bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6317: Developing a fully human heavy chain only Claudin-18.2-specific CAR-T cell therapy
Authors: Noon Jason , Lu Yao , Kaushik Itishree , Ay Oyku , Luk Adam , Delaney Bradley , DeMeritt David , Bao Musheng
Published: March 2024

Camelid VHH/nanobodies have been widely applied as the antigen binding domain of CAR-T cell therapies due to a smaller size and simpler structure compared to the traditionally used single chain variable fragment (scFv). Additionally, camelid VHH/nanobodies provide greater flexibility for dual-targeting CAR-T cell therapies. However, camelid VHH/nanobodies require laborious and costly humanization and still have a significant risk of inducing anti-drug antibodies when administered to patients. Transgenic mice exclusively harboring the most stable human VH genes (HCAb Harbour Mice®) provide one of the best solutions to get fully human heavy chain only antibodies (HCAbs). To efficiently identify lead HCAb VHs for Claudin-18.2-targeting CAR-T cells, we developed an innovative direct CAR-function based HCAb library screening platform. From Claudin-18.2-immunized HCAb Harbour Mice®, we constructed a total VH library in a CAR lentiviral vector that we developed in-house. After producing the lentiviral particle library, we transduced a Jurkat-NFAT-GFP reporter cell line and stimulated with Claudin-18.2 overexpression cell line. Subsequently, GFP positive reporter cells, representing Claudin-18.2-specific CARs, were isolated through FACS sorting, expanded, and further stimulated with Claudin-18.2-positive NUGC-4 tumor cells. Then, Next Generation Sequencing and abundance ranking was performed on VHs recovered from sorted CAR-reporter cells. Lead VH candidates were cloned into the CAR-P2A-RFP lentiviral vector, and many of these VH clones exhibited superior Claudin-18.2-specific functionality compared to a clinically-validated reference scFv in both reporter and primary CAR-T cells. Many lead VH clones also exhibited excellent developability as soluble HCAbs. These data suggest that this innovative method of combining our fully human HCAb and CAR-based library screening platform will provide new avenues for CAR-T cell therapies.

Citation Format: Jason Noon, Yao Lu, Itishree Kaushik, Oyku Ay, Adam Luk, Bradley Delaney, David DeMeritt, Musheng Bao. Developing a fully human heavy chain only Claudin-18.2-specific CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6317.
Abstract 6360: A novel CAR-NK cell therapy to target lung adenocarcinoma cells
Authors: Zolov Sergey , Chuikov Sergei , Katkam Shiva Krishna , Shield Delaney , Keshamouni Venkateshwar G.
Published: March 2024

Cell Adhesion Molecule (CADM1) is a Bonafede tumor suppressor with 40% of NSCLC tumors lose CADM1 expression either by loss of heterozygosity or promoter hypermethylation. The tumor suppressive ability of CADM1 is attributed to its cell adhesion function. However, we previously demonstrated that CADM1 also mediates its tumor suppression through regulation of NK-mediated immunosurveillance. In tumors that do not lose CADM1, it is expressed on cell surface and up regulated during TGF-β-induced EMT making it a biomarker for potential therapeutic targeting. Here we report the design and development of T and NK specific CAR that would specifically target lung cancer cells in CADM1 dependent manner. Anti-CADM1 single chain variable fragment (CADM1-scFv) was fused to NK or T cell specific transmembrane and co-stimulatory domains. The resulting CAR constructs were cloned into a retroviral vector, from which viral stocks were generated to transduce human and mouse cells. Next, we introduced anti-CADM1-CAR-NK construct into NK92 cell line and tested it in a cytotoxicity assay using A549-GFP-ffluc cell line as a target. We observed a significant increase in the killing of target cells with anti-CADM1-CAR-NK cells compared with non-transduced NK92 cells. This effect was significantly reduced when A549- GFP-ffluc-CADM1 KO cells were used as target. Next, we generated anti-CADM1-CAR-T cells by transducing human CD3+ T-cells purified from human PBMCs. Similar to anti -CADM1-CAR-NK, anti-CADM1-CAR-T cells showed increased T-cell specific cytotoxicity in a CADM1-dependent manner. Next, we evaluated the efficacy of anti-CADM1-CAR-T cells through adoptive transfer into NSG mice xenografted with human lung adenocarcinoma cells into mouse lungs. The administration of anti-CADM1-CAR-T cells markedly reduced tumor size and enhanced the survival rate of the mice. Importantly this effect was dependent on CADM1 expression, as mice xenografted with human CADM1 KO cells showed neither tumor size reduction nor increased survival. Finally, using IHC staining, we demonstrated that anti-CADM1-CAR-T cells directly target xenografted tumors in the mouse lungs. In conclusion, our findings indicate that CAR-NK and CAR-T cells targeting CADM1-expressing lung tumors have a significant therapeutic potential for the treatment of NSCLC.

Citation Format: Sergey Zolov, Sergei Chuikov, Shiva Krishna Katkam, Delaney Shield, Venkateshwar G. Keshamouni. A novel CAR-NK cell therapy to target lung adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6360.
Abstract 6404: GRB2 stabilizes RAD51 at reversed replication forks restricting genomic instability and associated innate immune response
Authors: Ahmed Zamal , Ye Zu , Xu Shengfeng , Shi Yin , Cheng Xueqian , Zhang Yuan , Roy Sunetra , Namjoshi Sarita , Longo Michael A. , Link Todd , Schlacher Katharina , Peng Guang , Yu Dihua , Wang Bin , Tainer John
Published: March 2024

Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adaptor for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination.

Citation Format: Zamal Ahmed, Zu Ye, Shengfeng Xu, Yin Shi, Xueqian Cheng, Yuan Zhang, Sunetra Roy, Sarita Namjoshi, Michael A. Longo, Todd Link, Katharina Schlacher, Guang Peng, Dihua Yu, Bin Wang, John Tainer. GRB2 stabilizes RAD51 at reversed replication forks restricting genomic instability and associated innate immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6404.
Abstract 6458: Clinical significance of cancer genome profiling tests for gastrointestinal cancer patients with liver metastasis
Authors: Sakuma Takashi , Yashiro Masakazu , Fukui Yasuhiro , Maruo Koji , Tsujio Gen , Yamamoto Yurie , Kasashima Hiroaki , Maeda Kiyoshi
Published: March 2024

Introduction: Distant metastasis is one of critical prognostic factors for patients with gastrointestinal cancer. Especially, liver metastasis shows high effects on prognosis of patients with gastrointestinal cancer. In Japan, cancer genome profiling (CGP) testing have been available for patients who have no selective standard treatment options since 2019. Based on the genetic mutations by CGP test, therapeutic drugs with evidence have been discussed at expert panels. CGP data has been accumulated in around 60,000 cases. It is necessary to evaluate the clinical significance of CGP tests for gastrointestinal cancer patients with liver metastasis to receive high benefits of CGP testing through C-CAT. Aims: We aimed to analyze the clinical significance CGP tests for gastrointestinal cancer patients with liver metastasis. Materials and methods: A total of 3,738 patients of gastrointestinal cancer with liver metastases including 215 cases of esophageal cancer, 347 cases of gastric cancer, and 3,176 cases of colorectal cancer, was enrolled in this study. Three types of CGP tests, including NCC Oncopanel, FoundationOne CDx, and FoundationOne Liquid, were performed, and the CGP data was collected to Center for Cancer Genomics and Advanced Therapeutics in Japan (C-CAT) database. Adequate treatment for each patient was discussed at the expert panel meeting according to the results from the CGP tests. Results: A total of 2,882 cases (77.1%) in 3,176 cases have been revealed druggable. However, only 216 cases (13.3%) were medicated by anti-cancer treatments. In the 216 treated cases, disease control case, complete response (CR), partial response (PR), or stable disease (SD) was only 52 cases. In 52 cases, six cases (4 of SD cases and 2 of PR cases) was administered encorafenib-based chemotherapies based on BRAF gene mutation. Five genes, ZNF217, SRC, ARFRP1, BARD1, FGF10, were significantly more frequently expressed on liver metastasis cases, in compared to other metastasis cases in gastrointestinal cancer. Conclusion: Around 13% of gastrointestinal cancer patients with liver metastasis might have a druggable benefit by CGP testing. BRAF mutation might be promising target for patients with liver metastasis. ZNF217, SRC, ARFRP1, BARD1, FGF10 might be associated with metastatic process to liver in gastrointestinal cancer.

Citation Format: Takashi Sakuma, Masakazu Yashiro, Yasuhiro Fukui, Koji Maruo, Gen Tsujio, Yurie Yamamoto, Hiroaki Kasashima, Kiyoshi Maeda. Clinical significance of cancer genome profiling tests for gastrointestinal cancer patients with liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6458.
Abstract 6463: Highly frequent gene mutations and co-occurring genes analysis in colorectal cancer
Authors: Zha Qinqin , Scarpa Frank , Rengaraj Madhu , Juncker-Jensen Anna
Published: March 2024

Background: Colorectal cancer (CRC) is the second most leading cause of cancer-related deaths worldwide. Understanding the molecular alterations that drive CRC is critical for improving patient outcomes. Next-generation sequencing (NGS) has revolutionized CRC molecular profiling by simultaneous analysis of multiple genes with high accuracy and sensitivity. In this study, we analyzed a cohort of CRC formalin-fixed paraffin-embedded (FFPE) samples to identify highly frequent gene mutations and co-occurring genes in CRC.

Methods: FFPE samples from CRC patients tested from January 2020 to May 2023 were analyzed using NeoTYPE® Colorectal Tumor Profile. This profile analyzes 44 biomarkers through a combination of NGS focusing on a panel of 36 genes known to be frequently mutated in CRC, as well as 3 FISH and 3 IHC biomarkers. Gene mutation frequency, co-occurring mutation genes and clinical significance were explored and analyzed. In addition, immunotherapy markers, Microsatellite Instability (MSI) and Tumor Mutation Burden (TMB) were assessed.

Results: A total of 2,920 patients (1,577 men and 1,343 women) with CRC were included in this study. Average sequence coverage was 2386X with mean minor allelic frequency of 30%. A higher average number of variant allele frequency were observed in metastatic versus primary colorectal cancer (P < .001). We found that APC, TP53, KRAS, PIK3CA and ARID1A were the most frequently mutated genes. APC truncated mutation p. R1450* were the most common mutations. KRAS, PIK3CA and TP53 missense mutations are the prevalent consequence. Notably, a subset of patients exhibited high microsatellite instability (MSI-H) or TMB high, indicating potential eligibility for immune checkpoint inhibitor therapy. In addition, we found APC mutations co-occurring with TP53 or KRAS mutations (p <.001) in our cohort.

Conclusion: Our results provide insight into the genetic landscape of CRC and highlight the importance of understanding co-occurring mutations in this disease. Specifically, APC mutations frequently coincide with TP53 mutations, jointly contributing to the onset of CRC. TP53 often play a role in promoting genomic instability and evading mechanisms that regulate cell growth. The identification of highly frequent mutations and co-occurring genes may have important implications for the development of targeted therapies and personalized treatment approaches for CRC patients.

Citation Format: Qinqin Zha, Frank Scarpa, Madhu Rengaraj, Anna Juncker-Jensen. Highly frequent gene mutations and co-occurring genes analysis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6463.
Abstract 6493: CRISPR-directed prime editing sensitizes EGFR inhibitor by correcting oncogenic KRAS mutants In NSCLC
Authors: Fang Qingxiao , Yang Chao , Huang Chongbiao , Wang Hongwei , Yu Jun , Hao Jihui
Published: March 2024

EGFR inhibitor has been demonstrated with huge therapeutic potential in advanced non-small cell lung cancer (NSCLC), while the patients carrying KRAS mutation had poor response to EGFR inhibitor. KRAS mutation is the most frequent molecular alteration found in advanced NSCLC, which is associated with a poor prognosis without available targeted therapy. Previous research showed that CRISPR-directed prime editor could induce unconstrained correction for KRAS mutants (KRASG12D, KRASG12C, KRASG12V). Accordingly, it would be a promising strategy to combine PE-based genetic therapy with EGFR inhibitor for enhanced therapy in NSCLC. Here,we selected several PE variants with reported highly efficient reverse transcriptase, among which eEc48-based PE6a showed the highest editing efficiency toward KRAS mutation. The lentivirus-mediated PE6a editing inhibited cell proliferation and invasion in vitro and in vivo. After that, EGFR inhibitor combined with PE6a was employed in patient-derived organoid (PDO) model carrying diverse KRAS mutants. We found that PE6a editing significantly inhibited PDO proliferation compared to EGFR inhibitor alone and control. Correspondingly, the transcriptome sequencing also showed that the cell apoptosis, cell growth and cell motility signaling were inhibited upon PE6a editing. Our findings demonstrated that the PE-directed genetic therapy could sensitize EGFR inhibitor in NSCLC, providing an alternative choice for NSCLC patients carrying KRAS mutant.

Citation Format: Qingxiao Fang, Chao Yang, Chongbiao Huang, Hongwei Wang, Jun Yu, Jihui Hao. CRISPR-directed prime editing sensitizes EGFR inhibitor by correcting oncogenic KRAS mutants In NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6493.
Abstract 6583: Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein
Authors: Jossart Jennifer , Gu Long , Haratipour Pouya , Li Caroline , Lingeman Robert , Hickey Robert , Malkas Linda , Perry John J.
Published: March 2024

Proliferating cell nuclear antigen (PCNA) is a proliferation biomarker in a variety of human tumors and has critical cellular functions in DNA replication and repair. We previously identified a unique cancer-associated isoform of the protein, termed caPCNA, which allows for structure-based drug discovery studies to develop inhibitors for selective therapeutic targeting of cancer cells. Distinct strategies have been used to develop caPCNA targeting agents, which includes peptide and small molecule-based compounds, although the success in developing therapeutically tractable compounds has been limited so far. Here, we present the crystal structures and corresponding biochemical characterizations of our novel small molecule-based caPCNA inhibitors, which bind into the PIP-box binding pocket of PCNA. Our lead compound selectively kills cancer cells, and it appears to induce replication stress, apoptosis and increase cancer cell sensitivity to genotoxic agents, while these effects are not observed in non-malignant cell controls. This caPCNA inhibitor is orally administrable, metabolic stable and it suppresses tumor growth as a monotherapy or as a combination treatment and is now an investigational new drug in Phase 1 clinical trials. PCNA plays a critical role enabling the replication fork to proceed from transcription replication conflict sites, by temporarily dislodging RNA polymerase II. Thus, structure-based drug discovery against caPCNA may open a novel therapeutic avenue for the development of selective chemotherapeutics, through targeting a cancer cell’s vulnerability to unresolved transcription replication conflicts.

Citation Format: Jennifer Jossart, Long Gu, Pouya Haratipour, Caroline Li, Robert Lingeman, Robert Hickey, Linda Malkas, John J. Perry. Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6583.
Abstract 6612: Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress
Authors: Li Melvin , Amend Sarah R. , Pienta Kenneth J.
Published: March 2024

Therapy resistance is responsible for 90% of cancer-related deaths. We identified a novel mechanism of therapy resistance in which cancer cells enter an endocycling cell state, undergoing repeated S and G phases without dividing. As a result, resistant endocycling cells are 40 times larger than mitotic cancer cell controls. Interestingly, we observed that these non-proliferative endocycling cells can eventually give rise to proliferative progeny weeks after chemotherapy is removed, modeling cancer recurrence. We hypothesize that this endocycling cancer cell state is a critical driver of therapy resistance and cancer lethality.

We evaluated oxidative stress in endocycling cells that survived the days and weeks following chemotherapy treatment using DCF-DA staining. We found increased levels of reactive oxygen species (ROS) in endocycling prostate cancer cells compared to untreated mitotic cells when normalized to cell size. Given that mitochondria are major producers and regulators of ROS, we hypothesize that cells surviving in this resistant state alter mitochondrial structure and function in response to increased oxidative stress. We performed immunofluorescence and confocal microscopy on mitochondria in untreated cells and cells following chemotherapy treatment to observe their morphology. Surviving endocycling cells had increased mitochondrial fragmentation when compared to the untreated group, a result that was further supported by reduced expression of phospho-DRP1 Ser637 via western blot.

To assess changes in glucose metabolism, we then utilized isotope tracing-based metabolomics with [U-13C]glucose on untreated and surviving endocycling cells following chemotherapy. Endocycling cells had decreased labeling of 13C in TCA cycle metabolites, suggesting a decrease in mitochondrial function when compared to untreated cells. Ongoing work involves further studying metabolic reprogramming as cells enter this resistant state with parallel labeling experiments using [1,2-13C]glucose and [U-13C]glutamine to perform mathematical modeling-based 13C-metabolic flux analysis. In addition, we engineered a cell line to track mitochondria in real-time to quantify mitochondrial dynamics underlying the morphology observed in untreated and surviving cells.

Our goal is to characterize how this therapy-resistant cell state alters its mitochondrial structure and function to survive therapeutic stress. Identifying vulnerabilities of this phenotype will enable new approaches for eliminating drug resistance in cancer.

Citation Format: Melvin Li, Sarah R. Amend, Kenneth J. Pienta. Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6612.
Abstract 668: Enhancing chemosensitivity of lung adenocarcinoma via Y-box binding protein 1 (YB-1) inhibition
Authors: Lee Jee Min , Tailor Dhanir , Garcia-Marques Fernando J. , Bermudez Abel , Pitteri Sharon , Malhotra Sanjay V.
Published: March 2024

Background: Y-box binding protein 1 (YB-1, YBX1) is a multifunctional nucleic acid binding protein that regulates the expression of various genes by binding to their mRNA and stabilizing them. In over 20 cancer types, including lung adenocarcinoma (LUAD), YBX1 expression is significantly elevated in tumor samples compared to normal tissue. Within cancer cells, YB-1 promotes the expression of genes involved in tumorigenesis and chemoresistance. Consequently, elevated YBX1 expression in tumor is associated with poor clinical outcomes and significantly reduced overall survival in LUAD patients. Therefore, YB-1 emerges as a promising therapeutic target for LUAD treatment. Utilizing a small molecule YB-1 inhibitor, CET056, we performed a series of in vitro and in vivo analyses to assess the therapeutic efficacy of YB-1 inhibition and unravel YB-1’s role in tumorigenesis and disease progression in LUAD.

Methods: Four human and one mouse LUAD cell lines were treated with CET056 and/or chemotherapeutic drugs and analyzed for the in vitro therapeutic activity of CET056. Using xenograft and syngeneic LUAD mouse models, the preclinical therapeutic efficacy of CET056 on disease progression of LUAD was also tested.

Results: CET056 was shown to effectively decrease YB-1 expression in both human and mouse LUAD cell lines and exert inhibitory effects on LUAD in vitro and in vivo. This treatment delayed tumor progression and metastasis in CMT-64 syngeneic mouse model. Proteomics analysis revealed that translational machinery-associated pathways were down-regulated in LUAD cells after CET056 treatment whereas apoptosis and cell death-associated pathways were up-regulated. In alignment with this finding, CET056 treatment in combination with cisplatin showed chemo-sensitizing effect on LUAD in vitro and in vivo.

Conclusion: Our studies suggest that YB-1 could be a promising target for combating chemoresistance in LUAD. Inhibition of YB-1 with a small molecule drug as a monotherapy or combination therapy could an effective strategy to treat LUAD.

Citation Format: Jee Min Lee, Dhanir Tailor, Fernando J. Garcia-Marques, Abel Bermudez, Sharon Pitteri, Sanjay V. Malhotra. Enhancing chemosensitivity of lung adenocarcinoma via Y-box binding protein 1 (YB-1) inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 668.
Abstract 6686: The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors
Authors: Lin Nina Yi-Tzu , Fukuoka Shota , Motooka Daisuke , Shigeno Yuko , Irie Takuma , Tourlousse Dieter M. , Murotomi Kazutoshi , Sekiguchi Yuji , Tamaki Hideyuki , Shindo Mariko , Tsuji Takahiro , Wake Hiroaki , Goto Koichi , Doi Toshihiko , Shitara Kohei , Watanabe Keisuke , Maeda Yuka , Nakamura Shota , Benno Yoshimi , Nishikawa Hiroyoshi , Koyama Shohei
Published: March 2024

Gut microbiota is associated with antitumor efficacy of immune checkpoint blockade therapies, yet the mechanism is not largely understood. Here, we show that a novel bacterial strain of Ruminococcaceae isolated from the feces of patients who respond to PD-1 (programmed cell death 1) blockade efficiently primes/activates tumor-specific CD8+ T cells through effective stimulation/maturation of CD103+ dendritic cells (DCs) during migration from the gut to the tumor microenvironment (TME). The administration of this bacteria strain with PD-1 blockade significantly inhibited tumor growth, even when adding them to fecal transplantation of non-responder patients in animal models. Mechanistically, the bacterial strain promoted the IRF8-driven differentiation of CD103+ DCs by stimulating multiple Toll-like receptors and the accumulation of these DCs in tumors and tumor-draining lymph nodes. These CD103+ DCs prolonged engagement with cognate antigen-specific CD8+ T cells, which induced the activation of CD8+ T cells specific for a wide variety of tumor antigens and fostered their PD-1 expression through enhanced T-cell activation signals with NFATc1 nuclear translocation, thereby leading to abundant PD-1+CD8+ T cells, a key effector cell population in PD-1 blockade, with a broader T-cell receptor repertoire in the TME. Our findings with novel single bacterium isolated from feces of responder patients demonstrate the mode of action by gut microbiota to augment antitumor immunity.

Citation Format: Nina Yi-Tzu Lin, Shota Fukuoka, Daisuke Motooka, Yuko Shigeno, Takuma Irie, Dieter M. Tourlousse, Kazutoshi Murotomi, Yuji Sekiguchi, Hideyuki Tamaki, Mariko Shindo, Takahiro Tsuji, Hiroaki Wake, Koichi Goto, Toshihiko Doi, Kohei Shitara, Keisuke Watanabe, Yuka Maeda, Shota Nakamura, Yoshimi Benno, Hiroyoshi Nishikawa, Shohei Koyama. The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6686.
Abstract 6692: Intermittent fasting-remodeled gut microbiota improves anti-tumor immunity in pancreatic cancer
Authors: Li Le , Chandra Vidhi , Le Roux Olivereen , Howell Rian Morgan , Tahan Virginia , Putluri Nagireddy , Petrosino Joseph , Sreekumar Arun , Kolls Jay K. , White James , McAllister Florencia
Published: March 2024

Pancreatic ductal adenocarcinoma (PDAC) has become the third leading cause of cancer-related death in the United States with high resistance to therapies, presentation at late stages and aggressive biology. Immunotherapy has revolutionized cancer therapy by offering survival benefit in many solid malignancies, however, it is still not effective in pancreatic cancer. Host factors are crucial modulators of responses to immunotherapy. The roles of gut microbiome in modulating the immune system and determining cancer outcomes have been well established. Eating patterns can alter the gut microbiota and metabolic output, both of which affect anti-tumor immune responses. Intermittent fasting (IF), a form of dietary restriction which is characterized by limited feeding with unlimited calorie consumption during a certain time window. IF has been shown to affect gut microbiota composition, modulate immunity, and metabolic processes. Despite the well-known beneficial role of fasting in pancreatic conditions, including pancreatitis and diabetes, its effect in the treatment of pancreatic cancer has not been explored. Our study found that IF reverses immunotherapy resistance in PDAC. IF can reshape gut microbiota composition and reprogram the metabolism of host. We found that IF improves anti-tumor immunity through modulating gut microbiota. Depletion of gut microbiota can abrogate the beneficial effect of IF on anti-tumor response, while fecal microbiota transplantation from mice exposed to IF can sensitize the anti-tumor effects of immunotherapy. Using 16S rRNA sequencing, metabolic profiling and transcriptomic sequencing, our findings elucidate the mechanism of IF-remodeled gut bacteria influencing metabolic molecules which drive immune cells activation. Our study suggests that IF-based interventions have potential utility for the prevention and treatment of PDAC.

Citation Format: Le Li, Vidhi Chandra, Olivereen Le Roux, Rian Morgan Howell, Virginia Tahan, Nagireddy Putluri, Joseph Petrosino, Arun Sreekumar, Jay K. Kolls, James White, Florencia McAllister. Intermittent fasting-remodeled gut microbiota improves anti-tumor immunity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6692.
Abstract 6707: IgG based B7-H3xCD3 bispecific antibody for treatment of gynecological tumors
Authors: Greiner Sarah Maria , Mauermann Jonas , Lutz Martina , Hagelstein Ilona , Tobias Engler , Koch André , Andreas Hartkopf , Sara Brucker , Zekri Latifa , Märklin Melanie
Published: March 2024

Ovarian cancer is characterized by late stage disease and a poor prognosis. Endometrial cancer is a heterogeneous disease with different prognoses depending on the molecular subtype. Novel treatment strategies are needed to improve patient survival in both entities.

T cell-based immunotherapy has significantly improved the treatment options for many malignancies. B7-H3 (CD276) is an interesting target for immunotherapeutic approaches because it is overexpressed on a wide range of tumor entities and has characteristics of immune checkpoint molecules. Its expression not only on tumor cells but also on the tumor vasculature allows improved infiltration of immune effector cells into the tumor to overcome the major limitation of T cell-based therapy of solid tumors, which is the access of immune cells to the tumor site. A novel bispecific antibody in an IgG-based format called CC-3 with B7-H3xCD3 specificity has already been shown to efficiently induce T cell responses against gastrointestinal malignancies.

Here we show that B7-H3 is highly expressed on endometrial and ovarian cancer cell lines. Treatment with CC-3 induced T cell activation and degranulation as well as secretion of IL-2, IFNy and perforin. These results demonstrate CC-3 induced T cell reactivity against ovarian and endometrial cancer cells. In addition, CC-3 induced efficient T cell proliferation and formation of T cell memory subsets. This resulted in potent target cell lysis.

Taken together, our results underscore the potential of CC-3, which is currently in GMP manufacturing, to enable clinical evaluation for the treatment of gynecologic malignancies.

Citation Format: Sarah Maria Greiner, Jonas Mauermann, Martina Lutz, Ilona Hagelstein, Engler Tobias, André Koch, Hartkopf Andreas, Brucker Sara, Latifa Zekri, Melanie Märklin. IgG based B7-H3xCD3 bispecific antibody for treatment of gynecological tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6707.
Abstract 6721: AI-guided generation of novel GUCY2C-targeting T-cell engagers for the treatment of colorectal cancer
Authors: Tian Liang , Hou Jinxiu , Su Chenpeng , Xu Xiaoou , Chen Xiaoqian , Liu Dandan , Tian Jiyuan , Shen Rui , Pan Yi , Yuan Yuan , Dong Lisha , Gu Tingting , Peng Jian
Published: March 2024

Colorectal cancer (CRC) ranks as one of the most lethal cancers worldwide. Given the advances in immunotherapy and targeted therapy, the survival time in CRC has been improving recently. However, the development of novel and effective therapies for CRC still remains an unmet medical need. Guanylyl cyclase C (GUCY2C) is a transmembrane receptor expressed on the surface of intestinal epithelial cells. Recent studies have demonstrated that GUCY2C is causally linked to intestinal inflammation, dysbiosis, and tumorigenesis. To this end, GUCY2C has emerged as a promising target for immunotherapy and a diagnostic marker of primary and metastatic cancer. Here we describe the discovery and optimization of a novel panel of GUCY2C-targeting T cell engagers (TCEs) developed via our streamlined AI-guided TCE development platform. We have identified a number of anti-GUCY2C antibodies with various binding epitopes on GUCY2C, and subsequently generated a panel of novel GUCY2C-targeting multispecific antibodies with favorable developability and functionalities via our AI-driven engineering technology. Our data demonstrate that a biparatopic GUCY2C x CD3 TCE engineered as an asymmetric ‘1+2’ format can recognize two distinct epitopes on GUCY2C. This biparatopic GUCY2C x CD3 TCE shows enhanced tumor cell binding affinity and superior T cell-mediated cytotoxicity compared to the ‘1+1’ GUCY2C x CD3 TCE and ‘1+2’ bivalent GUCY2C x CD3 TCE where the two GUCY2C targeting arms can only bind to an identical epitope on GUCY2C. In addition, we have engineered a novel GUCY2C × CD3 x CD28 trispecific TCE, in which the CD3-targeting arm in tandem with the co-stimulating CD28-targeting arm mediate the interaction with T cells. This anti-GUCY2C trispecific TCE induces favorable synergistic T cell activation and tumor cell killing in vitro compared to the bispecific counterparts. In vivo anti-tumor activity assessments of these multispecific TCE molecules in various tumor xenograft mouse models are currently ongoing.

Citation Format: Liang Tian, Jinxiu Hou, Chenpeng Su, Xiaoou Xu, Xiaoqian Chen, Dandan Liu, Jiyuan Tian, Rui Shen, Yi Pan, Yuan Yuan, Lisha Dong, Tingting Gu, Jian Peng. AI-guided generation of novel GUCY2C-targeting T-cell engagers for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6721.
Abstract 6765: A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response
Authors: Crouchet Emilie , Almeida Nuno , Durand Sarah C. , Parnot Marie , Oudot Marine A. , Giannone Fabio , Gadenne Cloé , Cherradi Sara , Roehlen Natascha , Saviano Antonio , Felli Emanuele Emanuele , Pessaux Patrick , Chayama Kazuaki , Duong Hong Tuan F. , Baumert Thomas F. , Schuster Catherine
Published: March 2024

Hepatocellular carcinoma (HCC) is the third leading and fastest rising cause of cancer-related death worldwide. The discovery and preclinical development of compounds targeting HCC is hampered by the absence of authentic tractable systems recapitulating the heterogeneity of patient HCC tumors and the tumor microenvironment (TME). Here, we established a novel, simple and robust patient-derived multicellular tumorspheroid model based on clinical HCC tumor tissues combined with the autologous patient serum. The model was robust and reproducible independently from cancer etiology and grade recapitulating key functional features of original tissue. We show that the use of autologous patient serum was essential for the TME function. The tumorspheroids comprised the main cell compartments including epithelial cancer cells as well as all major cell populations of the TME (cancer-associated fibroblasts, macrophages and T cells). Cell type proportions were variable between tumorspheroids reflecting HCC heterogeneity. Moreover, we observed differential responses to FDA HCC approved drugs (including tyrosine kinase and checkpoint inhibitors as well as VEGF-targeting agents) between donors as in patients. This patient HCC serum-tumorspheroid model provides novel opportunities for drug discovery and development as well mechanism of action studies including compounds targeting the TME. In conclusion, this HCC serum-tumorspheroid platform will contribute to improve the dismal outcome of patients with HCC.

Citation Format: Emilie Crouchet, Nuno Almeida, Sarah C. Durand, Marie Parnot, Marine A. Oudot, Fabio Giannone, Cloé Gadenne, Sara Cherradi, Natascha Roehlen, Antonio Saviano, Emanuele Emanuele Felli, Patrick Pessaux, Kazuaki Chayama, Hong Tuan F. Duong, Thomas F. Baumert, Catherine Schuster. A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6765.
Abstract 6770: Acquired temozolomide resistance instructs patterns of glioblastoma cell motility and proteome expression within gelatin hydrogels
Authors: Kriuchkovskaia Viktoriia , Eames Ela , Riggins Rebecca , Harley Brendan
Published: March 2024

Acquired drug resistance in glioblastoma (GBM) presents a major clinical challenge and is one of the key factors contributing to the abysmal prognosis with less than 15 months median overall survival. The aggressive chemotherapy with the frontline therapeutic, temozolomide (TMZ), ultimately fails to kill residual, highly invasive tumor cells after surgical resection and radiotherapy. Here, we present a robust three-dimensional (3D) in vitro hydrogel model of acquired temozolomide (TMZ) resistance using isogenically-matched glioblastoma (GBM) cell lines. We demonstrate distinct drug response patterns between TMZ-sensitive and TMZ-resistant GBM cells, emphasizing our model's promise for future pre-clinical investigations aimed at developing novel therapies targeting drug-resistant GBM. Importantly, our model detects apoptosis-related protein changes in response to TMZ, validating its ability to study drug-induced molecular changes. Furthermore, our study delves into the migratory potential of GBM cells within the 3D extracellular matrix environment. Additionally, we uncover significant alterations in cytokine expression associated with matrix remodeling and angiogenesis, providing new avenues for therapeutic investigation. Although our findings are derived from a specific set of isogenically-matched cell lines, this model highlights the potential to explore personalized treatment strategies. Ongoing research in our lab is dedicated to gaining a more profound understanding of matrix remodeling and the migratory potential of TMZ-resistant GBM cells. Moreover, our future work will aim to expand our study to include a wider range of cell lines and patient-derived xenografts within our advanced microphysiological systems that include key elements of GBM tumor microenvironment such as perivascular niche. This endeavor seeks to establish connections between observed therapeutic behaviors and clinical outcomes, ultimately contributing to the development of more effective treatments for GBM patients.

Citation Format: Viktoriia Kriuchkovskaia, Ela Eames, Rebecca Riggins, Brendan Harley. Acquired temozolomide resistance instructs patterns of glioblastoma cell motility and proteome expression within gelatin hydrogels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6770.
Abstract 6775: 3D culture aggregation morphology depends on cell phenotype and gene expression profiles in lung cancer
Authors: Hayashi Naohiro , Tsudaka Shimpei , Suzawa Ken , Higashihara Tomoaki , Thu Yin Min , Matsuoka Atsushi , Mizuno Daisuke , Yoshichika Ryo , Mukohara Fumiaki , Yoshikawa Mao , Shien Kazuhiko , Yamamoto Hiromasa , Toyooka Shinichi
Published: March 2024

Background: Three-dimensional (3D) cell culture allows for the reproduction of tissue structure and function in a state more akin to the in vivo environment compared to two-dimensional (2D) cell culture. It is established that 3D culture more faithfully replicates cell morphology, differentiation, proliferation, gene and protein expression, and responsiveness to stimulation, as well as drug metabolism. Focusing on the observation that lung cancer cell lines exhibit different clustering morphologies in 3D cell culture, we conducted a comparative study on the relationship between these clustering morphologies and the characteristics of the lung cancer cell lines.

Methods: We cultured 30 lung cancer cell lines using 3D culture plate, and classified the types of cell clustering morphologies formed. We evaluated the molecular and cellular biology characteristics of all cell lines. Additionally, comprehensive gene expression analysis using RNA sequencing was performed on the 30 3D-cultured cell samples, and the characteristics of gene expression between different clustering types were compared.

Results: The 3D culture aggregation morphology of 30 lung cancer cell lines were classified into three types (monolayer sheet, NOS, spheroid). Monolayer sheet-type cells exhibited higher proliferation, migration, and invasion capabilities compared to spheroid-type cells. In the transcriptomic bioinformatics analysis of the acquired gene expression data, GSEA analysis using Hallmark gene sets showed that the enrichment of epithelial-mesenchymal transition (EMT) and cancer metastasis-related genes in the monolayer type cells significantly compared to the spheroid type cells. Conversely, cell adhesion and intercellular binding-related gene sets were highly enriched in the spheroid type cells.

Conclusion: 3D culture aggregation morphology represents phenotypic properties and transcriptomic profiles in non-small cell lung cancer cells.

Citation Format: Naohiro Hayashi, Shimpei Tsudaka, Ken Suzawa, Tomoaki Higashihara, Yin Min Thu, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. 3D culture aggregation morphology depends on cell phenotype and gene expression profiles in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6775.
Abstract 6777: An ex-vivo organotypic culture platform of mucinous carcinomatosis peritonei identifies CDK4-6 inhibition as a novel treatment
Authors: Weitz Jonathan , Nishizaki Daisuke , Patel Jay , Ng Isabella , Sun Siming , Ramms Dana , Zou Jingjing , Baumgartner Joel , Kelly Kaitlyn , Patel Hitendra , White Rebekah , Veerapong Jula , Vu Peter , Gutkind Silvio , Tiriac Herve , Kato Shumei , Lowy Andrew
Published: March 2024

Concomitant mutations in KRAS and GNAS have been linked to mucinous histology in gastrointestinal neoplasms of the appendix, colon, and pancreas (IPMN). Upon progression, these tumors are characterized by metastasis which favors the peritoneal surface, resulting in mucinous carcinomatosis peritonei (MCP). Recently we have described a novel technique which allows for long term culture and drug intervention of human MCP tumors ex-vivo. With evidence of durable clinical benefit of CDK4-6 inhibition in a single patient with chemo refractory MCP of appendiceal origin, we sought to further investigate anti-tumor responses using CDK4-6 inhibition in ex-vivo tumor slices, in order to identify candidates for a personalized clinical therapy trial. Here, in a comparison of ex-vivo patient tumor slices from 18 individual donors, treatment using CDK4-6 inhibitors revealed a significant reduction in cancer cell specific proliferation in mucinous GNAS-mut tumors compared to non-mucinous cancers. Anti-tumor responses of CDK4-6 were determined to be tumor-cell intrinsic, as CDK4-6 inhibition blocked proliferation of stromal cells in the TME independently of patient tumor mutational and mucin status. Based on these finding a personalized clinical trial was conducted where we report that 13 of 16 patients (81%) enrolled with MCP treated with palbociclib had at least a 10% decrease in CEA, as compared to historical chemotherapy responses rates reported from 14-30%. These results indicate that CDK4/6 inhibition is a novel and efficacious treatment for patients with MCP.

Citation Format: Jonathan Weitz, Daisuke Nishizaki, Jay Patel, Isabella Ng, Siming Sun, Dana Ramms, Jingjing Zou, Joel Baumgartner, Kaitlyn Kelly, Hitendra Patel, Rebekah White, Jula Veerapong, Peter Vu, Silvio Gutkind, Herve Tiriac, Shumei Kato, Andrew Lowy. An ex-vivo organotypic culture platform of mucinous carcinomatosis peritonei identifies CDK4-6 inhibition as a novel treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6777.
Abstract 6824: Secretion of MAGE-A3 and MAGE-A4 in cell lines and lung cancer
Authors: Gilmore Bailey , Kong Dehe , Gonzalez Rachel , Yom Jina , Han Andy , Qu Tianli , Strom Alex , Zhao Xiaoxiao , Zewdu Eden , Guo Hailey , Guo Zhaoying , Zhang Ranran , Hu Xiaomin , Ren Qi , Liu Xuan , Fu Wei
Published: March 2024

Melanoma-associated antigen gene A (MAGE-A) family of proteins have previously been shown to express in a variety of tumors, having different roles in cancer pathogenesis. MAGE-A3 and MAGE-A4 have both been shown to inhibit the p53 pathway which leads increased cell growth by stopping apoptosis. Both of these proteins have been associated with cancer progression and poor prognosis. In our studies, we have evaluated multiple MAGE-A3 and MAGE-A4 antibodies using CytoSections. While previous research had only pointed to these proteins being intracellular, our overexpression studies in the development of CytoSections indicated that some MAGE-A proteins were secreted. In this study, ELISA binding assays corroborated that MAGE-A3 and MAGE-A4 were secreted from cells overexpressing these proteins, and serum MAGE-A4 was detected in 24% of lung cancer patients. Flow cytometry data was then collected to see that exogenous MAGE-A proteins were binding to cells in a pattern consistent with secretion data from IHC experiments. Variants of MAGE-A4, differing by only a handful of amino acids, showed very different levels of secretion from one another. MAGE-A3 and MAGE-A6 share 95% homology and were also extremely different in secretion shown between IHC, flow cytometry, and ELISA. This study validates that certain members of the MAGE-A family are secreted from cells.

Citation Format: Bailey Gilmore, Dehe Kong, Rachel Gonzalez, Jina Yom, Andy Han, Tianli Qu, Alex Strom, Xiaoxiao Zhao, Eden Zewdu, Hailey Guo, Zhaoying Guo, Ranran Zhang, Xiaomin Hu, Qi Ren, Xuan Liu, Wei Fu. Secretion of MAGE-A3 and MAGE-A4 in cell lines and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6824.