Format: Nicole D. Barth, Floris J. van Dalen, Camille Duran, Utsa Karmakar, Lorena Mendive-Tapia, Takanori Kitamura, Martjin Verdoes, John S. Condeelis, Maja Oktay, Marc Vendrell, Valerie Brunton, David Entenberg. Does the metastatic immunoenvironment determine poor immunotherapy responses. [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 3591.
Abstract 3601: Tumor MK2 signaling regulates cell migration and invasion in head and neck squamous cell carcinoma
Authors: Okwuone Dakota D. , Morgan Deri , Smith Hannah M. , Millington Grace , Berggren Kiersten L. , Lominska Christopher E. , Thomas Sufi M. , Gan Gregory N.
Published: June 2023

Poor 5-year overall survival in head and neck cancer (HNC) can be attributed to high rates of locoregional and distant metastasis. We have previously demonstrated that high phosphorylation/activation of MAPK-activated protein kinase 2 (MK2), a stress-activated kinase directly downstream of p38 MAPK, in head and neck squamous cell carcinoma (HNSCC) is associated with worse overall survival and inhibition of the MK2 pathway enhances in vivo HNSCC radiosensitivity. Our prior work also demonstrates that radiotherapy-induced epithelial-to-mesenchymal transition (EMT) gene expression can be suppressed in cells when MK2 is inhibited. Several studies have shown that EMT plays a prominent role in HNSCC tumor invasion, treatment resistance, and locoregional metastases to lymph nodes. In this study, we investigate the impact of MK2 activity on HNSCC tumor progression by examining its contribution to cancer cell morphology. We used lentiviral-shRNA and the CRISPR-Cas9 system to knock down/out the MK2 gene in multiple human and murine HNSCC cell lines. We measured tumor cell migration and invasion in both classic 2D in vitro culture as well as with 3D spheroids. We performed unbiased analysis via RNAseq in human HNSCC cell lines comparing MK2 shRNA vs scramble cell lines. Substantial gene and pathway alterations were examined using a combination of immunoblot and RT-qPCR. Our data shows knocking down/out MK2 in HNSCC cells significantly diminishes cell migration and invasion in 2D and 3D in vitro culture assays. RNAseq analysis demonstrated loss of MK2 leads to a significant increase in many cell adhesion molecules, including E-cadherin, and a decrease in N-cadherin, suggesting a shift from mesenchymal to more epithelial phenotype. Furthermore, we note expression of multiple matrix metalloproteinases and EMT markers (i.e., Snail) are substantially reduced in MK2 shRNA and KO cell lines. These results indicate that the MK2 pathway is involved in HNSCC motility and invasion. We also observe the loss of MK2 contributes to a shift in the epithelial:mesenchymal ratio potentially by suppressing cancer EMT plasticity. All in all, this study provides evidence suggesting tumor MK2 activation mediates HNC progression.

Citation Format: Dakota D. Okwuone, Deri Morgan, Hannah M. Smith, Grace Millington, Kiersten L. Berggren, Christopher E. Lominska, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 signaling regulates cell migration and invasion 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 3601.
Abstract 3730: A new type of transcriptional reprogramming by an IRF4 mutation in lymphoma
Authors: Cauchy Pierre
Published: June 2023

Disease-causing mutations in genes encoding transcription factors (TFs) are a recurrent finding in hematopoietic malignancies and might involve key regulators of lineage adherence and cellular differentiation1-3. Such mutations can affect TF-interactions with their cognate DNA-binding motifs4,5. Whether and how TF-mutations impact upon the nature of binding to TF composite elements (CE) and influence their interaction with other TFs is unclear. Here, we report a new mechanism of TF alteration in human lymphomas with perturbed B cell identity. It is caused by a recurrent somatic missense mutation c.295T>C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cell-differentiation and -activation6,7. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF composite elements (CEs). Furthermore, IRF4-C99R thoroughly modifies IRF4 function, by blocking IRF4-dependent plasma cell induction, and up-regulating disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single arginine mutation creates a complex switch of TF specificity and gene regulation. These data open the possibility of designing specific inhibitors to block the neomorphic, disease-causing DNA-binding activities of a mutant transcription factor.

Citation Format: Pierre Cauchy. A new type of transcriptional reprogramming by an IRF4 mutation in lymphoma. [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 3730.
Abstract 3798: lncRNA LIMp27 promotes p53-defective cancer pathogenesis
Authors: La Ting , Chen Song , Zhao Xiao Hong , Zhang Yuan Yuan , Feng Yu Chen , Zhang Xu Dong , Jin Lei
Published: June 2023

Inactivation of p53 occurs in approximately 50% of human cancers, where p53-mediated p21 activation is devoid and p27 becomes essential for the establishment of the G1/S checkpoint upon DNA damage. Here, we show that the E2F1-responsive lncRNA LIMp27 selectively represses p27 expression and promotes proliferation, tumorigenicity, and treatment resistance in p53-defective colon adenocarcinoma (COAD) cells. LIMp27 competes with p27 mRNA for binding to cytoplasmically localized hnRNA0, which otherwise stabilizes p27 mRNA, leading to cell cycle arrest at the G0/G1 phase. In response to DNA damage, LIMp27 is upregulated in both wild-type and p53-mutant COAD cells, whereas cytoplasmic hnRNPA0 is only increased in p53-mutant COAD cells due to translocation from the nucleus. Moreover, high LIMp27 expression is associated with poor survival of p53-mutant but not wild-type p53 COAD patients. These results uncover a lncRNA mechanism that promotes p53-defective cancer pathogenesis and suggest that LIMp27 may constitute a target for the treatment of such cancers.

Citation Format: Ting La, Song Chen, Xiao Hong Zhao, Yuan Yuan Zhang, Yu Chen Feng, Xu Dong Zhang, Lei Jin. lncRNA LIMp27 promotes p53-defective cancer pathogenesis. [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 3798.
Abstract 3802: miR-199a and miR-199b stimulate the progression of diffuse gastric cancer through direct targeting of Frizzled-6
Authors: Park Jihye , Lee Sieun , Hong Soon Auck , Ko Yoon Ho , Ahn Young-Ho
Published: June 2023

There is no pathological difference between early and advanced diffuse gastric cancer (DGC), particularly in the gastric mucosa. In this study, we tried to identify microRNAs (miRNAs) as diagnostic biomarkers that can differentiate between early and advanced DGC. miRNA expression profiling was performed by NanoString nCounter method in human DGC tumors. miR-199a and miR-199b (miR-199a/b) were particularly up-regulated in advanced DGC compared with early DGC. Ectopic expression of miR-199a/b accelerated growth, viability, and motility of SNU601, human GC cells, and expedited tumor development in a mouse xenograft model. miR-199a/b also inhibited cell adhesion. Through 3’-UTR luciferase assay, Frizzled-6 (FZD6) was confirmed as a direct target of miR-199a/b. siRNA-mediated depletion of FZD6 increased cell growth and motility and addback of FZD6 restored cell growth, motility, and adhesion. To explore their clinicopathological roles in patients, miR-199a/b levels were measured by in situ hybridization in human DGC tumor sections. High miR-199a/b was correlated with advanced lymphovascular invasion, advanced T stages, and lymph node metastasis. Collectively, miR-199a/b promote the progression of DGC via targeting FZD6. These results imply that miR-199a/b can be used as diagnostic and prognostic biomarkers of DGC.

Citation Format: Jihye Park, Sieun Lee, Soon Auck Hong, Yoon Ho Ko, Young-Ho Ahn. miR-199a and miR-199b stimulate the progression of diffuse gastric cancer through direct targeting of Frizzled-6. [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 3802.
Abstract 3859: Alterations in hormone receptors regulome following response to kinase inhibitors in lethal prostate cancer
Authors: Adelaiye-Ogala Remi M. , Gulla Surendra , Bard Jonathan E. , VanderWeele David J. , Kelly Kathleen
Published: June 2023

The androgen receptor (AR) signaling axis is critical for prostate cancer (PCa) pathogenesis and all subsequent phases of disease progression. Although initial success with androgen deprivation therapy and AR-targeted agents such as enzalutamide or abiraterone, most patients inevitably relapse due to therapeutic resistance. Previous studies have shown that genomic AR is unchanged despite resistance to AR antagonists, and its gene expression is unaltered. Therapeutic resistance is broadly classified into two categories; restoration of AR activity and tumor progression despite AR blockade. Indeed, others have noted an extensive reprogramming of the AR cistrome in clinical and preclinical specimens, where a shift in canonical to non-canonical AR cistrome is associated with an increase in Gleason grade. In previous studies, we observed increases in known AR target genes following exposure to kinase inhibitors which was marked by a decrease in tumor growth. We hypothesized that restoration of canonical AR cistrome can be mediated through kinase inhibition and is associated with re-sensitization to AR-targeted therapy. In addition, we treated cell lines and patient-derived xenograft models of advanced prostate cancer with kinase signaling pathway inhibitors, AR signaling inhibitors, or a combination of both for 24 hours (in vitro) or five days (in vivo). In addition, samples were collected and processed for RNA-seq and ChIP-seq. Our transcriptomic analysis showed enrichment of previously published non-canonical AR target genes in cells resistant to AR-targeted agents. When resistant cells were exposed to kinase inhibitors, we observed an enrichment of canonical AR target genes. In addition, we identified unique AR-enriched peaks at active sites associated with resistance. Through GSEA of gene annotated peaks, we found unique regulatory transcription factor targets, some of which are associated with cell proliferation and differentiation and may play a functional role in AR cistrome adaptation to resistance and survival. Overall, our data suggest that AR transcription adapts to the therapeutic resistance of AR antagonists via the enrichment of non-canonical AR cistrome. Furthermore, restoration of canonical AR cistrome is achieved following kinase signaling pathway inhibition and is associated with re-sensitization to AR-targeted therapy.

Citation Format: Remi M. Adelaiye-Ogala, Surendra Gulla, Jonathan E. Bard, David J. VanderWeele, Kathleen Kelly. Alterations in hormone receptors regulome following response to kinase inhibitors in lethal prostate 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 3859.
Abstract 4128: AJ17 potentiates agonist CD40 antibody efficacy and attenuates agonist CD40 induced PD-L1 expression and associated toxicity in murine tumor models
Authors: Gaur Vidit , Barnwal Anjali , Ateeq Bushra , Bhattacharyya Jayanta
Published: June 2023

Agonist CD40 antibody (αCD40) has shown excellent anti-tumor efficacy in both pre and early clinical studies. However, in clinics, αCD40 has failed to prove its potential as αCD40 is associated with dose-limiting toxicity due to cytokine release syndrome (CRS), grade 3 to 4 hematological and liver toxicities. In addition, αCD40 induces immune checkpoint proteins (PD-L1, CTLA-4) which makes the tumor microenvironment (TME) immunosuppressive. In this context, this study investigates the therapeutic efficacy of a novel combination of αCD40 and AJ17, a small molecule inhibitor of receptor tyrosine kinase. Combination treatment showed a significant delay in the tumor growth and improved overall survival in mice bearing B16-F10 melanoma and 4T1 orthotopic tumor by increasing the population of CD8+ T cells and lowering the expression of PD-L1, Foxp3+, and arginase in both tumor and spleen. Most interestingly, AJ17 lowered the in vivo toxicity associated with αCD40 monotherapy by lowering the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and IL-6 in serum. In conclusion, this combination can be further explored in clinics to improve the in vivo anti-tumor efficacy of αCD40 while lowering the associated toxicity.

Citation Format: Vidit Gaur, Anjali Barnwal, Bushra Ateeq, Jayanta Bhattacharyya. AJ17 potentiates agonist CD40 antibody efficacy and attenuates agonist CD40 induced PD-L1 expression and associated toxicity in murine tumor 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 4128.
Abstract 4132: Effect of mechanical cues on T cell killing of cancer cells
Authors: Dada Hannah , Do Kun , Cartagena-Rivera Alexander , Altan-Bonnet Grégoire
Published: June 2023

Cancer immunotherapies have focused on strategies to strengthen immune cell activation and effector function. These treatment strategies enhance the effector function of immune cells, particularly cytotoxic T cells (CTL), driving tumor eradication. Methods to improve the killing mechanisms of CTLs are not well studied and largely focused on assessing/strengthening the antigenic responses of T cells. We hypothesize that the mechanical landscape of CTL-tumor interactions can also drastically impact CTL cytotoxicity. By altering the physical context for T cell recognition of tumors, we measured cytotoxicity changes and subsequently used these changes in killing capacities to train and generate a quantitative parametrization of T cell killing. CTLs utilize cytokine expression to kill and shape the inflammatory milieu. Taking advantage of a robotic platform that enables the automatic collection of cytokines, we assembled datasets of T cell attacks of tumor cells grown on substrates with varying stiffness levels. The stiffness levels were altered using polyacrylamide (PAA) gels and ranged from 0.2kPa to approximately 113 kPa. Preliminary results from two different models (i.e., mouse and human TCR-transgenic T cells) suggest that there exists an optimal stiffness value for maximal response by T cells. These results suggest that CTLs utilize biophysical cues in addition to molecular cues (antigen) to properly elicit an immune response—modeling and fine-tuning these mechanical cues can be a novel and effective approach to improving cancer immunotherapies.

Citation Format: Hannah Dada, Kun Do, Alexander Cartagena-Rivera, Grégoire Altan-Bonnet. Effect of mechanical cues on T cell killing of 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 4132.
Abstract 4167: Translational control of T cell exhaustion in melanoma, perspectives in therapeutics
Authors: Muhammadnejad Samad , Biswas Biswendu , Guemiri Ramdane , Benannoune Naima , Lavigne Jérémy , Quidville Virginie , Scoazec Jean-Yves , Vagner Stephan , Robert Caroline
Published: June 2023

Background: Eukaryotic initiation factor 4F (eIF4F) complex plays a pivotal role in the selective translational control of protein expression in cancer. We previously showed that the inhibition of eIF4F had both a direct antitumor effect that could overcome resistance to BRAF inhibition in BRAFV600 melanoma and an indirect immune-mediated antitumor effect via PD-L1 down regulation resulting from STAT1 decreased translation. We now explore further the translational control of immune checkpoints (ICs) on T cell and its link with lymphocyte exhaustion.

Methods: We performed polysome profiling (PP) on peripheral blood-derived activated and resting T lymphocytes as well as tumor-infiltrating lymphocytes (TILs) isolated from melanoma specimens in the presence or absence of silvestrol as an eIF4A (the helicase component of eIF4F) inhibitor. We studied the expression of ICs and exhaustion-associated transcription factors on lymphocytes at the various stages of exhaustion. The tumor cell lysing capacity of PBMC was evaluated with and without silvestrol. We also assessed the effect of silvestrol on tumor microenvironment in the BP syngeneic melanoma tumor model.

Results: Silvestrol decreased the translation of multiple ICs in activated CD4+ and CD8+ T cells, including TOX, NFAT1, NFAT2, LAG3, and TIM3 (the two latter being also transcriptionally regulated). FACS analysis confirmed the dose-dependent decrease of protein expression of exhaustion-associated transcription factors TOX, NFATc1, TCF1 and T-bet in activated lymphocytes. This effect was more prominent in more exhausted T cells. Silvestrol decreased T cell proliferation but, in co-culture experiments, it increased immune cell killing of melanoma cells by T-lymphocytes. In the BP murine model, the antitumor effect of silvestrol was associated with denser T cell infiltration and less exhausted TILs than control tumors.

Conclusion: Our findings demonstrate the role of eIF4F in the orchestrated regulation of functionally related proteins involved in T cell exhaustion. They highlight the drugability of eIF4A for the cancer treatment and suggest that eIF4A inhibitors could be useful with IC inhibitors in combination or sequentially.

Citation Format: Samad Muhammadnejad, Biswendu Biswas, Ramdane Guemiri, Naima Benannoune, Jérémy Lavigne, Virginie Quidville, Jean-Yves Scoazec, Stephan Vagner, Caroline Robert. Translational control of T cell exhaustion in melanoma, perspectives in therapeutics. [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 4167.