Cell Patient-derived Xenograft Research Articles

IMMU-26. TARGETING CHK2-YBX1/3 INTERACTION TO OVERCOME IMMUNOSUPPRESSION IN GLIOMAS

Abstract Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cells are less studied. We performed an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cells. The screen revealed a DNA damage response gene-checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are in the clinical trials, in combination with PD-1/PD-L1 blockade, led to survival benefit in preclinical glioma models. Analysis of human GBMs demonstrated that Chek2 expression is inversely associated with antigen presentation and T-cell activation. However, the mechanism underlying the immunosuppressive function of Chek2 in gliomas is unknown. Here, we identified that glioma tumor cell intrinsic Chek2 interacts with Y-Box Binding Protein 1 and 3 (Ybx1&3) in ex vivo gliomas using immunoprecipitation followed by mass spectrometry. Independently, YBX3 was also identified as one of the most significantly phosphorylated protein upon Chek2 knockout using phosphoproteomics analysis. The YBX1&3 are DNA/RNA binding proteins implicated in transcriptional regulation of immune-modulatory genes. In silico docking using Alphafold and PIPER tool in Schrodinger revealed that the YBX1 cold-shock domain interacted near the ATP/ADP binding site in CHK2. This interface was also part of the CHK2-CHK2 dimer interface. Consistent with our model, disruption of CHK2 dimerization using prexasertib led to increased colocalization of CHK2-YBX1 in human GBM patient derived xenograft cells. Further, targeting of CHK2-YBX1 interaction with YBX1 inhibitor SU056 reversed the repression of IFNγ response genes in glioma cells. Together this study unveils a kinase independent (non-catalytic) function of CHK2, providing insight into the non-canonical immunosuppressive role of CHK2 in gliomas.

TMET-40. UPREGULATION OF URIDINE SALVAGE IN MESENCHYMAL GLIOBLASTOMA STEM-LIKE CELLS

Abstract Glioblastoma is a heterogenous tumor comprised of multiple molecular subtypes of cancer cells and cancer stem-like cells. Of particular importance are the cancer stem-like cell niche population as they are believed to be more resistant to existing therapies and re-seed the tumor following initial response to standard-of-care treatment. To better understand glioblastoma stem-like cells we completed an untargeted proteomic analysisof 35 patient derived glioblastoma stem cell lines. This system driven analysis revealed an increase in several important proteins, in the mesenchymal subtype, involved in the uridine salvage pathway. A subset of these proteins, including ecto-5’-nucleotidase (NT5E/CD73), uridine phosphorylase (UPP1), phosphoglucomutase-2-like-1 (PGM2L1), nicotinamide phosphoribosyltransferase (NAMPT), and nicotinamide N-methyltransferase (NNMT), suggest an augmented pathway for utilization of UMP and uridine as a fuel source and to replenish NAD+. Uridine and UMP are present in human serum and are enriched in the tumor microenvironment providing a sustainable resource to resupply the tumor. The upregulation of this pathway was validated in established cell lines and orthogonal patient-derived xenograft cell lines not included in the proteomics study. Further work is underway to selectively inhibit or exploit this pathway with small molecule inhibitors.

TMET-35. UPREGULATION OF THE URIDINE SALVAGE PATHWAY IN MESENCHYMAL GLIOBLASTOMA STEM-LIKE CELLS

Abstract Glioblastoma is a heterogenous tumor comprised of multiple molecular subtypes of cancer cells and cancer stem-like cells. Of particular importance are the cancer stem-like cell niche population as they are believed to be more resistant to existing therapies and re-seed the tumor following initial response to standard-of-care treatment. To better understand glioblastoma stem-like cells we completed an untargeted proteomic analysis of 35 patient derived glioblastoma stem cell lines. This revealed an increase in several important proteins involved in the uridine salvage pathway, in the mesenchymal subtype. A subset of these proteins, including ecto-5’-nucleotidase (NT5E/CD73), uridine phosphorylase (UPP1), phosphoglucomutase-2-like-1 (PGM2L1), nicotinamide phosphoribosyltransferase (NAMPT), and nicotinamide N-methyltransferase (NNMT), suggest an augmented pathway for utilization of UMP and uridine as a fuel source and to replenish NAD+. Uridine and UMP are present in human serum and are enriched in the tumor microenvironment, providing a sustainable resource to resupply the tumor. The upregulation of this pathway was validated in established cell lines and orthogonal patient-derived xenograft cell lines not included in the proteomics study. Further work is underway to selectively inhibit or exploit this pathway with small molecule inhibitors.

Establishment and characterization of a patient-derived metastatic extraskeletal Ewing sarcoma cell line ES-ZSS-1.

The methods available for treating metastatic Ewing sarcoma (ES) are inadequate; thus, innovative therapeutic approaches need to be developed. However, the lack of clinically relevant ES models has hindered the discovery of drugs for this disease. In this study, we established and characterized a patient-derived xenograft (PDX) cell line model, which was constructed using tumor tissue from a patient with metastatic extraskeletal ES. The cells were found to recapitulate the morphological and histopathological features of the patient tumor and were designated as ES-ZSS-1. The cells harbor the characteristic EWSR1-FLI1 infusion and underwent successive passages in vitro. By performing gene expression profiling, we found that the mutation in STAG2 was the most frequent. An increase in Twist1 and epithelial-to-mesenchymal transition (EMT) was recorded. These genetic features might be relevant to metastasis and resistance to chemotherapy. To summarize, the novel patient-derived ES cell line we developed closely mimics the phenotype and genotype of patient tumors, making it a reliable tool for research on metastatic ES.

Spheroid culture to select theoretical therapeutic drugs in intravascular large B-cell lymphoma.

Intravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal large B-cell lymphoma that is characterized by the proliferation of lymphoma cells in the lumina of small vessels. Recent progress uncovering the genetic characteristics associated with MYD88/CD79B mutations has stimulated interest in the use of drugs targeting B-cell receptor signaling, including Bruton's tyrosine kinase. However, difficulties in culturing exvivo IVLBCL cells has hampered research on the development of novel therapies. In the present study, we demonstrated the establishment of an exvivo culture system of IVLBCL cells obtained from patient-derived xenograft (PDX) models. The spheroid culture enabled us to culture IVLBCL PDX cells for more than 10 days and to explore the efficacy of drug treatments acting on these cells. We found that carfilzomib and ibrutinib were effective for treating IVLBCL in exvivo experiments and conducted invivo analyses to assess the efficacy of these drugs. Although the efficacy of carfilzomib was difficult to confirm due to its toxicity in our models, ibrutinib showed comparable efficacy to a standard combination of chemotherapy drugs. Together, our data provide a new culture method for IVLBCL PDX cells and a rationale for translating ibrutinib to clinical use in IVLBCL patients.

Single-cell transcriptional atlas of human breast cancers and model systems.

Breast cancer's complex transcriptional landscape requires an improved understanding of cellular diversity to identify effective treatments. The study of genetic variations among breast cancer subtypes at single-cell resolution has potential to deepen our insights into cancer progression. In this study, we amalgamate single-cell RNA sequencing data from patient tumours and matched lymph metastasis, reduction mammoplasties, breast cancer patient-derived xenografts (PDXs), PDX-derived organoids (PDXOs), and cell lines resulting in a diverse dataset of 117 samples with 506719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 positive (HER2+), and triple-negative breast cancer (TNBC) subtypes, including isogenic model pairs. Herein, we delineated similarities and distinctions across models and patient samples and explore therapeutic drug efficacy based on subtype proportions. PDX models more closely resemble patient samples in terms of tumour heterogeneity and cell cycle characteristics when compared with TNBC cell lines. Acquired drug resistance was associated with an increase in basal-like cell proportions within TNBC PDX tumours as defined with SCSubtype and TNBCtype cell typing predictors. All patient samples contained a mixture of subtypes; compared to primary tumours HR+ lymph node metastases had lower proportions of HER2-Enriched cells. PDXOs exhibited differences in metabolic-related transcripts compared to PDX tumours. Correlative analyses of cytotoxic drugs on PDX cells identified therapeutic efficacy was based on subtype proportion. We present a substantial multimodel dataset, a dynamic approach to cell-wise sample annotation, and a comprehensive interrogation of models within systems of human breast cancer. This analysis and reference will facilitate informed decision-making in preclinical research and therapeutic development through its elucidation of model limitations, subtype-specific insights and novel targetable pathways. Patient-derived xenografts models more closely resemble patient samples in tumour heterogeneity and cell cycle characteristics when compared with cell lines. 3D organoid models exhibit differences in metabolic profiles compared to their in vivo counterparts. A valuable multimodel reference dataset that can be useful in elucidating model differences and novel targetable pathways.

BIRC5 upregulation enhances DNMT3A-mutant T-ALL cell survival and pathogenesis

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm. Although the prognosis of pediatric T-ALL has improved with intensified chemotherapy regimens, this benefit has largely not translated to the adult T-ALL population. Development of new treatments requires understanding the mechanisms driven by specific mutations. DNMT3A mutations are identified in ∼10% to 18% of adult patients with T-ALL and are associated with poor clinical outcomes. Here, using primary human specimens, we show that cells from patients with T-ALL with DNMT3A mutations are resistant to apoptosis and certain chemotherapies. Elevated JAK/STAT signaling drove prosurvival programs in patients with mutant DNMT3A, and JAK/STAT inhibition restored sensitivity to chemotherapy. The prosurvival gene BIRC5 was upregulated in patients with DNMT3A-mutant T-ALL, and these cells were specifically sensitive to the Baculoviral IAP Repeat Containing 5 (BIRC5) inhibitor YM155. Genetic inhibition of BIRC5 in vivo lead to rapid depletion of DNMT3A-mutant T-ALL cells in patient-derived xenografts, positioning BIRC5 as a precision medicine target for these patients.

Anti-HDGF antibody targets EGFR tyrosine kinase inhibitor-tolerant cells in NSCLC patient-derived xenografts.

Constitutively active mutant epidermal growth factor receptor (EGFR) is one of the major oncogenic drivers in NSCLC. Targeted therapy using EGFR tyrosine kinase inhibitor (TKI) is a firstline option in patients that have metastatic or recurring disease. However, despite the high response rate to TKI, most patients have a partial response, and the disease eventually progresses in 10 to 19 months. It is believed that drug-tolerant cells that survive TKI exposure during the progression-free period facilitate the emergence of acquired resistance. Thus, targeting the drug-tolerant cells could improve the treatment of NSCLC with EGFR mutations. We demonstrated here that EGFR mutant patient-derived xenograft (PDX) tumors responded partially to osimertinib despite near complete inhibition of EGFR activation. Signaling in AKT/mTOR and MAPK pathways could be reactivated shortly after initial inhibition. As a result, many tumor cells escaped drug killing and regained growth following about 35 days of continuous osimertinib dosing. However, when an antibody to hepatoma-derived growth factor (HDGF) was given concurrently with osimertinib, tumors showed complete or near-complete responses.There was significant prolongation of progression-free survival (PFS) of tumor-bearing mice as well. Immunohistochemistry and western blot analysis of tumors collected in the early stages of treatment suggest that increased suppression of the AKT/mTOR and MAPK pathways could be a mechanism that results in enhanced efficacy of osimertinib when it is combined with anti-HDGF antibody.

A brain organoid/ALL coculture model reveals the AP-1 pathway as critically associated with CNS involvement of BCP-ALL

AbstractCentral nervous system (CNS) involvement remains a clinical hurdle in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The disease mechanisms of CNS leukemia are primarily investigated using 2-dimensional cell culture and mouse models. Given the variations in cellular identity and architecture between the human and murine CNS, it becomes imperative to seek complementary models to study CNS leukemia. Here, we present a first-of-its-kind 3-dimensional coculture model combining human brain organoids and BCP-ALL cells. We noticed significantly higher engraftment of BCP-ALL cell lines and patient-derived xenograft (PDX) cells in cerebral organoids than non-ALL cells. To validate translatability between organoid coculture and in vivo murine models, we confirmed that targeting CNS leukemia–relevant pathways such as CD79a/Igα or C-X-C motif chemokine receptor 4–stromal cell-derived factor 1 reduced the invasion of BCP-ALL cells into organoids. RNA sequencing and functional validations of organoid-invading leukemia cells compared with the noninvaded fraction revealed significant upregulation of activator protein 1 (AP-1) transcription factor–complex members in organoid-invading cells. Moreover, we detected a significant enrichment of AP-1 pathway genes in PDX ALL cells recovered from the CNS compared with spleen blasts of mice that had received transplantation with TCF3::PBX1+ PDX cells, substantiating the role of AP-1 signaling in CNS disease. Accordingly, we found significantly higher levels of the AP-1 gene, jun proto-oncogene, in patients initially diagnosed as CNS-positive BCP-ALL compared with CNS-negative cases as well as CNS-relapse vs non–CNS-relapse cases in a cohort of 100 patients with BCP-ALL. Our results suggest CNS organoids as a novel model to investigate CNS involvement and identify the AP-1 pathway as a critical driver of CNS disease in BCP-ALL.

Understanding clonal heterogeneity and mechanisms of methotrexate resistance in two osteosarcoma PDX models.

e23512 Background: Osteosarcoma is a primary malignant bone tumor that is known to have significant intratumoral heterogeneity with branched cancer evolution. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % children and young adults. It has been revealed that the metastatic ability and resistance of cancers including osteosarcoma might be due to the presence of a subset of cells within the intra-tumorally heterogeneous population. We investigated the emergence of resistant clonal subpopulations following targeted methotrexate treatment in two patient derived xenograft (PDX) models in vitro and in vivo. Our aim was to not only track the subclonal population of cells that establish in the lungs but also to identify clonal drivers and the mutational signature of the genes and pathways involved in MTX resistance. Methods: We previously established 20 barcoded osteosarcoma PDX models and demonstrated that the application of methotrexate (MTX-IC95) treatment as a selective pressure in seven models produces clonal subpopulations that were more resistant to MTX. In the current work we used barcoded next generation and RNA sequencing techniques to map the clonal and transcriptomic architecture of MTX resistant single clones in M36 and OS252 PDX models. We next injected the barcoded PDX cells intratibially in twenty severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize to the lungs. Following primary tumor growth, limb amputation was carried out and mice were treated with methotrexate or vehicle for two weeks. Primary tumor, blood and lung samples were collected after death from metastasis or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -800C. DNA and RNA was extracted and analyzed by PCR, NGS or WES to map clonality, mutational and evolutionary profiles. Results: MTX treated single clones showed diversity and barcode abundance when compared to vehicle; barcode abundance reduced in the resistant clonal subpopulations when compared to the vehicles. Ten unique subclones were shared in both vehicle and MTX resistant clones in M36 while OS252 had only one shared subclone respectively. More than 50% of the injected mice developed primary tumors and matched lung micrometastases. We are currently analyzing both targeted and RNA sequencing results to further understand clonality, the mutational signature of the genes (DHFR, MTR, FPGS, GGH) and pathways involved in MTX resistance. Conclusions: We investigated mechanism of methotrexate resistance in two barcoded PDX models in vitro and in vivo using cytotoxicity, next generation and RNA sequencing techniques. Targeted amplicon sequencing of key genes involved in antifolate pathway will yield new information on clonal evolution and mechanisms of resistance.

Effect of oncolytic herpes simplex virus on fusion-positive rhabdomyosarcoma to radiotherapy.

10063 Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite multimodal interventions including surgery, chemotherapy, and radiotherapy, nearly a third of patients with localized disease will experience tumor recurrence. Patients with tumors harboring an oncogenic FOXO1 fusion are at greater risk of relapse and are most in need of novel therapies. Oncolytic viruses are being explored as treatments for pediatric tumors, and data suggest that oncolytic viruses may enhance responses to chemoradiotherapy. M002 is a genetically engineered oncolytic herpes simplex virus (oHSV) that exhibits aneurovirulence in normal cells. M002 selectively replicates in malignant cells, eliminating these cells through cellular burst during the lytic cycle. In the present study, we explored the efficacy of the M002 virus alone and in combination with low-dose ionizing radiotherapy in a human patient-derived xenograft (PDX) model of fusion-positive RMS (FP-RMS). Methods: A human FP-RMS PDX was established and maintained by injection into the quadriceps femoris muscle of athymic nude mice under Institutional Animal Care and Use Committee guidelines (IACUC-09363). Tumors were dissociated and cells were treated with increasing multiplicity of infection (MOI) of either the oHSV, R3659 or its genetically engineered variant, M002. A colorimetric assay evaluated the cytotoxicity of oHSV treatments on the RMS PDX cells after 24, 48, and 72 hours of infection. In separate experiments, cells treated with vehicle (control) or infected with M002 were exposed to irradiation (2 Gy), and viability was determined at 24, 48, 72, and 96 hours. Results: Treatment with M002 or its parent construct, R3659, significantly reduced RMS PDX cell viability in a dose-dependent manner at all time points. M002 showed slightly higher cytotoxicity than R3659 as measured by lethal dose 50% (LD50, 37±1.25 vs 44±0.95 PFU/cell, M002 vs R3659, at 24 hours, p=0.02). When sub-lethal doses of M002 were combined with low-dose irradiation, RMS PDX cell viability was significantly decreased compared to cells treated with either M002 or irradiation alone (26% decrease at 10 PFU/cell). Conclusions: Our preliminary data highlight that M002 exhibits cytotoxicity in a PDX model of FP-RMS. Treatment with M002 further increased the cytotoxicity of ionizing radiation compared to individual treatments, suggesting that oHSV sensitized the cells to radiotherapy. Future studies in vivo may provide more pre-clinical evidence for the radio-sensitizing and therapeutic potential of M002 in FP-RMS.

Synergistic effects of the combination of trametinib and alpelisib in anaplastic thyroid cancer with BRAF and PI3KCA co-mutations

BackgroundAnaplastic thyroid cancer (ATC), a rare and aggressive malignancy with a poor prognosis, has shown promise with the approved dabrafenib/trametinib combination for BRAFV600E mutation. Co-occurring PI3KCA mutations, identified as negative prognostic factors in lung cancer with BRAFV600E mutation, emphasize the need to target both pathways. Exploring trametinib and alpelisib combination becomes crucial for ATC. MethodsA patient-derived xenograft (PDX) and primary cell line were obtained from an ATC patient with BRAF and PI3KCA co-mutation. Individual testing of targeted therapies against BRAF, MEK, and PI3KCA was followed by a combination treatment. Synergistic effects were evaluated using the combination index. Immunoblotting assessed the efficacy, with validation performed using a PDX model. ResultsIn this study, the ATC0802 cell line and PDX were established from a refractory ATC patient. NGS revealed BRAF and PI3KCA co-mutations pre- and post-dabrafenib/trametinib treatment. Trametinib/alpelisib combination showed synergy, suppressing both pERK and pAKT levels, unlike monotherapies or BRAF knockdown. The combination induced apoptosis and, in the PDX model, demonstrated superior tumor growth inhibition compared to monotherapies. ConclusionsThe combination of trametinib and alpelisib showed promise as a strategy for treating ATC with co-mutations in BRAF and PI3KCA, both in vitro and in vivo. This combination offers insights into overcoming resistance to BRAF-targeted treatments in ATC with mutations in BRAF and PI3KCA.

Abstract 937: Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe

Abstract Translation of precision oncology data into feasible precision therapies for hard-to-cure childhood, adolescent and young adult malignancies remains a significant challenge. Identifying therapeutic targets at the protein and pathway level and demonstrating treatment response in personalized models hold great promise, particularly for combination therapies, but may be considered complex and time consuming. Here, we present the case of an adolescent with metastatic, progressive spindle epithelial tumor with thymus-like differentiation (SETTLE) and evaluate how proteomics combined with rapid patient-derived models can identify treatment options not apparent at the genome or transcript level. Mass spectrometric proteome analysis of macro-dissected tumor and adjacent normal from formalin fixed paraffin embedded sections was completed within two weeks of biopsy and identified key proteins involved in one-carbon metabolism, including SHMT2 and DHFR as possible targets for single or combination therapy. Elevated SHMT2 levels were validated by immunohistochemistry and compared to levels across AYA tumors. Based on the suitability for an innovative therapy trial, we prioritized single-agent sertraline, a commercially available anti-depressant medication that inhibits SHMT2, and confirmed a positive drug response in both chicken chorioallantoic membrane (CAM) and larval zebrafish xenografts generated from the patient. Retrospective expansion in a murine xenograft enabled metabolic tracing on isolated SETTLE- patient-derived xenograft cells using 13C6-glucose confirming SHMT2 activity and response to in vitro treatment. Following failure of cytotoxic chemotherapy and second-line sorafenib treatment, a monotherapy trial of sertraline was initiated by the patient but stopped after 8 weeks after evidence of progressive disease. Possible combination therapies were evaluated further in the patient-derived models. Combining sertraline with the common antibiotic trimethoprim, resulted in enhanced growth inhibition of SETTLE cells in the larval zebrafish xenografts. Significance: Overall, we demonstrate that proteomics and personalized xenograft models may provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact clinical practice. Citation Format: Georgina D. Barnabas, Tariq A. Bhat, Verena Goebeler, Pascal Leclair, Nadine Azzam, Nicole Melong, Jason N. Berman, Jennifer A. Chan, Donna L. Senger, Seth Parker, Christopher A. Maxwell, Gregor S. Reid, Jonathan Bush, Caron Strahlendorf, Rebecca Deyell, C James Lim, Philipp F. Lange. Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe [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 937.

Abstract 5905: Propentofylline enhances GBM sensitivity to temozolomide via targeting TROY signaling

Abstract Glioblastoma (GBM) is a high grade glioma that accounts for the majority of primary malignant brain tumors. The standard of care for GBM consists of maximal surgical resection, concurrent radiation and temozolomide (TMZ), and adjuvant TMZ. However, this has only led to a modest increase in median patient survival. TROY (TNFRSF19) is an orphan member of the TNF receptor superfamily that is differentially upregulated in GBM with low expression in normal brain tissue. Previously, we have reported that TROY is overexpressed in GBM and signals to mediate cell invasion and therapeutic resistance. Propentofylline (PPF) is a well-studied synthetic methylxanthine derivative that has strong BBB penetrance. We have shown that PPF decreases TROY expression in gliomas, inhibits invasion, and sensitizes GBM to TMZ in vitro. Here we examined the efficacy of PPF in combination with TMZ using in vivo studies of intracranial models of GBM patient-derived xenograft (PDX) models in murine. We tested 3 models of GBM PDX cells with various TROY expression levels (GBM8-high TROY; GBM43-moderate TROY; GBM22-low/no TROY). Our intracranial PDX experiments showed that PPF treatment alone did not increase survival in all PDX cells. The combination of PPF and TMZ treatment did provide a survival benefit (p < 0.039) relative to TMZ alone (only in GBM8-high TROY and GBM43-moderate TROY) that was partially lost when treatment was discontinued. However, continuous PPF administration following a concomitant PPF and TMZ treatment course led to extended survival (p < 0.0015) compared to TMZ alone resulting in increased DNA damage (γH2AX) and apoptotic markers (Cleaved Caspase-3). Immunohistochemical analyses showed that TROY expression was decreased in tumors treated with PPF. In addition, PPF-treated tumors showed a differential myeloid population with well circumscribed tumors as compared to control-treated or TMZ-treated tumors. Thus, our data demonstrates that PPF may provide a pharmacological approach to targeting TROY concomitant with standard-of-care to increase patient survival. Citation Format: Ryan Eghlimi, David Nascari, Angad Beniwal, Drake Alton, Dustin Grief, Joseph Loftus, Melanie Haluska, David Coleman, Nhan Tran. Propentofylline enhances GBM sensitivity to temozolomide via targeting TROY signaling [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 5905.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [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 1392.

Abstract 6914: Genomic landscape and stability of preclinical models in NCI's patient-derived models repository

Abstract Background: Patient-derived xenograft (PDX) models serve as a powerful tool for cancer translational research. However, it is unclear to what extent PDX models reflect the genomic intratumoral heterogeneity and genomic aberrations present within the original tumor sample. The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov/) consisting of PDX, organoids (PDOrg), and tumor cell cultures (PDC) from patients with diverse cancer histologies. We have conducted an in-depth investigation into the genomic stability of PDXs at early passaging and tumor heterogeneity in a large set of preclinical models including rare cancers. Methods: Tumor specimens were used to establish 1114 models from 1034 patients. For whole exome sequencing (WES) and RNASeq analysis, 1059 models with at least 1 PDX sample and 55 models with only a PDC or PDOrg were used. Analyzed specimens represent the original patient specimens, PDX passages P0, P1, P2, and P3+ and in vitro models. 80% of the PDX specimens were within passages P0 to P2. Results: Genomic stability in PDMR models was maintained through early passages (P0 - P2) and across independent lineages in PDMR models based on the following observations: 1) variant allele frequencies (VAF) of somatic driver mutations showed no major deviations (exceeding ±10%) when using the stromal fraction corrected VAF from originator data over PDX passages; 2) the median change in the fraction of genome impacted by copy number alterations (CNA) from the originator specimens through passages P0 - P2 was statistically inconsequential, although there was a significant increase in CNA fraction, from 5.3% at P0 initially to 18% by P3; 3) gene expression profiles of specimens within a given PDX model formed clusters in principal component analysis (PCA) plots and showed high correlation (>99.5% models with a Spearman coefficient of >= 0.8). The majority of the PDC and PDOrg specimens exhibited similar findings when compared to the original tumor samples. Furthermore, the positive percent agreement (PPA) was performed to measure the similarity between the patient's tumor and all available derived PDX specimens, with a median PPA over 90%, indicating a high level of retained heterogeneity within the PDX models. Lastly, 69% of models in NCI PDMR had predictive biomarkers, ranging from OncoKB level of evidence 1-4, which could be used to evaluate targeted therapeutics in preclinical studies. Conclusion: The NCI PDMR has a large and histologically diverse cancer representation. In this analysis, the PDXs exhibited genomic stability within early passages and preserved the majority of tumor heterogeneity observed in the patient specimens. NCI PDMR thus represents a valuable resource for researchers interested in preclinical drug screening or other investigations. Citation Format: Ting-Chia Chang, Biswajit Das, Li Chen, Peter I-Fan Wu, Yvonne A. Evrard, Rini Pauly, Dianne L. Newton, Shahanawaz Jiwani, Sergio Y. Alcoser, Luke H. Stockwin, Corinne E. Camalier, Tomas Forbes, Nikitha Nair, Alyssa Chapman, Lindsay Dutko, Michael Mullendore, Tara Grinnage-Pulley, Kimberly Klarmann, Sarah B. Miller, Chris A. Karlovich, Alice P. Chen, P Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. Genomic landscape and stability of preclinical models in NCI's patient-derived models repository [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 6914.

Abstract 2860: High-resolution mapping of oncogenic structural changes in osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify cancer-specific oncogenic alterations for treatment of osteosarcomas. Osteosarcoma is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. The functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments to affect gene expression. SVs can also lead to the formation of extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers yet they remain poorly understood. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), we studied SVs in osteosarcoma with the goal of elucidating their contribution to cancer development. We used a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies as well as genomically via whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in osteosarcoma, we integrated OGM and WGS data for all 11 cell lines to describe detailed genome-wide SVs including ecDNA formation. PDX lines assayed using OGM exhibited between 106 and 704 translocations each (versus zero detected in germline controls) and we identified dozens of potential ecDNA amplicons, including one amplicon which may explain exceptionally high expression of YAP1 and BIRC3 transcripts. Further, we used HiC to describe the topological environment resulting from these SVs and observed potential enhancer hijacking events affecting dozens of known oncogenes including MYC and CDK4. HiC interaction maps among six cell lines reveal genomic compartments and TAD boundaries in detail, with 4,167 unique TAD boundaries identified among six cell lines using 50kb genomic bins. Despite the observed complexity of these genomes, we note that 71% of TAD boundaries are consistent in at least four of the six HiC assayed cell lines, and 44% are present in all six lines. Changing TAD boundaries are associated with numerous SVs and/or copy number discrepancies, and are associated with genomic regions that contain genes with dysregulated RNA transcript levels. Overall, OGM and HiC modalities add considerably to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew Scott Clugston, Stan Leung, Eunice Fuentes, Leanne Sayles, Megan Ostrowski, Coco Wu, Christina Curtis, Zhicheng Ma, Yanding Zhao, Georgi K. Marinov, Vijay Ramani, Marcus Breese, Alejandro Sweet-Cordero. High-resolution mapping of oncogenic structural changes in osteosarcoma [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 2860.

Abstract 3390: Development of hypoxia responsive targeted polymersomes drug therapy validated using patient derived xenograft of triple negative breast cancer

Abstract Introduction: Eliminating triple-negative breast cancer (TNBC) resistance to neoadjuvant chemotherapy is a critical unmet clinical need. TNBC accounts for 20% of breast cancer and is more aggressive due to increased metastasis, high recurrence rate, and significant resistance to chemotherapy (e.g., Doxorubicin, DOX). Napabucasin (NAPA, a small organic molecule) is recognized to kill cancer stem cells by targeting the transcription factor STAT3 pathway and is currently in clinical trials. Our previous in vitro results showed significant effects with hypoxia-responsive targeted polymersomes compared to control in MDA-MB-231 and Patient-Derived Xenograft (PDX) TNBC cells. Further, we observed increased NRP-1 expression in TNBC patient-derived xenograft (PDX) cells under hypoxic (0.2% oxygen) conditions compared to normoxia (21% oxygen). Hence, we hypothesize that targeted hypoxia-responsive polymersome drug therapy would reduce the tumor growth of the PDX mouse model of triple-negative breast cancer in vivo. Methods: We have established the TNBC PDX mouse model with two major phases, initial propagation (F1-F3) and final study phase (F3-F6), for in vivo study (TNBC tumor samples received from Sanford Broadway Clinic, Fargo, IRB approved). We utilized 3-6 months old female adult NSG mice (Jackson Lab) weighing 18-25 g. PDX tumor-bearing female NSG mice were administered polymersome-encapsulated drug (doxorubicin and other groups) by intravenous injection twice a week for 4 weeks and then observed the animals for an additional week. The percentage of tumor volume growth was calculated on these treatment groups and compared to vehicle. The tumors from all the groups were excised and dissected after the treatment and fixed in 10% formalin for histological evaluation. Tumors from all the groups after treatment were stained using hematoxylin and eosin to analyze the tumor core necrosis, and the average area of necrosis was compared among all the groups. Results: The TNBC PDX mouse model treated with DOX alone and a combination of encapsulated DOX with NAPA showed significant weight loss compared to the control (over 4 weeks). DOX-alone treated mice have shown reduced tumor growth and exhibited a significant difference compared to the control group. Notably, the targeted DOX-encapsulated polymersome demonstrated a much lower tumor volume and displayed a marked difference compared to non-targeted vehicles, free-DOX, and saline. In the initial analysis, we observed a higher necrosis in the tumor of the targeted DOX-encapsulated polymersome compared to the control. Summary and Conclusion: Overall, the hypoxia-responsive targeted polymersomes showed potent antitumor activity in the novel TNBC PDX animal model. With further developments, the targeted polymersomes might have translational potential as drug carriers for treating TNBC. Citation Format: Yogaraj S Ramakrishnan, Shubhashri Ambhore, Connor Edvall, Jiyan Mohammed, Sanku Mallik, Daniel M Tuvin, Venkatachalem Sathish. Development of hypoxia responsive targeted polymersomes drug therapy validated using patient derived xenograft of triple negative breast 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 3390.

Abstract 6902: Development of a stable luciferase expressing PDX-derived cell line for translational in vitro and in vivo testing

Abstract Despite much effort invested in developing novel cancer therapies, more than 90% of clinical studies fail due to toxicity or lack of efficacy in patients. This can mainly be ascribed to lack of relevant and translational preclinical models. A common model used in preclinical studies is the xenograft model using immortalized cancer cells. Cell line-based models are cost efficient and can be genetically manipulated but are often criticized for lacking translational value. A murine model with higher clinical relevance is the patient derived xenografts (PDX) model established by implanting patients’ tumor tissue onto immunodeficient mice. Knowledge of patient therapy response, mutational status and tumor heterogeneity makes it a relevant model. However, PDX models are costly, not applicable for in vitro assays or subjective to genetic manipulation. To more closely mimic in vivo tumors we have established a PDX-derived cell line (PDXc) and characterized it extensively in a head-to-head comparison to the parental PDX model. Cells were isolated from a subcutaneous (SC) glioblastoma (GBM) PDX tumor (ST146), processed into single cells and cultivated in vitro. Once the PDXc were stable over several passages the cells were re-implanted into mice. Tumor take and growth rate were evaluated against the parental PDX model and common characteristics of the model such as EFGR and Vimentin expression were determined by IHC. We furthermore generated a stable Luciferase expressing PDXc using 3rd generation lentiviral particles and implanted the cells in an intracranial (IC) model of GBM. Tumor growth was evaluated using bioluminescence imaging (BLI) and MRI. Upon multiple passages and freeze cycles the cell line (ST146c) exhibited a stable in vitro doubling time (2.6 ± 0.2 days) with a morphology like other GBM cell lines with formation of neurospheres. STR analysis confirmed a genetic identical profile to ST146. ST146c implanted into mice had similar tumor take and growth rates to the parental PDX when injected IC (>90%, Td=3 d) and SC (>75%, Td=6 d). We also showed that ST146c had more homogeneous tumor growth. Ex vivo analysis showed similar expressions of EGFR (-), Vimentin (+++) and Ki67 (++) between the models. The BLI signal obtained from luciferase expressing ST146c implanted orthotopically was correlated to tumor volume obtained by MRI. We developed a PDX cell line that preserved critical in vitro and in vivo characteristics of the PDX model such as doubling time, tumor take rate and marker expression making it feasible to use the PDXc model in future preclinical studies. This will allow in vitro screens with patient derived tumor cells. Furthermore, the expression of luciferase in the PDXc enables intracranial and metastatic implantation with tumor monitoring using BLI. This platform consisting of in vitro and in vivo PDX models could be used as a predictive translational platform for testing new cancer therapies. Citation Format: Sigrid Cold, Rikke N. Bøge, Emma Papin, Ida T. Michler, Lotte K. Kristensen, Trine B. Engel, Andreas Kjaer, Carsten H. Nielsen, Sebastian Gnosa. Development of a stable luciferase expressing PDX-derived cell line for translational in vitro and in vivo testing [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 6902.

Abstract 3358: Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM cells

Abstract Glioblastoma (GBM; WHO grade 4) is the most aggressive form of glioma with a dismal 5-year survival rate. The current therapies are largely ineffective and therapy resistance is commonly encountered. We have recently shown that TRIB1, a Ser/Thr pseudokinase contributes to therapeutic resistance in GBM cells by activating both ERK and Akt pathways. Here we show that, increased expression of TRIB1 contributed to RT/TMZ resistance by activating pro-survival Akt signaling resulting in increased viability of GBM cells. High level of Akt phosphorylation/activation is correlated with poor prognosis of GBM patients. Many pan Akt inhibitors have been developed which are currently in various phases of clinical trials. The three Akt isoforms (1, 2 and 3) are known to play a role in cancer during proliferation, transformation and metastasis. A previous report has shown that TRIB1 interacts with Akt1 in Drosophila and blocks its activation. In this study we show that Akt binds and forms a complex with TRIB1 in GBM cells and this interaction could be isoform and cell line specific. We utilized patient derived xenograft (PDX) GBM cell lines and established GBM cell lines in this study. Stable TRIB1 overexpressing cell lines were generated by antibiotic selection. Co-immunoprecipitation (Co-IP) was performed utilizing Anti-FLAG or anti-c-Myc magnetic agarose beads to evaluate protein-protein interactions. Western blotting was done to detect protein levels. The FLAG tagged TRIB1 deletion mutants were a generous gift from Dr. Takuro Nakamura (Division of Carcinogenesis, The Cancer Institute, Tokyo, Japan). Cells were transfected using Lipofectamine 2000 reagent. Co-IP studies showed that TRIB1 formed a complex with Akt in PDX GBM cell lines. We then transiently transfected respective TRIB1 deletion mutants lacking either N and C terminal residues in HEK 293 cells and performed Co-IP with Anti-FLAG magnetic agarose beads. We observed that all TRIB1 deletion mutants except TRIB1-ΔN2 (lacking first 160 amino acids) were able to pull down Akt. The western blot results showed that each Akt isoform had a variable protein expression across different cell lines. Co-IP studies in the PDX cell lines showed that TRIB1 interacted with all three Akt isoforms in T08-387 cells whereas TRIB1 consistently interacted with only Akt3 but not Akt1/2 in GBM30 cells that lack wild-type p53 expression. Our data suggest that TRIB1 forms a complex with Akt in PDX GBM cell lines. The potential Akt binding site on TRIB1 lies between 90-160 amino acids. TRIB1 can interact with different Akt isoforms in a cell type dependent manner thereby playing a role in their activation and consequent tumorigenic processes. Therefore, targeting TRIB1 mediated Akt activation could be considered a better therapeutic option over targeting Akt directly, which may decrease Akt’s oncogenic activities without risking the toxicity associated with available Akt- specific inhibitors. Citation Format: Karnika Singh, Chunhua Han, Heather Manring, Saikh Jaharul Haque, Arnab Chakravarti. Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM 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 3358.