Patient-derived Xenograft Lines Research Articles

Abstract 6897: Defining critical features for successful preclinical use of glioblastoma patient-derived xenografts (PDX) models for therapeutic assessment and clinical trial development

Abstract Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients. This large collection of PDXs provides a broad representation of genetic heterogeneity seen in patients. GBM PDXs that perform well in both in vitro and in vivo settings can be extensively utilized in the preclinical setting for downstream analysis. Based on 20 years of experience, we have defined the attributes that best enable preclinical use of our GBM PDX models. Among them are short-term in vitro culturing performance, large scale genomic evaluation data, benchmarked responses to standards of care, and median time to moribund for untreated orthotopic tumors. PDXs with prolonged survival timelines delay study readouts and, in some situations, confound efficacy results due to age-related animal decline. Setting median survival thresholds as part of the upfront experimental design allows for more timely study readouts that are unaffected by these confounding variables. Our preferred cutoff for the Mayo GBM PDX collection is 70 days median survival (n=64). Using this 64 PDX line panel, regimens expected to double survival would produce results within 4-5 months. This timeframe is well within the range of robust health for athymic nude mice. This 64-line collection features both newly diagnosed (nGBM; n=42) and recurrent (rGBM; n=22) tumors. MGMT promoter methylation status was determined by qMS-PCR (25 methylated, 37 unmethylated, 2 indeterminate). Available WES (n=62) and RNAseq (n=42) data, along with our tissue microarray (n=42), are critical resources for biomarker-based evaluations aimed at defining patient populations that are most likely to benefit from a given therapeutic. From these data, the molecular subtypes available include 26 classical, 14 proneural, and 3 mesenchymal. Twenty-seven of these 64 PDX models have also been successfully cultured from fresh as well as cryopreserved cell stocks to further expand their experimental value. Correlative clinical data including diagnosis and treatments are also available (n=61). Current standards of care for GBM include radiation, temozolomide, and bevacizumab. Defining sensitivity to these therapies is an important first step for evaluating the integration of novel therapeutics with these standards. Since the creation of our GBM PDX collection, efficacy of these therapies has been evaluated as follows: RT 26 PDXs, TMZ 20 PDXs, and Avastin 23 PDXs. The Mayo Clinic GBM PDX collection provides an important platform for preclinical evaluation of novel therapeutics. Through careful selection of PDX lines with favorable growth characteristics and known genetic features, researchers can select the most relevant models for their study. This approach will enable more meaningful preclinical work in the hopes of providing more tangible clinical benefits for GBM patients. Citation Format: Danielle M. Burgenske, Ann C. Mladek, Katrina K. Bakken, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. Defining critical features for successful preclinical use of glioblastoma patient-derived xenografts (PDX) models for therapeutic assessment and clinical trial development [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 6897.

Abstract 691: Preclinical evaluation of valproic acid in combination with radiation in orthotopic models of glioblastoma

Abstract Background: Valproic acid (VPA) is an FDA-approved anti-seizure medication that has been demonstrated to have an anti-tumor and radiosensitization effect on multiple cancer cells including aggressive brain cancer, glioblastoma (GBM). VPA is thought to exert its anti-tumor effects in part through inhibition of histone deacetylases (HDACs), upregulation of apoptosis and autophagy, and disruption of DNA repair mechanisms. Overall survival of patients treated with VPA in combination with TMZ and radiation therapy (RT) was significantly improved compared to historical controls (mOS 29.6 months). Hence, to establish a preclinical benchmark, we investigated the effectiveness of VPA alone and in combination with the radiotherapy in patient-derived xenograft (PDX) models of GBM in vitro and in vivo. Methods: Anti-proliferative effect of VPA in combination with radiation was assessed across multiple GBM cells lines (long-term cultures and PDX lines). Athymic mice were implanted with PDX GBM cells orthotopically and were randomized into four cohorts: vehicle, VPA, IR (6 gy), VPA + IR (6 gy). Animals were treated with 300mg/kg/day of VPA alone or in combination with 6Gy of radiation. Treatments were performed either via intraperitoneal route for 3 consecutive days or via a gastric gavage 5 days on/2 days off for the duration of the study. For pharmacodynamics (PD), animals were sacrificed after 3 days of drug treatment at 2hr, 24hr and 4 days post last dose and processed for immunohistochemical (IHC) analysis for acetylated H3K9/14, KI67, pH2AX, and cleaved caspase 3. Results: VPA treatment demonstrated a dose-dependent decrease in GBM cell viability and enhancement of radiation sensitivity. Pharmacodynamic analysis of tumor-bearing mice demonstrated a significant increase in acetylated H3K9/14 levels upon treatment with combination of VPA and irradiation. Elevation of acetylated H3K9/14 levels was present 2 hours after the administration of the last dose of VPA and it persisted for 4 days. Treatment of mice with irradiation upregulated levels of the apoptosis marker, cleaved caspase 3, which was further enhanced upon VPA and irradiation combination treatment. No reduction in tumor volume or survival benefit was seen in VPA alone or combination cohorts. Conclusion: Consistent with previous reports, treatment of PDX GBM line with VPA reduced cell viability in vitro, increased histone acetylation, and cell death. However, these changes were not sufficient to provide survival benefit in the intracranial model and warrants further investigation into the genetic requirements for radiation sensitization by VPA. Citation Format: Mariya Stavnichuk, Zorana Opachich, Dylan Huttenlocher, James McNamara, Jennifer Molloy, Xu He, Sonam Patel, Nader Sanai, An-Chi Tien, Shwetal Mehta. Preclinical evaluation of valproic acid in combination with radiation in orthotopic models of glioblastoma [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 691.

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 458: Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer. Despite aggressive therapy consisting of surgery followed by radio/chemotherapy GBM recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. We have recently developed PBAE nanoparticle formulations optimized for in vivo miRNA delivery to brain tumor stem cells, or GSCs. Carefully selecting one miRNA to target several mRNAs dysregulated within a pathway can allow the targeting of multiple nodes using a single agent, mimicking combinatorial therapies. To test this hypothesis, we performed bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens combined with gene set enrichment analysis (GSEA) and identified TGFBR2 signaling as a targetable pathway in rGBM. We show that SMAD2 signaling, a downstream effector of TGFBR2, is enriched in rGBM and that patients with elevated TGFBR2 transcripts respond poorly to current standard of care. Utilizing patient-derived xenograft lines from rGBM, we show that blocking TGFBR2 signaling using siRNAs or ITD1, a selective TGFBR2 inhibitor, decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM cells to temozolomide (TMZ) in-vitro. In vivo, we show that ITD1 is modestly effective in subcutaneous and ineffective in orthotopic rGBM models. Not surprisingly, these tumors exhibited classical growth patterns associated with acquisition of resistance. To overcome this obstacle, we performed a miRNA-based network analysis in rGBM and identified miR-590-3p as a putative tumor suppressor that targets SMAD2 signaling at multiple nodes in rGBM. In-vitro, transgenic miR-590-3p robustly decreased the expression of 37 putative oncogenes simultaneously concurrent with decreasing stem cell capacity, cell viability and re-sensitizing rGBM PDX cells to TMZ. In vivo delivery of miR-590-3p to established PDX-derived rGBM orthotopic tumors using novel PBAE nanocarriers robustly decreased tumor sizes and increased tumor necrosis. These results show that the promiscuous nature of miRNAs, in combination with cutting-edge nucleic acid delivery vehicles, can be leveraged as advanced targeted therapeutics for rGBM bypasses the resistance that is developed to standard of care and available small molecules. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes [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 458.

ARF4-mediated retrograde trafficking as a driver of chemoresistance in GBM.

Cellular functions hinge on the meticulous orchestration of protein transport, both spatially and temporally. Central to this process is retrograde trafficking, responsible for targeting proteins to the nucleus. Despite its link to many diseases, the implications of retrograde trafficking in glioblastoma (GBM) are still unclear. To identify genetic drivers of TMZ resistance, we conducted comprehensive CRISPR-knockout screening, revealing ADP-ribosylation factor 4 (ARF4), a regulator of retrograde trafficking, as a major contributor. Suppressing ARF4 significantly enhanced TMZ sensitivity in GBM patient-derived xenograft (PDX) models, leading to improved survival rates (p<0.01) in both primary and recurrent lines. We also observed that TMZ exposure stimulates ARF4-mediated retrograde trafficking. Proteomics analysis of GBM cells with varying levels of ARF4 unveiled the influence of this pathway on EGFR signaling, with increased nuclear trafficking of EGFR observed in cells with ARF4 overexpression and TMZ treatment. Additionally, spatially-resolved RNA-sequencing of GBM patient tissues revealed substantial correlations between ARF4 and crucial nuclear EGFR (nEGFR) downstream targets, such as MYC, STAT1, and DNA-PK. Decreased activity of DNA-PK, a DNA repair protein downstream of nEGFR signaling that contributes to TMZ resistance, was observed in cells with suppressed ARF4 levels. Notably, treatment with DNA-PK inhibitor, KU57788, in mice with a recurrent PDX line resulted in prolonged survival (p<0.01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking. Our findings demonstrate that ARF4-mediated retrograde trafficking contributes to the development of TMZ resistance, cementing this pathway as a viable strategy to overcome chemoresistance in GBM.

Glioblastoma Cells Use an Integrin- and CD44-Mediated Motor-Clutch Mode of Migration in Brain Tissue.

Glioblastoma (GBM) is an aggressive malignant brain tumor with 2year survival rates of 6.7% (Stupp et al. in J Clin Oncol Off J Am Soc Clin Oncol 25:4127-4136, 2007; Mohammed et al. in Rep Pract Oncol Radiother 27:1026-1036, 2002). One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue (Lefranc et al. in J Clin Oncol Off J Am Soc Clin Oncol 23:2411-2422, 2005; Hoelzinger et al. in J Natl Cancer Inst 21:1583-1593, 2007). To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Several models of cell migration have been proposed, including the motor-clutch, bleb-based motility, and osmotic engine models. Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. We found that nearly all cell-vasculature interactions reflected pulling, rather than pushing, on vasculature at the cell leading edge, a finding consistent with a motor-clutch mode of migration, and inconsistent with an osmotic engine model or confined bleb-based migration. Reducing myosin motor activity, a key component in the motor-clutch model, was found to decrease migration speed at high doses for all cell types including U251 and 6 low-passage patient-derived xenograft lines (3 proneural and 3 mesenchymal subtypes). Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor-to-clutch ratio. Targeting of molecular clutches including integrins and CD44 slowed migration of U251 cells. Overall we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch. The online version contains supplementary material available at 10.1007/s12195-024-00799-x.

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

MODL-30. UTILIZING SERUM-FREE CO-CULTURE MODELS FOR IN-VITRO MODELING OF TUMOR-ASSOCIATED MACROPHAGES IN GLIOBLASTOMA

Abstract BACKGROUND The GBM tumor microenvironment consists of various cancerous and non-cancerous cells that influence GBM growth, invasion, angiogenesis, and resistance to chemoradiation. In-vitro models of GBM often lack multiple cell types. Some addressed this by using multicellular models with macrophages and astrocytes; however, these models employ serum-containing media, which causes GBM brain-tumor initiating cells to lose their stem-like properties. Moreover, these models use IL-4/13 polarized macrophages (M2), despite tumor-associated macrophages (TAMs) not adhering to the M2 phenotype. Thus, there is a need to develop physiologically accurate in-vitro models of GBM. METHODS We transitioned THP-1 monocytes from serum-containing RPMI 1640 to serum-free TheraPEAKTM X-VIVOTM-15 media. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing with GBM patient-derived xenograft (PDX) lines using transwell inserts. Macrophage polarization was characterized through cytokine arrays, invasion, angiogenesis, and phagocytosis assays, comparing them to resting (M0), pro-inflammatory (M1), and M2 macrophages. RESULTS THP-1 monocytes grown in X VIVO-15 media exhibited similar proliferation rates to those in RPMI 1640 for up to 48 hours. However, at 72 hours, there was a slight decrease in proliferation rates in X VIVO-15 media (p&amp;lt; 0.0001). Macrophages polarized by the GBM PDX line, JX39P-RT, did not exhibit the cytokine expression profile of any other group. M2 macrophages displayed higher phagocytic capacity (25%) of JX39P-RT compared to M0, M1, and TAMs (&amp;lt; 15%, p&amp;lt; 0.01). M1 macrophages showed significantly higher invasion rates than M0, M2, and JX39P-polarized TAMs. CONCLUSIONS We have demonstrated a serum-free method by which we can polarize macrophages towards a GBM-supportive phenotype that differs molecularly and functionally from M0, M1, and M2 macrophages. This high-fidelity method of modeling TAMs in GBM will aid in the development and testing of innate immunomodulatory therapeutics that may one day enter the clinic in hopes of improving GBM outcomes

Resistance to PRMT5-targeted therapy in mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma, and patients who relapse on targeted therapies have poor prognosis. Protein arginine methyltransferase 5 (PRMT5), an enzyme essential for B-cell transformation, drives multiple oncogenic pathways and is overexpressed in MCL. Despite the antitumor activity of PRMT5 inhibition (PRT-382/PRT-808), drug resistance was observed in a patient-derived xenograft (PDX) MCL model. Decreased survival of mice engrafted with these PRMT5 inhibitor–resistant cells vs treatment-naive cells was observed (P = .005). MCL cell lines showed variable sensitivity to PRMT5 inhibition. Using PRT-382, cell lines were classified as sensitive (n = 4; 50% inhibitory concentration [IC50], 20-140 nM) or primary resistant (n = 4; 340-1650 nM). Prolonged culture of sensitive MCL lines with drug escalation produced PRMT5 inhibitor–resistant cell lines (n = 4; 200-500 nM). This resistant phenotype persisted after prolonged culture in the absence of drug and was observed with PRT-808. In the resistant PDX and cell line models, symmetric dimethylarginine reduction was achieved at the original PRMT5 inhibitor IC50, suggesting activation of alternative resistance pathways. Bulk RNA sequencing of resistant cell lines and PDX relative to sensitive or short-term–treated cells, respectively, highlighted shared upregulation of multiple pathways including mechanistic target of rapamycin kinase [mTOR] signaling (P < 10-5 and z score > 0.3 or < 0.3). Single-cell RNA sequencing analysis demonstrated a strong shift in global gene expression, with upregulation of mTOR signaling in resistant PDX MCL samples. Targeted blockade of mTORC1 with temsirolimus overcame the PRMT5 inhibitor–resistant phenotype, displayed therapeutic synergy in resistant MCL cell lines, and improved survival of a resistant PDX.

Spatially Resolved Whole Transcriptome Analysis of Histologically-Characterized Tissue Microarray of Patient-Matched Primary and Recurrent Glioblastomas to Identify Underlying Mechanisms of Treatment Resistance

Glioblastoma multiforme (GBM) is a lethal CNS malignancy. Radiation therapy increases overall survival, but tumors often recur in high-radiation dose regions. Additionally, recent investigations have underscored the importance of intra-tumoral heterogeneity as a driver of GBM biology. The purpose of this investigation is to characterize transcriptome differences in primary and recurrent GBM patient clinical samples using a digital spatial profiling approach to better appreciate treatment resistance mechanisms. To address the lack of understanding of molecular mechanisms of resistance in GBM, patient-matched primary and recurrent GBM pathological specimens were identified within the brain tissue biorepository and tissue cores were selected for generation of a tissue microarray (TMA). Hematoxylin and eosin staining with histomorphological (cellular tumor, pseudopalisading necrosis, invasive edge, and perivascular inflammation) scoring were performed in a blinded fashion for every core. This array was then molecularly characterized using digital spatial profiling of the transcriptome. Quality assurance including filtering of lowly expressed genes followed by downstream analyses of the data were performed using the manufacturer's recommended methods within their Bioconductor library. Gene Set Enrichment Analysis (GSEA) was then performed on the ranked gene lists. After recommended filtering, 6171 genes and 248 regions of interest remained for downstream analysis representing 22 unique patients across four different tumor histomorphological types. Significance testing revealed 679 genes that were differentially expressed between primary and recurrent tumor samples (at FDR<1%). On GSEA analysis, the chromosomal positional locus that contains genes most strongly up-regulated is 12q14, a locus that was previously identified as genomically amplified in multiple patient-derived xenograft lines after radiation selection. Additionally, recurrent tumors display a transcriptional profile more similar to the mesenchymal subtype, whereas primary tumors have a more classical transcriptional phenotype. The epithelial-to-mesenchymal transition pathway is particularly strongly up-regulated in recurrent tumors. Recurrent selection at previously identified genomic loci and molecular pathways underscores a possible conserved set of pathways for treatment resistance. This analysis has yielded a set of gene and molecular pathways that will guide future work in our lab targeting treatment resistance using novel therapeutics and radiation techniques in GBM. Future directions include assessing the feasibility of mapping these clinical samples onto our previously generated panel of comprehensively characterized patient-derived xenograft lines.

Multi-omic profiling and real time ex vivo modelling of imatinib-resistant dermatofibrosarcoma protuberans with fibrosarcomatous transformation.

Dermatofibrosarcoma protuberans (DFSP) is a rare and indolent cutaneous sarcoma, with the risk of aggressive fibro-sarcomatous transformation. Limited effective options are available for un-resectable or metastatic DFSP beyond targeting the oncogenic PDGF pathway with imatinib therapy. We established a patient-derived xenograft (PDX) and cell line model (designated MDFSP-S1) of imatinib-resistant DFSP with fibro-sarcomatous transformation. Whole genome sequencing identified high-level amplification at chromosomes 17 and 22, whilst homozygous deep deletion was demonstrated at chromosome 9 (CDKN2A, CDKN2B, MTAP). RNA sequencing followed by Sanger sequencing confirmed the pathognomonic COL1A1-PDGFB t (17;22) rearrangement in the original tumour, PDX and cell line model. Immunohistochemistry profiles of the PDX model were consistent with the patient's tumour sample (CD34 + /MIB1 + /SOX10- ). Gene set enrichment analysis highlighted top-scoring Hallmark gene sets in several oncogenic signalling pathways, including potentially targetable MTORC1 signalling and angiogenesis pathways. Antiangiogenic agents (sunitinib, regorafenib, pazopanib, axitinib) and the third-generation irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor osimertinib exhibited modest anti-proliferative activity in the cell line, with IC50 values between 1 and 10µM at 72h. No significant activity was observed with imatinib, palbociclib, everolimus, olaparib, gefitinib and erlotinib (IC50 all > 10µM). In conclusion, we established MDFSP-S1, a new PDX and cell line model of imatinib-resistant DFSP with fibro-sarcomatous transformation.

Identification of the epigenetic mechanism for colorectal cancer stem cell regulation through the comprehensive microRNA expression profiling of human colorectal cancer tissues.

42 Background: MicroRNAs (miRNAs) are key regulators of cancer stem cell (CSC) functions, including self-renewal and differentiation. However, little is known about miRNAs that regulate such properties in human colorectal cancer. Methods: We compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (non-tumorigenic cancer cells (NTCs)) sorted in parallel from human primary colorectal cancers by multiplex semi-quantitative PCR. Results: Comparison of the expression levels of miRNAs in the CSCs and NTCs resulted in the identification of 10 miRNAs upregulated (miR-221, miR-218, miR-500 and miR-195) or downregulated (miR-185, miR-19a, miR-337, miR-10a, miR-21 and miR-24) in CSCs compared to NTCs. Among them, miR-221 was most highly expressed in CSCs; and its expression level was very low in NTCs and normal colon epithelial cells (n=6, p&lt;0.05). Kaplan-Myer estimates and univariate and multivariate analyses showed that higher miR-221 expression was an independent prognosis factor for poorer outcome. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. miR-221 directly targeted Quaking (QKI) 5, the most abundant splicing isoform of the human QKI gene, and suppressed its protein level. Knockdown of miR-221 suppressed the clonogenicity of colorectal CSCs in vitro in a QKI-expression dependent manner and ability to form tumors in vivo. Conclusions: Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer and proposes that miR-221 will be a promising marker and a specific therapeutic target for human colorectal CSCs.

Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas.

Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Bi-specific killer cell engagers (BiKEs) are novel immunotherapies designed to engage natural killer (NK) cells against cancer. We designed a BiKE molecule consisting of a single-domain CD16 antibody, an interleukin-15 linker, and a single-chain variable antibody against the glioma-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2). Recombinant BiKE protein was expressed in HEK cells and purified. Flow cytometric analysis of co-cultures of peripheral blood-derived NK cells with GBM6 and GBM39 patient-derived xenograft lines revealed significantly increased activation of NK cells (CD25+CD69+) and increased glioma cell killing following BiKE treatment compared to controls (n = 4, p < 0.01). Glioma cell killing was also confirmed via immunofluorescence staining for cleaved caspase-3 (p < 0.05). In vivo, intracranial delivery of NK cells with BiKE extended median survival in mice bearing GBM6 (p < 0.01) and GBM12 (p < 0.01) tumors compared to controls. Finally, histological analysis of brain tissues revealed a higher frequency of peritumoral NK cells in mice treated with BiKE than with NK cells alone (p < 0.05). In conclusion, we demonstrate that a BiKE generated in a mammalian expression system is functional in augmenting NK cell targeting of IL13Rα2-positive gliomas.

UBE2C expression is elevated in hepatoblastoma and correlates with inferior patient survival.

Hepatoblastoma (HB) is the most common malignant liver tumor among children. To gain insight into the pathobiology of HB, we performed RNA sequence analysis on 5 patient-derived xenograft lines (HB-243, HB-279, HB-282, HB-284, HB-295) and 1 immortalized cell line (HUH6). Using cultured hepatocytes as a control, we found 2,868 genes that were differentially expressed in all of the HB lines on mRNA level. The most upregulated genes were ODAM, TRIM71, and IGDCC3, and the most downregulated were SAA1, SAA2, and NNMT. Protein-protein interaction analysis identified ubiquitination as a key pathway dysregulated in HB. UBE2C, encoding an E2 ubiquitin ligase often overexpressed in cancer cells, was markedly upregulated in 5 of the 6 HB cell lines. Validation studies confirmed UBE2C immunostaining in 20 of 25 HB tumor specimens versus 1 of 6 normal liver samples. The silencing of UBE2C in two HB cell models resulted in decreased cell viability. RNA sequencing analysis showed alterations in cell cycle regulation after UBE2C knockdown. UBE2C expression in HB correlated with inferior patient survival. We conclude that UBE2C may hold prognostic utility in HB and that the ubiquitin pathway is a potential therapeutic target in this tumor.

Bone Marrow Microenvironment-Induced Chemoprotection in KMT2A Rearranged Pediatric AML Is Overcome by Azacitidine–Panobinostat Combination

Advances in therapies of pediatric acute myeloid leukemia (AML) have been minimal in recent decades. Although 82% of patients will have an initial remission after intensive therapy, approximately 40% will relapse. KMT2A is the most common chromosomal translocation in AML and has a poor prognosis resulting in high relapse rates and low chemotherapy efficacy. Novel targeted approaches are needed to increase sensitivity to chemotherapy. Recent studies have shown how interactions within the bone marrow (BM) microenvironment help AML cells evade chemotherapy and contribute to relapse by promoting leukemic blast survival. This study investigates how DNA hypomethylating agent azacitidine and histone deacetylase inhibitor panobinostat synergistically overcome BM niche-induced chemoprotection modulated by stromal, endothelial, and mesenchymal stem cells and the extracellular matrix (ECM). We show that direct contact between AML cells and BM components mediates chemoprotection. We demonstrate that azacitidine and panobinostat synergistically sensitize MV4;11 cells and KMT2A rearranged pediatric patient-derived xenograft lines to cytarabine in multicell coculture. Treatment with the epigenetic drug combination reduced leukemic cell association with multicell monolayer and ECM in vitro and increased mobilization of leukemic cells from the BM in vivo. Finally, we show that pretreatment with the epigenetic drug combination improves the efficacy of chemotherapy in vivo.

Abstract PR006: PMR-116, a novel inhibitor of ribosome biogenesis with antitumor activity in preclinical models of prostate cancer

Abstract Advanced prostate cancer is characterised by mutations and amplifications of genes involved in regulating protein synthesis. PTEN-loss stimulates activity of the mTOR pathway, while amplification of MYC leads to increased ribosome biogenesis and elevated mRNA translation rate. Our previous work has demonstrated the efficacy of co-targeting ribosome biogenesis, via inhibition of RNA Pol I activity, and 4E-BP1 phosphorylation to suppress prostate cancer growth in vivo in GEMM of PCa and in patient-derived xenografts(1-3). In a collaboration with Pimera Inc., we investigated the efficacy of their new lead RNA Pol I inhibitor PMR-116 in models of prostate cancer. PMR-116 is well tolerated in vivo in mice and can be given at 300mg/kg weekly. Using the Hi-MYC mouse model of PCa we show that dosing 6-month-old mice once weekly for 4 weeks can decrease the incidence of invasive lesions by up to 85% compared to vehicle control while reverting glands to patterns of low grade intraepithelial neoplasia. PMR-116 rapidly inhibits proliferation in the Hi-MYC model with a 50% decrease in Ki67 observed 12 hours after oral administration. Conversely, PMR-116 showed minimal anti-tumour efficacy in the PTEN-null model of PCa suggesting that elevated MYC signalling may be required for optimal response. To further validate our promising GEMM results in more clinically relevant human-derived models, we used patient-derived xenografts lines we established from multidrug-resistant, metastatic PCa(4). PMR-116 treatment decreased tumour volume in all PDX tested including complete response in a line in which tumour volume decreased by ~90% compared to baseline. We believe this new RNA Pol I inhibitor shows promising results in a wide range of preclinical models of androgen receptor dependent and independent PCa and may exert higher efficacy in tumours expressing high levels of MYC. PMR-116 is currently in Phase I dose escalation trial in patient with solid tumours (ACTRN12620001146987).

Dual inhibition of PARP and ATR induces homologous recombination repair deficiency and leads to synthetic lethality in prostate cancer with CDK12 alteration.

e17042 Background: CDK12 is considered as a gene involved in homologous recombination repair (HRR); however, recent clinical trials have shown poor benefit of poly(ADP-ribose) polymerase(PARP) inhibitors in prostate cancer (PCa) patients with CDK12 alterations. Since CDK12 is the third most commonly altered gene in the HRR pathway in PCa, development of a new treatment strategy is urgently needed. Methods: To understand the biological consequence of CDK12 alteration, CDK12 was knocked out in LNCaP using CRISPR-Cas9 system to generate CDK12KO cells. Cell growth and cell cycle were evaluated as well as HRR efficiency. Downstream genes were evaluated by RT-PCR and western blots. Combination therapy by a PARP inhibitor and ATR inhibitors were tested using two lines of patient derived xenografts (PDX) established from tissues of patients with deleterious CDK12 variants. Three different ATR inhibitors were tested to confirm reproducibility. Results: In LNCaP, CDK12 knockout enhanced cell cycle progression until the M phase, however, cell cycle was stalled at the M phase with accumulation chromosomal aberration, suggesting that rapidly progressive CDK12 altered tumors clinically encountered harbor a mechanism of escaping the M phase checkpoint without DNA repair. γH2AX was accumulated in CDK12KO after treatment with CPT11, consistent with impaired HRR in CDK12 KO cells. Analysis of HRR associated genes showed that in PCa, CDK12 knockout leads to transcriptional downregulation of ATM but not the other HRR associated genes. Expression patterns of downstream genes after double strand break (DSB) induction in CDK12 and ATM knockout cells suggested that in PCa, CDK12 regulates HRR through regulation of ATM. Since synthetic lethality of PARP and ATR inhibition has been reported in ATM deficient PCa, 2 PDX lines with CDK12 alterations were administered the combination therapy. Tumor growth suppression and accumulation of γH2AX in tumor tissue was observed when treated by the combination therapy, but not with either drug alone. Either drug alone or in combination did not affect the growth of a PDX established from a tissue of a patient with wild-type CDK12. Conclusions: In PCa, CDK12 acts through transcriptional regulation of ATM, and combination treatment with PARP inhibitor and ATR inhibitor could be a potential treatment strategy in PCa with CDK12 alteration.

Abstract 4567: Transcriptomic analysis of a 3D engineered cancer model recapitulating stage-dependent heterogeneity in colorectal PDX tumors

Abstract Colorectal cancer (CRC) is the third-most leading cause of cancer-related deaths in the United States. To advance the understanding of CRC tumor progression, models which mimic the tumor microenvironment (TME) and have translatable study outcomes are urgently needed. CRC patient-derived xenografts (PDXs) are promising tools for their ability to recapitulate tumor heterogeneity and key patient tumor characteristics, such as molecular characteristics. However, as in vivo models, CRC PDXs are costly and low-throughput, which leads to a need for equivalent in vitro models. To address this need, we previously established an in vitro model using a tissue engineering toolset with CRC PDX cells. However, it is unclear whether tissue engineering has the capacity to maintain patient- and/or cancer stage-specific tumor heterogeneity. To address this gap, we employed three PDX tumor lines, originated from stage II, III-B, and IV CRC tumors, in the formation of 3D engineered CRC PDX (3D-eCRC-PDX) tissues and performed an in-depth comparison between the 3D-eCRC-PDX tissues and the original CRC-PDX tumors. To form the tissues, CRC-PDX tumors were expanded in vivo and dissociated. The isolated cells were encapsulated within poly(ethylene glycol)-fibrinogen hydrogels and remained viable and proliferative post encapsulation over the course of 29 days in culture. To gain molecular insight into the maintenance of PDX line stage heterogeneity, we performed a transcriptomic analysis using RNA seq to determine the extent to which there were similarities and differences between the CRC-PDX tumors and the 3D-eCRC-PDX tissues. We observed the greatest correspondence in overlapping differentially expressed human genes, gene ontology, and Hallmark gene set enrichment between the 3D-eCRC-PDX tissues and CRC-PDX tumors in the stage II PDX line, while the least correspondence was observed in the stage IV PDX line. The Hallmark gene set enrichment from murine mapped RNA seq transcripts was PDX line-specific which suggested that the stromal component of the 3D-eCRC-PDX tissues was maintained in a PDX line-dependent manner. Consistent with our transcriptomic analysis, we observed that tumor cell subpopulations, including human proliferative (B2M+Ki67+) and CK20+ cells, remained constant for up to 15 days in culture even though the number of cells in the 3D-eCRC-PDX tissues from all three CRC stages increased over time. Yet, tumor cell subpopulation differences in the stage IV 3D-eCRC-PDX tissues were observed starting at 22 days in culture. Overall, our results demonstrate a strong correlation between our in vitro 3D-eCRC-PDX models and the originating in vivo CRC-PDX tumors, providing evidence that these engineered tissues may be capable of mimicking patient- and/or cancer stage-specific heterogeneity. Citation Format: Yuan Tian, Iman Hassani, Benjamin Anbiah, Bulbul Ahmed, William Van Der Pol, Elliot J. Lefkowitz, Peyton C. Kuhlers, Nicole L. Habbit, Martin J. Heslin, Elizabeth A. Lipke, Michael W. Greene. Transcriptomic analysis of a 3D engineered cancer model recapitulating stage-dependent heterogeneity in colorectal PDX tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4567.

Abstract 40: NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline

Abstract The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a national repository of Patient-Derived Models (PDMs) comprised of patient-derived xenografts (PDXs), in vitro patient-derived tumor cell cultures (PDCs) and cancer associated fibroblasts (CAFs) as well as patient-derived organoids (PDOrg). These PDMs are clinically annotated with molecular information available in an easily accessible database for the extramural community. A key effort in developing these models is to develop matched models sets allowing for larger scale screening efforts using 2D or 3D models to prioritize selection of PDX models for preclinical translational research. To date, over 220 model sets with a PDX, PDOrg, and PDC from a single patient have been developed; 40 of these have matched CAF models allowing for exploration of research questions focused on tumor microenvironment. The largest model sets are in colorectal cancer (COADREAD, n=76), gynecologic cancers (n=33), pancreatic adenocarcinoma (PAAD, n=29), melanoma (MEL, n=19), and head and neck squamous cell carcinomas (HNSCC, n=17). Every model undergoes several quality control assessments that serve as go/no-go criteria including pathology assessment, STR validation, NGS concordance assessment and for PDXs, human:mouse DNA content assessment. It should be noted that not every model is successful in the development or QC phase so additional model sets with only one or two of the model types are also available for researcher requests, for example there are over 125 PDX/PDOrg matched model sets. The NCI is currently performing parallel preclinical screening of PDXs and PDCs or PDOrgs to determine the ability of the in vitro lines to predict in vivo activity. Genetic and histopathologic assessment of these matched model sets have demonstrated a high degree of stability by somatic mutation, copy number alteration (CNA) and gene expression data. Gene expression correlation analysis shows that mean of Spearman r between PDXs 0.89, between matched PDC/PDXs 0.79, and between matched PDOrg/PDXs 0.82. As expected, some variation at the gene expression level when comparing PDX to in vitro cultures by t-SNE can be observed, likely due to the differences in culture conditions. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Li Chen, Sergio Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Ting-Chia Chang, Alice Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Ahalt-Gottholm, Tara Grinnage-Pulley, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Shahanawaz Jiwani, Tiffanie Miner, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Cindy R. Timme, Dianne L. Newton, Paul Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 40.

Abstract 214: Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify patient-specific oncogenic alterations for treatment of pediatric osteosarcomas. Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults, with over 500 cases identified each year in the United States. OS is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. These structural variants (SVs) can alter gene expression stoichiometrically, but 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 affecting transcription. SVs can also form 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. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), our goal is to detect complex tumor-specific SVs that will inform and refine pediatric OS subtypes, revealing new tumor-specific chemotherapy treatment options. We have generated a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies using whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in OS, we have integrated OGM and WGS data for six cell lines, and OGM, WGS, and HiC data for two. SVs were detected using OGM, HiC, and WGS modalities, and we observed 59-61% concordance between SVs called using OGM and HiC, versus 22-45% when comparing with WGS calls. PDX lines assayed using OGM exhibit between 5,135 and 7,514 SVs each, and translocation numbers ranged from 212 to 1,422. These lines exhibit between dozens of potential ecDNA amplicons, including one amplicon which may explain that line’s specific and exceptionally high expression of YAP1 and BIRC2 transcripts. HiC interaction maps for two PDX lines reveal genomic compartments and TAD boundaries in detail, with 3,713 unique TAD boundaries identified between them using 50kb genomic bins. We observe that 60% of these boundaries are consistent between these lines, and 27% exhibit differences that suggest boundary loss or gain. These changing boundaries are associated with numerous SVs and/or copy number discrepancies, and with genomic regions containing genes with dysregulated RNA transcript levels. Incorporating OGM and HiC modalities adds substantially to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew S. Clugston, Megan Ostrowski, Coco Wu, Leanne Sayles, Stanley G. Leung, Vijay Ramani, E. Alejandro Sweet-Cordero, Marcus R. Breese. Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 214.