Patient-derived Tumor Research Articles

Abstract 7203: Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft tumors

Abstract Mutant epidermal growth factor receptor (EGFR), a major oncogenic driver in non-small cell lung cancer (NSCLC), is found in the tumors of 15% (western countries) to 50% (Asian countries) of patients. EGFR-targeted therapy using tyrosine kinase inhibitors (TKIs) is a standard of care for those patients who developed metastatic or recurrent diseases. However, despite having an 80% initial response rate, most patients had partial tumor regression and their disease would progress when acquired resistance emerges. Thus, developing more effective treatment is an unmet need. The clinical response of these patients can be recapitulated in mouse models of patient-derived xenograft (PDX) tumor bearing sensitizing EGFR mutations. In this study, treating established PDX tumors with osimertinib, a third generation EGFR TKI, induced partial tumor regression (50% to 70% size reduction from baseline) followed by progression with a median progression-free survival (PFS) of 26 to 28 days. Interestingly, co-administering an antibody against hepatoma-derived growth factor (HDGF) with osimertinib resulted in enhanced tumor regression (98% to 100% size reduction from baseline) which translated into significantly extended animal survival with a median PFS of 103 or 135 days. Immunohistochemical and western blot examination of tumors collected at different treatment timepoints indicated that AKT/mTOR and MAPK pathways were reactivated after initial suppression in TKI monotherapy arm despite persistent EGFR inhibition. Since AKT/mTOR and MAPK pathways are key survival and proliferative signaling pathways, their reactivation can promote TKI tolerance of tumor cells and the emergence of TKI resistance. However, blocking HDGF attenuated the reactivation of these pathways which could partially explain the enhanced efficacy of osimertinib plus anti-HDGF antibody combination treatment seen in this study. Our results suggest tumor-derived HDGF could play a crucial role in the rise of acquired resistance to osimertinib. Identifying the exact cells and molecular pathways utilized by HDGF to promote tumor cell tolerance to TKI will help us understand how TKI sensitive tumor cells survive EGFR blockade and emerge as resistant cells. Citation Format: Cindy Q. Zhou, Ariel Li, Kaoru Ri, Ahmed Sultan, Hening Ren. Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft 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 7203.

Abstract 4608: Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids

Abstract The alternative splicing of mRNA precursors allows one gene the capacity to yield multiple protein isoforms, each with distinct functions. Although this intricate process is subject to stringent regulation under normal physiological conditions, aberrant alternative splicing can generate atypical proteins and contribute to various diseases, including cancer. By activating splicing factors, Cdc-like kinases (CLKs) serve as pivotal regulators of alternative splicing and are considered promising therapeutic targets for various tumors. In this study, we investigated the activity of two CLK inhibitors, cirtuvivint (SM08502) and CC-671, in combination with precision oncology agents or conventional chemotherapeutics. Thirty highly characterized patient-derived cancer cell lines were selected from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm). The cell lines were derived from a range of cancer types including head and neck, bladder, pancreas, endometrial, colon, and melanoma, and contained clinically relevant variants of KRAS (G12C, G12D). Multi-cell type tumor spheroids were grown from a ratio of 6:2.5:1.5 malignant cells, endothelial cells, and mesenchymal stem cells, respectively. Following three days of growth, the spheroids were treated with single agents or combinations at concentrations up to their clinical Cmax value, if reported. After seven days of continuous drug exposure, cell viability was assessed using the CellTiter-Glo 3D assay. As a single agent, cirtuvivint showed concentration-dependent activity in all spheroid types with 1 – 3 logs of cytotoxicity, whereas CC-671 did not show activity in all spheroid types and achieved no more than 1 log of cytotoxicity. Among the standard chemotherapeutic drugs, doxorubicin and SN-38 demonstrated additive and greater-than-additive cytotoxicity in various spheroid types when combined with either CLK inhibitor. Several of the targeted oncology drugs exhibited noteworthy additive and greater-than-additive cytotoxicity when combined with a CLK inhibitor. These agents included the XPO1 inhibitor, eltanexor, the MCL-1 inhibitor, tapotoclax, the α-isoform-specific PI3K inhibitor, inavolisib, and the pan-PI3K inhibitor, copanlisib. Notably, combinations of the CLK inhibitors with the KRAS G12D variant-specific inhibitor, MRTX-1133, showed selective activity against all tumor cell lines harboring this genetic variant. Interestingly, a strong antagonistic interaction between paclitaxel and CC-671 was observed in all thirty spheroid types, while no such interaction was observed with cirtuvivint. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Nathan P. Coussens, Steven D. Gore, James H. Doroshow, Beverly A. Teicher. Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids [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 4608.

Abstract 6903: Treatment efficacy of transcatheter hepatic arterial infusion chemotherapy with cisplatin in a severe immunodeficient rat model with patient-derived primary pancreatic ductal adenocarcinoma xenografted into the liver

Abstract Purpose: Liver metastases of pancreatic ductal adenocarcinoma (PDAC) is a highly fatal disease; most patients die within 5 years. There is a critical need to identify appropriate anticancer treatments for these patients. A patient-derived tumor xenograft (PDX) model in severe immunodeficient rat with primary PDAC xenografted into the liver is suitable for evaluating transcatheter hepatic arterial infusion chemotherapy. Materials and Methods: The purpose of this study was to develop a severe immunodeficient rat model with patient-derived primary PDAC xenografted into the liver, and to assess the treatment efficacy of hepatic arterial infusion chemotherapy with cisplatin in this model. Three patient-derived tumors of PDAC were transplanted into the liver of 21 rats each, totaling 63 rats. Four weeks after transplantation, the 21 rats were randomly assigned into three treatment groups of seven rats each: hepatic arterial infusion, systemic venous infusion, and control group. Tumor volumes were evaluated on CT twice at pre-treatment and 4-weeks post-treatment. Following euthanasia of the rats, resected tumor specimens were pathologically analyzed. Results: The liver-implanted PDAC PDX rat model was established in all 63 rats. hepatic arterial infusion chemotherapy and systemic venous infusion chemotherapy with cisplatin were able to be performed in the assigned rats. The hepatic arterial infusion group significantly inhibited tumor growth and reduced Ki67 expression, indicating lower cell proliferation, compared to the control group in three PDX tumors. Conclusion: A rat-based preclinical platform was established by liver-implanted PDAC PDX rat model. Arterial cisplatin infusion chemotherapy would be one of potential therapies for PDAC liver metastases. Citation Format: Ken Kageyama, Akira Yamamoto, Masanori Ozaki, Kenjiro Kimura, Shinpei Eguchi, Ryota Tanaka, Mizue Terai, Takami Sato, Yukio Miki. Treatment efficacy of transcatheter hepatic arterial infusion chemotherapy with cisplatin in a severe immunodeficient rat model with patient-derived primary pancreatic ductal adenocarcinoma xenografted into the liver [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 6903.

Abstract 5409: Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion

Abstract Although head and neck squamous cell carcinomas (HNSCCs), including tongue, vocal cord, and buccal cancers, are one of the most common cancers worldwide with a mortality rate of more than 40%, there have been no solid standard biomarkers for effectively diagnosing HNSCCs at early stages and no effective protocols available to support in vitro expansion of HNSCC cells. To establish a cell culture method to support in vitro self-renewal of HNSCC cells, patient-derived tumor was enzymatically digested, and the tongue cancer epithelial cells were seeded onto feeder cells with the Rho kinase inhibitor (Y-27632). Additionally, to test our hypothesis that free thiol groups in the cells, generated by a mild reduction of cell surface proteins using the reducing agent tris(2-carboxyethyl) phosphine hydrochloride (TCEP), would modulate cancer cell adhesion and migration, we assessed the expression levels of integrins, focal adhesion kinase (FAK), and phosphorylated FAK (pFAK) of tongue cancer epithelial cells cultured on both soft and rigid polyacrylamide (PAAm) hydrogel-based cell culture substrates. Patient-derived tongue cancer epithelial cells exhibited self-renewal ability and maintained cancerous phenotypes, with high expressions of P53, Ki67, E-cadherin, CD44, and AHDH1A1. Treatment with the reducing agent (TCEP) increased the degree of cell adhesion through the significantly upregulated integrin α5β1-mediated phosphorylation of FAK, resulting in inhibited single-cell and collective cell migration as well as invasion. Additionally, when these cells were cultured on stiff PAAm hydrogels mimicking the tumor microenvironment, the cells exhibited accelerated cell migration behaviors, compared to cells on soft hydrogels. These results highlight the synergetic contributions of feeder cells and Rho kinase inhibitor to support long-term self-renewal of tongue cancer patient-derived epithelial cells. Citation Format: Joo Hyun Kim, Laurensia D. Anggradita, Sung Sik Hur, Jae Hong Park, Sang-Heon Kim, Myung Jin Ban, Yongsung Hwang. Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion [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 5409.

Abstract 3210: Raptamer-drug conjugates as molecularly targeted cancer therapeutics

Abstract The use of aptamers as a therapeutic targeting moiety holds the promise of building on the success of antibody-drug conjugates (ADCs). While ADCs are an attractive modality, they face limitations, including limited penetration into solid tumors and a complicated production process for large-scale manufacturing. Aptamers have several potential advantages over antibodies as a targeting moiety including: (i) a smaller size that enables better tumor penetration; (ii) chemical synthesis which allows rapid scale-up and ease of incorporating modifications; (iii) a superior safety profile from lack of immunogenicity and (iv) room temperature stability. Therefore, the use of aptamers in conjunction with drugs (similar to ADCs) holds promise for development of targeted therapeutics. We have identified a novel therapeutic target for certain cancers (COF-01) using proteomics data and immunohistochemistry from patient-derived tumors. Both the COF-01 protein and related mRNA expression was shown to be significantly increased in non-small cell lung cancer, head and neck cancer and pancreatic cancer. Here, we present a new therapeutic approach that targets COF-01-positive cancer cells using next generation aptamer conjugates. We performed a cell- and protein-based selection using our proprietary bead-based combinatorial libraries to generate next-generation base-modified ssDNA aptamers (Raptamers) against COF-01. Binding affinities of the COF-01 Raptamer candidates were determined by concentration curves using biolayer interferometry. The dissociation constants for the COF-01 Raptamers ranged from 80 nM - 200 nM. COF-01-specific Raptamer-drug conjugates (Rap-DCs) were developed by conjugating the Raptamers to monomethyl auristatin E via a cleavable linker. In vitro efficacy data showed the COF-01 Rap-DC can selectively induce cytotoxicity in different types of COF-01-positive human cancer cells (A431, CAL 27, BxPC-3). There was a significant decrease in cell viability of COF-01-positive cells when compared to COF-01-negative control cells (IC50 values ranged from 5.19 nM - 14.11 nM). In vivo efficacy testing in a COF-01-postive cell-derived xenograft mouse model showed a significant decrease in tumor volume in animals treated with the COF-01 Rap-DC when compared to saline- and erlotinib-treated controls. This provides proof-of concept for the use of high-affinity Raptamers against novel molecular targets, such as COF-01. This platform can be used to develop Rap-DCs against emergent neo-antigens on cancer cells that have escaped chemotherapy. Citation Format: Stephanie P. Vega, Nancy E. Ward, Uksha Saini, Leniher Castan-Chibas, Garima Kaushik, Daniel Ciznadija, Dev Chatterjee, Michael J. Heffernan, Atul Varadhachary, Michael Ritchie. Raptamer-drug conjugates as molecularly targeted cancer therapeutics [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 3210.

Abstract 7480: Human LINE-1 ORF1p retrotransposition independent function in pancreatic cancer

Abstract The aberrant expression of repeat elements is a common feature found in both mouse models and human pancreatic ductal adenocarcinoma (PDAC). The majority of these are non-coding RNAs that have shown the ability to stimulate a viral-like innate immune response. The long interspersed nuclear element 1 (LINE-1) retrotransposon is one of the few protein coding repeat elements in the human genome. LINE-1 has two open reading frames, ORF1 and ORF2. The former (ORF1p) encodes a ribonucleotide protein and the latter (ORF2p) a reverse transcriptase and endonuclease. Although nuclear ORF1p and ORF2p is required for retrotransposition, ORF1p is primarily localized in the cytoplasm, which suggests the possibility of ORF1p function independent of LINE-1 retrotransposition. Here, we show that ORF1p has a function to shield repeat RNAs from activating pathogen recognition receptor (PRR) mediated antiviral response in PDAC cells. LINE-1 ORF1p-targeting shRNA significantly inhibits patient-derived PDAC tumor growth both in vitro and in vivo. Total RNA sequencing (RNA-seq) reveals distinct transcriptional profiles with ORF1p deficiency. Gene set enrichment analysis (GSEA) identified high enrichment of innate immune response-related pathways including IL6-JAK-STAT3 signaling, inflammatory response, interferon response, and apoptosis in ORF1 knockdown PDAC cell lines. These immune related pathways are suppressed by deletion of RIG-I and MAVS, two components of viral RNA sensing. Mechanistically, we demonstrated that ORF1p is associated with repeat RNAs including LINE-1 RNA (L1HS) in processing bodies (PBs), which are cytoplasmic membraneless ribonucleoprotein (RNP) granules. In the PBs, ORF1p interacts with MOV10 RNA helicase, an essential enzyme for dsRNA unwinding. Using MOV10 truncated mutants, we found that the ORF1p-MOV10 interaction is important in attenuation of PRR sensing and apoptosis upon repeat RNA exposure. Finally, we evalulated the activation of the innate immune response in the tumor microenvironment by ORF1p depletion. These results suggest that ORF1p loss induces a desmoplastic reaction driven by Acta2+ myofibroblastic CAFs in response to inflammatory cytokines. Collectively, our results demonstrate a critical and previously uncharacterized role of ORF1p in evasion of endogenous repeat RNA-triggered antiviral immune response in PDAC cells with implications in the tumor microenvironment. Citation Format: Eunae You, Bidish Patel, Alexandra Rojas, Natalie Ho, Siyu Sun, Michael Raabe, Yuhui Song, Ildiko Phillips, Katherine Xu, Peter Richieri, Linda Nieman, Benjamin Greenbaum, David Ting. Human LINE-1 ORF1p retrotransposition independent function 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 7480.

Abstract 217: Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity

Abstract Background: Cancer-associated fibroblasts (CAFs) are heterogeneous, with CAF subtypes being localized in spatially defined areas of tumors. However, it remains unclear which CAF subsets specifically play a role in fundamental processes such as tumor growth, invasion, and response to therapy. This study aims to establish a novel in vitro model that recapitulates the in vivo tumor-stroma spatial relationship to investigate CAF heterogeneity and its impact on tumor cell growth in non-small cell lung cancer (NSCLC). Methods: The in vitro model was established using NSCLC patient-derived CAFs and tumor organoids as a 6:1 ratio. Tumor cells originating from xenograft-derived organoids (XDOs) were embedded in a hydrogel containing CAFs (Day 0) and co-cultured for 5 days. Confocal microscopy was utilized to characterize and measure the proliferation of the XDO tumor cells in the co-culture setup, and the proliferation rate was defined as differences in tumor spheroid area between Day 1 and 5. Single-cell RNA-sequencing (scRNAseq) and gene set enrichment analysis (GSEA) were then used to assess the gene expression and signaling pathways dominant in mono- and co-culture to characterize CAF heterogeneity in this model. Results: Initially, the culture media formulations and the matrix for the growth of both CAFs and tumor cells were screened to assess the compatibility of different culture elements. Both CAFs and XDO tumor cells proliferated well in reduced organoid media (M27RD). This reduced medium and the novel matrix composition (50% Matrigel/50% Rat Tail type I collagen) also allowed for maintaining CAF contractility and XDO proliferation. Utilizing these harmonized media and matrix conditions, a spatially defined co-culture model, which incorporated XDOs as a central cluster surrounded by CAFs, was generated. In confocal microscopy, the proliferation rate of tumor cells was significantly higher in co-cultures with CAFs (adjusted p-value < 0.01), while not significant in mono-culture of tumor cells. The GSEA using the Hallmark gene sets on scRNAseq data generated in three co-cultures identified the heterogeneity among CAFs: two CAFs showed significantly higher effect on tumor cell proliferation with the most highly differential expressed genes (DEGs) enriched with inflammation and mitosis and the top high DEGs associated with epithelial-mesenchymal transition, while the other CAF has no effect. Conclusion: The harmonized co-culture conditions for NSCLC organoids and CAFs enabled the characterization of both cell phenotypes and the building of an informed model architecture to study tumor-stroma communication. The presence of CAFs increased tumor cell growth with enrichment in secreted proteins involved in epithelial-mesenchymal transition. Citation Format: Cheol-Kyu Park, Roya Navab, Elisa D'Arcangelo, Quan Li, Hiroyuki Ogawa, Nikolina Radulovich, Nhu-An Pham, Ming-Sound Tsao. Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity [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 217.

Abstract 636: Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts

Abstract The tumor microenvironment (TME) is well recognized for its role in tumor progression, chemoresistance, and recurrence. The TME consists of various components, such as extracellular matrix (ECM), chemical and mechanical cues, as well as stromal cells and their interactions. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have drawn a lot of attention. It has been reported that CAFs not only directly interact with cancer cells via surface proteins and cytokines, but also influence their behaviors by remodeling the ECM. However, due to their heterogenous characteristics and diverse functions, the influence of tumor-CAF interactions on drug response is not fully understood. Here, we present a coculture model for investigating CAF effects on patient-derived colorectal tumor organoids (CTOs) growth, differentiation, and drug response. It consists of normal colon fibroblasts, CCD18-Co, or patient-derived CAFs seeded with CTOs in a mixed hydrogel containing 90% basement membrane extract (BME) and 10% collagen type I. Media conditions were optimized (1:1 mix of CTO culture media plus reduced media excluding growth factors and inhibitors) to achieve similar organoid sizes in the monoculture and coculture conditions at the onset of drug testing. To support higher throughput investigations, our workflow incorporates a liquid handler for automated CTO seeding, media and drug exchange, and an automated microscope with a robotic arm for multi-plate imaging. Briefly, single cell CTOs and fibroblasts were seeded at a 1:1 ratio (3000 total cells/well) in 96-well plates. After 4 days in culture, anti-cancer drugs were administrated in reduced media to the samples. Brightfield images were acquired on days 0, 3, and 5 after drug administration. The collected images were analyzed with a neural network (NN) to dynamically monitor the growth and viability of individual organoids. The CellTiter-Glo assay was performed on day 5 to quantify the total CTO cell viability. The normalized drug response (NDR) was calculated from the NN processed images to determine the drug response. An immunostaining panel, which includes protein markers such as CK20, Ki67, α-SMA, Vimentin, and FAP, was used to evaluate the fibroblast phenotypes and CTO differentiation. Additionally, the cultures were subjected to fluorescent lifetime imaging microscopy (FLIM), a label-free imaging technique, to measure the metabolic adaptations of each cell population. Preliminary studies revealed a CAF-dependent increase in CTO growth and glycolytic signatures in the co-culture setting; however, minimal effects on drug response to common chemotherapy agents such as 5-fluorouracil and irinotecan were observed. In the future, we will use this integrated pipeline as a screening method to identify new treatment strategies aimed at targeting the stromal microenvironment in colorectal cancer. Citation Format: Yuyuan Zhou, Yu-Kai Huang, Emma J. Fong, Elizabeth Elton, Erin McManus, Brandon Choi, Seungil Kim, Shannon M. Mumenthaler. Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts [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 636.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate >70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [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 2500.

Abstract 6658: TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy

Abstract Background: Oncolytic viruses, such as the chimeric vesicular stomatitis virus variant VSV-GP have dual modes of action, direct tumor-cell killing, and activation of tumor-specific immunity. While tumor cells deficient in type I interferon (IFN) response are permissive to VSV-GP replication and killing, IFN competent tumor cells are protected from VSV-GP-mediated oncolysis. Our previous data demonstrated that VSV-GP therapy turns immunologically ‘cold’ tumors ‘hot’ by increasing infiltration of T cells into the tumor. The aim of this study was to use the VSV-GP platform to locally express a T cell engager (TcE) molecule to redirect these T cells to destroy non-permissive IFN competent tumor cells expressing the target of the TcE on their cell surface. Methods: We selected EpCAM as the target antigen and used a bispecific EpCAM-TcE molecule consisting of two variable single chain fragments against EpCAM and CD3, respectively. VSV-GP encoding EpCAM-TcE (VSV-GP-EpCAM-TcE) or an invariant control TcE (VSV-GP-TNP-TcE) were generated as previously described. T cells were co-cultured with human target cells expressing EpCAM on their cell surface, either infected with the VSV-GP-EpCAM-TcE virus or with supernatant from VSV-GP-EpCAM-TcE infected cells. T cell activation, proliferation, degranulation and cytokine secretion were analyzed by flow cytometry and multiplex cytokine assays as a measure of TcE mediated cross-linking of T cells to their target on cancer cells. Patient-derived head and neck squamous cell carcinoma (HNSCC) tumors were sectioned into approximately 200 µM slices and infected with the virus. T cell activation was measured by cytokine secretion. The therapeutic potential of VSV-GP-EpCAM-TcE was tested in vivo in cell-line derived xenograft (CDX) models implanted in immunodeficient mice humanized with donor-derived PBMCs or CD34+ cells. Results: Virus fitness was maintained for VSV-GP encoding the TcE molecule. Using co-culture assays, we demonstrated that EpCAM-TcE molecule is successfully expressed by VSV-GP and can activate both CD4+ and CD8+ T cells resulting in proliferation, degranulation, and cytokine secretion. Furthermore, we validated the concept using patient-derived HNSCC tumor slices which are representative of complex human tumor ecosystems. We could confirm that cancer cells infected by VSV-GP-EpCAM-TcE virus express functional TcE molecule and activates T cells to secrete IFNγ and TNFα. Finally, we tested the concept in two different CDX models where VSV-GP-EpCAM-TcE showed improved tumor growth inhibition due to a combined effect of oncolysis and T cell activation by EpCAM-CD3 TcE. Summary: The oncolytic virus VSV-GP can be successfully armed with a TcE to redirect tumor-infiltrating T cells to destroy tumor cells which are not permissive to viral replication. This resulted in an enhanced therapeutic efficacy compared to just the oncolytic effect. Citation Format: Aparna Ponnurangam, Theresa Schwaiger, Sophie Walter, Simone Bartl, Melissa Mayr, Monika Petersson, Tanja Schoenberger, Fabian Heinemann, Peter Gross, Wolfgang Rist, Srinath Kasturirangan, Charlotte Lempp, Jutta Petschenka, Krishna Das, Nolden Tobias. TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy [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 6658.

Abstract 7209: Overcoming platinum resistance of high-grade serous ovarian cancer targeting the activated JAK/STAT pathways via extracellular vesicles

Abstract High-grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer. While HGSOC usually responds to initial treatment, most cases eventually relapse and develop platinum-resistant ovarian cancer (PROC). The molecular definition of PROC is still unclear, and overcoming PROC has been the biggest challenges. Extracellular vesicles (EVs) have an essential role in cell-to-cell communication within both the primary and distant microenvironments. In this study. we aimed to identify molecular mechanisms of PROC via EVs to suggest novel therapeutic strategies. Platinum responses were categorized by primary treatment-free interval of platinum less than 6 months as PROC, and more than 36 months as platinum good response. Ascites and tumor tissues have been collected prior to initial chemotherapy in patients with HGSOC for analysis according to platinum response. mRNA sequencing in tumor tissues identified significantly upregulated JAK/STAT pathways in PROC. The JAK/STAT family was highly expressed in PROC than the good response group, and also increased than adjusted normal ovaries and fallopian tubes. In addition, PROC cell lines and PROC patient-derived xenograft model tumor tissues showed high JAK expression. Small RNA sequencing for HGSOC tissues and ascites-EVs revealed that miR-135a-5p and miR-221-5p were highly expressed in both PROC tissues and ascites, and the expression of the two miRNAs had a positive correlation between tissues and ascites. Overexpression of the two miRNAs in HGSOC cell lines resulted in higher JAK family expression and resistance to cisplatin. JAK inhibitors (JAKi) had equivalent efficacy for platinum-sensitive/resistant HGSOC cell lines and JAKi highly suppress tumor growth in the PROC mouse model. In addition, suppression of JAK family expression in PROC cell lines significantly increased sensitivity to cisplatin. JAKi also had a synergistic effect with cisplatin for PROC cell lines. IPA analyses in mRNA sequencing in tumor tissues, TGF-β was highly expressed in upstream regulators in PROC, and TGF-β stimulation resulted in the upregulation of the two miRNAs in mesothelial cells, but not in HGSOC cell lines and fibroblasts. In summary, we here demonstrate novel molecular mechanisms of PROC via EVs and provided the rationale for JAK-targeted therapy for PROC. EV-miRNAs can regulate the PROC activated JAK/STAT pathway and can be diagnostic and therapeutic biomarkers for PROC definition or JAKi indications, suggesting that patient stratification in precision medicine for initial treatment of HGSOC. Citation Format: Kazuhiro Suzuki, Akira Yokoi, Kousuke Yoshida, Yusuke Yamamoto, Hiroaki Kajiyama. Overcoming platinum resistance of high-grade serous ovarian cancer targeting the activated JAK/STAT pathways via extracellular vesicles [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 7209.

Abstract 6733: Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples

Abstract Background: Collective evidence highlights that agonism of the host STING pathway plays a critical role in eliciting robust and durable anti-tumor immunity. Dazostinag (TAK-676) is a novel systemically delivered STING agonist that ignites the innate immune system and mobilizes adaptive immunity. Preclinical studies with dazostinag demonstrated a strong, persistent CD8+ T cell anti-tumor response driven by innate immune derived type I IFN and pro-inflammatory cytokines. The role of CD8+ T cells in response to dazostinag was further supported by a previously demonstrated correlation between response and CD8+ T cell frequency in a patient-derived organotypic tumor spheroids (PDOTS) model of mesothelioma. Within the current work, we sought to determine the extent to which response to dazostinag in mesothelioma depends on CD8+ T cells versus the contribution of other immune cells. Methods: Surgical mesothelioma cases were collected along with clinicopathological variables at Brigham and Women’s Hospital under an IRB-approved protocol. PDOTS were generated as described previously. Ex vivo response was determined by fluorescent life/dead imaging, and orthogonally evaluated by multiplexed cytokine array, immunofluorescence, and flow cytometry; subset of tumors was studied using single cell RNA sequencing (scRNAseq). Results: Dazostinag-driven tumor cell killing was evaluated in 20 mesothelioma PDOTS. 11 samples were characterized as responders (R) with a cut-off of >30% reduction in live cell area; 9 as non-responders (NR). In 12 out of 20 PDOTS, CD8 neutralization effect was assessed: four were TAK-676 NR, and out of 8 R 4 had at least 20% killing abrogated by aCD8. Effect did not depend on the baseline amount of CD8+ T cells in the sample. scRNAseq analysis showed that R and NR T cells have different activation/exhaustion phenotypes and varying levels of interferon stimulated genes (ISG) in response to STING agonism. Other cell populations emerged as candidates for dazostinag resistance: NR cases carried remodeled immunosuppressive myeloid cells or matrix metalloproteinases-expressing fibroblasts. In general, myeloid cell abundance at baseline characterized by flow had a trend in negatively correlating with dazostinag response. Intrinsic expression of type I interferons shown by multiplexed cytokine array was linked to dazostinag sensitivity (R vs. NR IFN-β p=0.028; IFNα2a p=0.016) and can potentially be used as a predictor for response. Conclusion: We evaluated immune modulations in mesothelioma after treatment with a systemic STING agonist dazostinag using human tumor ex vivo culture. Innate interferon type I expression was higher in responding samples. TAK-676 efficacy was partially reduced in absence of effector lymphocytes, however other cells, such as immunosuppressive myeloid cells and fibroblasts, can have a negative impact on dazostinag response in mesothelioma. Citation Format: Elena Ivanova, Sung R. Park, Ari P. Zlota, Nathaniel Spicer, Minh Ha, Sophie Kivlehan, Iliana Gjeci, Simona Innocenti, Lauren Zasadil, Patrick Lizotte, Michael Y. Tolstorukov, Kai Ding, Adnan O. Abu-Yousif, Jeffrey Raizer, Vicky A. Appleman, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples [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 6733.

Abstract 5235: Combination STING agonist and NK cellular therapy in patient derived organotypic tumor spheroids

Abstract Background: Increasing the immunogenicity of solid tumors may potentiate durable anti-tumor immunity. Targeting innate pattern recognition receptor signaling is a promising strategy to repolarizing the suppressive tumor immune microenvironment (TME). A potential synergistic approach is the addition of cellular therapy. Tumor models expressing high levels of endogenous STING, but where the pathway is not active, were considered for evaluating STING agonism as a potential intervention. Additionally, prior work has demonstrated that STING induction is toxic to T cells; however, natural killer (NK) cells are unaffected, due to their differential regulation of autophagy. Here we investigate the immunomodulatory properties of STING agonism alone and in combination with NK cells using short-term microfluidic culture of fresh patient-derived organotypic tumor spheroids (PDOTS). Methods: Surgical cases from Brigham and Women’s Hospital under an IRB-approved protocol were studied. PDOTS were generated as previously described. Ex vivo response was assessed by fluorescent live/dead imaging, immunofluorescence (IF), multiplexed cytokine array, and single cell RNA sequencing (scRNAseq). Baseline immune phenotypes were analyzed by FACS from single cells isolated during tumor sample preparation. Results: Twelve explants were studied. Two specimens were excluded from viability assessment due to excess of fibrotic material and low viability. Using a cut-off of -40%, three cases were characterized as responders (R) and seven as non-responders (NR). We observed significant induction of CXCL10 in all samples treated with STING agonist. IFN-β, IFN-γ, TNF-α, and MIP1-α were also consistently induced by STING agonist treatment. More detailed analysis of two samples by scRNAseq revealed a strong interferon stimulated genes (ISG) signature in STING agonist conditions; the cellular source of CXCL10 secretion in STING agonist treatment was tumor cells and fibroblasts. We also observed profound changes in myeloid biology, indicating that these cells polarized to a more pro-inflammatory and antigen-presenting phenotype with STING agonism. Interestingly, NK-based cell therapy added to PDOTS cultures displayed better persistence and enhanced effector function relative to endogenous NK cells, with and without STING agonism. Conclusions: Using tumor explants and a variety of orthogonal techniques, we investigated the response to STING agonism and NK cellular therapy ex vivo. Our results indicate that STING agonism remodels the TME, creating a more immune-permissive environment. STING agonism induces secretion of chemoattractant CXCL10 by tumor cells and fibroblasts. STING agonism does not abrogate NK cell effector function and therefore, may synergize with NK cell therapies. Citation Format: Patrick H. Lizotte, Elena Ivanova, Sung Park, Nathaniel Spicer, Sophie Kivlehan, Iliana Gjeci, Stefan Kiesgen, Vicky Appleman, LeeAnn Talarico, Michael Y. Tolstorukov, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Combination STING agonist and NK cellular therapy in patient derived organotypic tumor spheroids [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 5235.

Abstract 5334: Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research

Abstract Background: The preclinical evaluation of novel immune therapies requires humanized immune system (HIS) mouse models. In previous studies we have demonstrated that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T and NK cells or CD34+ hematopoietic stem cells (HSC) can be used to establish a HIS model. With the development of next-generation NOG mice, lineage-specific differentiation of immune cell sub-populations of interest can be supported. Transplantation of cell line-derived (CDX) or patient-derived (PDX) tumor xenografts on HIS mice provides a full model for human tumors for the investigation of checkpoint inhibitors (CPI), as well as novel cell therapies and immune cell engagers. Methods: HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient mice. For humanization, NOG mice and next-generation NOG strains were used: NOG, NOG-EXL, hIL-2 NOG, hIL-6 NOG and FcResolv™ NOG mice were compared to each other for lineage-specific differentiation using single donors or a mixed HSC donor pool. Engraftment of immune cells was monitored by FACS analysis of blood every four weeks. In humanized NOG mice, CDX and PDX from different entities were s.c. transplanted and used to evaluate CPI. Results: Humanized hIL-2 NOG mice showed significantly decreased survival after HSC transplantation in comparison to the other mouse strains and had to be sacrificed within the first 6-8 weeks after HSC transplantation. In the other mouse strains, transplanted HSCs engrafted and differentiated mainly into B and T cells. NOG-EXL mice displayed the highest engraftment, with up to 80% of human cells in the blood, including a higher portion of myeloid cells. In humanized hIL-6 NOG mice a higher portion of monocytes could be determined. FcResolv™ NOG mice lack murine Fc gamma receptors in order to reduce confounding interactions between residual murine immune cells and antibody-based therapies, and they engraft and differentiate human HSCs similarly to the parent NOG strain. In humanized NOG mice, selected CDX and PDX tumors successfully engrafted without significant differences in tumor growth compared to non-humanized mice. CPI treatments induced tumor growth delay in selected models. Conclusions: Next-generation NOG mouse strains are characterized by a lineage-specific differentiation of immune cells depending on integrated human cytokines. Furthermore, we established a human tumor-immune-cell model for NOG mice using different entities of CDX or PDX in combination with CPI. Our human tumor-immune cell models allow preclinical translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Louise Baskin, Jens Hoffmann. Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research [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 5334.

Abstract 493: Unlocking solid tumors to nano-immunotherapy

Abstract Despite the remarkable efficiency of cancer immunotherapies, only a low percentage of patients achieve long-lasting clinical responses. Non-tumor cells within the tumor microenvironment (TME), including tumor vasculature and immune stromal cells, dictate therapeutic efficacy. The presence of germinal centers (GC) within the TME of cancer patients, including infiltrating T follicular helper (Tfh) cells and B cells, has been considered predictive of response to immunotherapies. We are developing nano-immunotherapies to induce immunological memory to control tumor relapse without any follow-up treatment.Nanomaterials co-incorporated tumor-associated antigens, clinically relevant toll-like receptor ligands, and regulators of tumor progression, namely the PD-L1/PD-1 expression and the secretion of TGF-β. Nanoparticle (NP) physicochemical properties were fully addressed. The immunotherapeutic potential of this nano-immunotherapy was addressed in melanoma, colorectal cancer (CRC), pancreatic cancer, and triple-negative breast cancer (TNBC) 3D models. These were created using patient-derived tumor cells, cancer-associated fibroblasts, and endothelial cells, to which paired patients' peripheral blood mononuclear cells were added to assess T-cell infiltration and tumor sprouting to identify the best nano-immunotherapy dosage/schedule. Our in vivo studies evaluated tumor volume, and animals’ survival, and characterized the tumor-infiltrating immune cells within TME, including the differentiation of Tfh cells and primary GC by flow cytometry.Our nano-immunotherapy remodeled the TME of B16F10 melanoma, MC38 CRC, 4T1 and E0771 TNBC, and KPC PDAC. Different TME subpopulations were identified as major blockers of anti-tumor immunity, and this knowledge guided the selection of combination approaches using modulators of those tumor immune evasion-related pathways (e.g., PD-L1, FAK). Our combination nano-immunotherapies tailored to address the tumor immune suppression profile found in vaccinated animals, significantly delayed tumor development, and increased disease-free survival rates. We found that the adjuvants delivered by our NP led to a ~4-fold increase in antibody production, being far more effective in generating GC responses. Thus, the effectiveness of combinational immunotherapy is at least partially related to the modulation of Tfh cells driving the formation of GC responses.Our nano-immunotherapy is a promising clinically translatable approach to defeating solid tumors. Acknowledgments: This work was supported by PTDC/BTM-SAL/4350/2021 and LCF/PR/HR22/52420016. Citation Format: Barbara Carreira, Rita C. Acúrcio, Ana I. Matos, Liane M. Moura, Ana C. Santos, Raquel Gouveia, Jéssica Cordeiro, Carina Peres, Daniela Vaskovich-Koubi, Ron Kleiner, Sabina Pozzi, Ronit Satchi-Fainaro, Helena F. Florindo. Unlocking solid tumors to nano-immunotherapy [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 493.

Abstract 4727: Large scale drug response comparison across different neuroblastoma cell model systems

Abstract Traditionally neuroblastoma research has predominantly used 2D cell lines, which, despite their simplicity, often fall short in mirroring the physiological complexity of actual tumors. Recently, 3D models like patient-derived tumor organoids (PDO) and patient-derived xenografts (PDX) have emerged as superior alternatives, offering more realistic predictions of patient treatment responses. This study aims to discern the differences in drug response between conventional 2D cell lines and the more advanced 3D neuroblastoma models - specifically, patient-derived organoids (nPDO) and patient-derived xenograft organoids (nPDXO). Employing high-throughput drug screening of 528 cancer drugs, we evaluated the drug responses across traditional 2D cell lines, nPDOs, and nPDXOs. Additionally, focused experiments with 29 selected drugs were conducted to determine the influence of culture topology on drug efficacy. The findings were compared to a study by Hansson et al.1 that used nPDXO. Our study discovered a pronounced difference in drug sensitivity between 2D cell lines and 3D models, with 3D models showing heightened sensitivity. Interestingly, 2D cell cultures had a more heterogenous drug response compared to 3D models. The drug response correlation between nPDOs and nPDXOs was relatively high (Spearman ~0.8), indicating consistent responses across these models. Our comparison underscores the impact of model system choice on drug response studies in neuroblastoma. The study highlights important differences in drug response between neuroblastoma 2D and 3D cell culture models. However, only subtle differences were found between PDO and PDX-derived organoid lines, despite differences in treatment centers and media compositions. This suggests that while the choice of cell culture topology greatly impacts drug response, PDO and PDX derived lines can largely be used interchangeably for drug screening experiments.

Abstract 6737: Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient tumors

Abstract Introduction: Immunogenic neoantigens derived from somatic tumor mutations are essential to initiate and sustain robust anti-tumor immune responses. Frameshift insertion/deletions (fs-indels) are a key source of immunogenic neoantigens. However, fs-indels often result in the introduction of premature termination codons (PTCs), leading to mRNA degradation by the nonsense-mediated mRNA decay (NMD) pathway. In human malignancies, NMD activity is increased in response to the heightened burden of somatic fs-indel mutations and effectively prevents the generation of highly immunogenic tumor-specific neoantigens, thereby promoting immune evasion. Approach: Here, we pharmacologically targeted SMG1, a core component of the NMD pathway, for the first time in patient tumor and normal samples as well as in a panel of human cancer cell lines and patient-derived organoids, to prevent the degradation of highly immunogenic frame-shifted transcripts. We analyzed the changes in transcriptome, proteome and immunopeptidome following SMG1 inhibition (SMG1i). We then made use of tumor-T cell co-cultures and Patient-Derived Tumor Fragments (PDTFs) to assess the effects of SMG1i on anti-tumor immunogenicity. Ex vivo immunological responses were measured by high-dimensional flow cytometry, cytometric bead array and single cell RNA and TCR sequencing. Results: Our data demonstrates a significant increase in T cell activation, expansion of tumor-reactive intratumoural CD8+ lymphocytes and cytokine secretion following SMG1 inhibition in ex vivo PTDFs (n=15) but not in matched normal-adjacent tissue (n=10). Mechanistically, SMG1 inhibition increased the abundance of frame-shifted mRNA transcripts, and aberrant HLA-presented peptides. Co-culture of tumor cells and patient-derived organoids with CD8+ T cells upon SMG1 inhibition induced potent MHC class I antigen-dependent T cell activation and tumor cell killing. Conclusion: Our findings, in a clinically relevant platform, highlight SMG1 inhibition as a novel cancer immunotherapy approach to promote neoantigen production and to induce anti-tumor immunogenicity irrespective of the tumor type or TMB status. Citation Format: Roberto Vendramin, Hongchang Fu, Yue Zhao, Shanila Fernandez-Patel, Danwen Qian, Lorena Ligammari, Gordon Beattie, Ronen Levy, Polina Greenberg, Osnat Bartok, Krupa Thakkar, Jun Murai, Despoina Karagianni, Chris Sng, Mansi Shah, Felipe Galvez-Cancino, Krijn Djikstra, Sergio Quezada, Yardena Samuels, TRACERx consortium, James Reading, Charles Swanton, Kevin Litchfield. Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient 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 6737.

Abstract 4461: Unveiling the links between methionine metabolism and epigenomic reprogramming in upper aerodigestive squamous cell carcinoma

Abstract Upper aerodigestive squamous cell carcinoma (UASCC) presents as a prevalent and aggressive malignancy, posing challenges in terms of effective therapeutic interventions. In this study, we explored the intricacies of amino acid metabolism within UASCC, revealing an unexpected observation that distinguishes UASCC among all human cancers, having the highest methionine levels, driven by the overexpression of its transporter LAT1. Notably, LAT1 exhibits peak expression levels in UASCC, regulated at the transcriptional level by UASCC-specific promoters and enhancers, co-regulated by SCC master regulators TP63/KLF5/SREBF1.Surprisingly, our unbiased bioinformatic screen identifies EZH2 as a pivotal downstream target of the LAT1-methionine pathway, establishing a direct link between methionine metabolism and epigenomic reprogramming. This cascade emerges as crucial for the survival and proliferation of UASCC patient-derived tumor organoids. Furthermore, we find that LAT1 expression closely correlates with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis.Significantly, the newly discovered LAT1-methionine-EZH2 cascade emerges as a promising target for intervention, with effective outcomes achievable through both pharmacological approaches and dietary interventions in vivo. In summary, this research unveils a novel mechanistic crosstalk connecting epigenomic reprogramming with methionine metabolism, revealing its biological significance in UASCC. Moreover, it identifies a unique tumor-specific vulnerability, providing avenues for exploitation through both pharmacological and dietary strategies Citation Format: Chehyun Nam. Unveiling the links between methionine metabolism and epigenomic reprogramming in upper aerodigestive squamous cell carcinoma [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 4461.

Abstract 942: Response to systemic therapies in patient-derived cell lines from primary and recurrent adult granulosa cell tumors

Abstract Introduction: In patients with adult-type granulosa cell tumor (aGCT), a rare subtype of ovarian cancer, surgery is the preferred treatment for both primary and recurrent disease. However, the probability of complete resection decreases after multiple recurrences, in which case systemic treatment is an alternative option. Response rates of systemic therapy are variable, resistance is a problem and it is difficult to predict which therapy will work in which patient. Drug screen testing on patient-derived aGCT cell lines may offer a solution. Methods: In a national prospective study on aGCT, fresh tissue was collected immediately after surgery. Tumor tissue was cultured and developed into 2D cell lines. Both clinically applicable and experimental drugs were tested on the patient-derived cell lines and two control cell lines. Dose-response curves and synergy were calculated using GraphPad Prism and Compusyn software. Results: We cultured 34 patient-derived cell lines from tissue of seven patients with primary aGCT and from 13 patients with recurrence. In eight patients multiple sites were cultured. A total of 27 drugs were tested, including 10 monotherapies and 17 combinations. Carboplatin/gemcitabine showed efficacy and synergy 33 of 34 patient-derived cell lines (97%), in contrast to the regular carboplatin/paclitaxel and carboplatin/etoposide combinations (50% and 74%, respectively). The experimental combinations alpelisib/fulvestrant and alpelisib/gemcitabine showed efficacy of more than 75%. Testing of anti-hormonal therapy was not sufficiently reliable with this technique. Conclusion: Drug screening on patient-derived tumor cell lines adequately reflects the reality of the variable response to systemic therapy in aGCT. In future research, this technique could be used to personalize the systemic treatment of aGCT patients. The response to carboplatin/gemcitabine in our patient-derived cell lines is striking. Citation Format: Geertruid Brink, Nizar Hami, Sander Mertens, Ward Hofhuis, Jurgen Piek, Cor de Kroon, Christianne Lok, Eva Maria Roes, Luc van Lonkhuijzen, Hans Nijman, Hugo Snippert, Jolijn Groeneweg, Petronella Witteveen, Ronald Zweemer. Response to systemic therapies in patient-derived cell lines from primary and recurrent adult granulosa cell 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 942.

Abstract 6219: Type V collagen promotes lung adenocarcinoma metastasis by regulating tumor mechanical stress and the implication for prognosis and therapeutic strategies

Abstract Introduction: The mechanical environment of the human body influences physiological and pathological processes, and understanding these biomechanical aspects holds promise for medical research. Tumor microenvironment changes, including extracellular matrix (ECM) alterations, result the changing of mechanical characteristics of the tissue, play a crucial role in cancer progression and metastasis, including lung adenocarcinoma (LUAD), one of a major histology type of lung cancer. This study focuses on elucidating how mechanical characteristics, especially those related to the ECM, influence LUAD and the signaling pathways involved. Methods: Cell culture, transfection, Western blot, immunofluorescence, immunohistochemistry, Transwell assay, scratch wound-healing assay were used to investigate the behavior changes of LUAD cells under different treatment. Bioinformatics analyses, and machine learning-based integrative approaches were employed to build the SVM_Score, which was built with relevant base membrane (BM) genes. Patient-derived tumor organoids, myofibroblasts, and co-culture systems were established. Sorafenib, which can inhibited collagen and fibronectin synthesis, was used to explore the proliferation and migration changing of lung cancer cells co-cultured with myofibroblasts. Results: Mechanical stress, simulated by matrix application, enhanced LUAD cell migration and invasion, correlating with ECM alterations and EMT pathway activation. SVM_Score predicted LUAD patient prognosis and EMT propensity across multiple datasets, revealing its robust prognostic capabilities. Lower SVM_Scores were associated with worse survival outcomes, increased cancer-related pathways, higher Tumor Mutation Burden and altered immune microenvironment characteristics. SVM_Score was also linked to myofibroblast-secreted COL5A1, a key marker for mechanical stress. Patient tissues with low SVM_Scores exhibited higher COL5A1 expression, enhanced EMT propensity, and increased internal mechanical stress. Sorafenib, by inhibiting COL5A1 secretion, attenuated the pro-tumor effects of myofibroblasts, inhibiting proliferation and migration of LUAD cells, and rendering LUAD cells more sensitive to chemotherapy. Conclusion: This comprehensive study unveils the intricate relationship between mechanical stress, ECM alterations, and LUAD progression. SVM_Score emerges as a potent prognostic tool, reflects tumor mechanical characteristics. Sorafenib intervention targeting COL5A1 secretion provides a potential therapeutic strategy to mitigate LUAD aggressiveness. These findings contribute to a deeper understanding of the biomechanical aspects of LUAD, offering insights for future research and clinical applications. Citation Format: Guangsheng Zhu, Yanan Wang, Yingjie Wang, Chen, Yongwen Li, Hongyu Liu, Jun Chen. Type V collagen promotes lung adenocarcinoma metastasis by regulating tumor mechanical stress and the implication for prognosis and therapeutic strategies [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 6219.