Patient-derived Orthotopic Xenograft Model Research Articles

Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of biological consequences beyond the classic DNA damage, we treated 9 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 5 PDOX models (P < 0.05) accompanied by γH2AX positivity in >95 % tumor cells in tumor core and >85 % in the invasive foci as well as ∼30 % apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species accumulation, were detected together with the enrichment of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies.

Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

BackgroundOvarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and...

Abstract 5113: Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Abstract Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of the biological activities beyond the classic DNA damage, we treated 6 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 4/6 PDOX models (P&amp;lt;0.05) and widespread γH2AX positivity in &amp;gt;95% tumor cells in tumor core and &amp;gt;85% in the invasive foci, accompanied by ~30% apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species buildup, were detected together with the accumulation of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies. Citation Format: Zi-Lu Huang, Zhi-Gang Liu, Qi Lin, Ya-Lan Tao, Xinzhuoyun Li, Patricia Baxter, Jack MF Su, Adekunle M. Adesina, Chris Man, Murali Chintagumpala, Wan Yee Teo, Yu-Chen Du, Yun-Fei Xia, Xiao-Nan Li. Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma [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 5113.

Abstract 4971: Integrating artificial intelligence and functional precision oncology for individualized cancer therapies

Abstract Precision oncology seeks to tailor cancer treatments to individual patients. Patient-derived xenografts (PDXs) have emerged as a promising platform for selecting efficacious, personalized therapies and developing new oncology drugs. Here, we present a workflow for selecting and validating treatments for patients with colorectal cancer (CRC) that integrates orthotopic PDX (O-PDX) models, molecular profiling, machine learning, and in vivo pharmacological validation. Of the nine tumor biopsies collected, seven were successfully developed into O-PDX models (78%) with a median time to establishment of 119 days. Models were molecularly profiled for gene expression and were serially passaged to perform orthotopic pharmacology studies to validate test agents selected by oncologists as well those suggested by CertisAITM, a novel ensemble of machine learning models for the prediction of drug response in human cancers. The correlation between CertisAI's predicted therapy responses and the actual observed tumor growth inhibition (TGI) was r = 0.7, encompassing 37 distinct treatments across studies from six patients. This research collectively illustrates that the integration of artificial intelligence with functional in vivo assays offers a powerful platform for precision oncology, enabling the identification and validation of tailored treatments for individual cancer patients. Citation Format: Fernando Eguiarte-Solomon, Rowan Prendergast, Bianca Carapia, Javier Rodriguez, Kristen Buck, Derrick Gorospe, Elizabeth Valencia, Jose Lopez-Ramos, Itzel Gutierrez, Rafaella Pippa, Yuan-Hung Chien, Warren Andrews, Long Do, Jantzen Sperry, Jonathan K. Nakashima. Integrating artificial intelligence and functional precision oncology for individualized cancer therapies [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 4971.

Abstract 3070: CDK12 is a novel therapeutic target in glioblastoma

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults carries a highly unfavorable prognosis of only one to two years despite treatment with the standard of care, involving surgery, temozolomide and radiation. Patient-derived xenograft (PDX) GBM cultures were assessed for their susceptibility to loss of CDK12 function, which was either mediated genetically (shRNA) or by the novel inhibitor, SR-4835. GBM metabolism was measured by utilizing extracellular flux analysis as well as LC/MS based carbon tracing analyses. In vivo efficacy was determined by orthotopic GBM models in mice. Based on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and shRNA library screens in multiple GBM cell cultures we identified CDK12 as essential for the growth of GBM. Consistently, loss of function of CDK12 (genetically and pharmacologically) led to a reduction of growth of GBM patient-derived xenograft (PDX) lines in vitro. Notably, SR-4835 reduced GBM growth in the low nanomolar range and synergistically enhanced the potency of temozolomide. Extracellular flux analysis and metabolite screening analyses, including carbon tracing (U13C-Glucose, U13C-Glutamine) suggested that loss of function of CDK12 elicited a substantial inhibition of cellular respiration, causing energy deprivation with subsequent cell death with apoptotic features. On the molecular level, loss of function of CDK12 increased ATF4 along with pro-apoptotic Noxa, whereas anti-apoptotic Mcl-1 decreased. In turn, adenoviral mediated ectopic expression of Mcl-1 inhibited cell death mediated by loss of function of CDK12 in GBM cells. In an orthotopic murine PDX model of GBM animals receiving treatment with SR-4835 had a significantly longer overall survival without induction of toxicity as compared to mice treated with vehicle. In summary, these findings suggest that CDK12 is a novel treatment target for GBM and that CDK12 is an important driver of energy metabolism in GBM to partially mediate apoptotic resistance. Moreover, SR-4835 enhanced the potency of temozolomide in GBM models. Therefore, the novel CDK12 inhibitor, SR-4835 should be tested in a clinical trial in patients suffering from GBM. Citation Format: Jeong-Yeon Mun, Chang Shu, Qiuqiang Gao, Mike-Andrew Westhoff, Georg Karpel-Massler, Markus D. Siegelin. CDK12 is a novel therapeutic target in 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 3070.

Abstract 1660: Tumor pharmacokinetics and pharmacodynamics assessment of TEAD inhibitor VT03989 in patient-derived xenograft models of glioblastoma

Abstract Background: The Hippo-YAP/TAZ pathway regulates cell proliferation, cell growth and migration. The major effectors of the Hippo pathway are the TEAD transcription factors, which are deregulated in several cancers, including glioblastoma (GBM). In this study, we evaluated the pharmacokinetics and pharmacodynamics of VT03989, a highly potent TEAD inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Methods: Mice with intracranial PDX GBM tumors were randomized into two cohorts to receive 10 mg/kg (oral) VT03989 or placebo for 4 days. VT03989 levels in plasma, tumor, and contralateral brain tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) at 2, 6, and 12 hours after the last treatment of VT03989. Unbound fractions were determined by equilibrium dialysis. Quantitative RT-PCR was performed to assess levels of TEAD target genes, CTGF and CYR61. Immunohistochemistry of Ki67 and cleaved caspase 3 (CC3) was performed. Results: The median unbound concentrations of VT3989 were 27 nM in plasma and 26 nmol/kg in the tumor. The median brain-to-plasma and tumor-to-plasma partition coefficients of unbound VT03989 were 0.65 and 0.99, respectively - indicating favorable brain and tumor penetration ability of VT03989. A decrease in CTGF and CYR61 indicated TEAD inhibition and was accompanied by an increase in CC3 (+) cells. Conclusion: VT03989 is well tolerated, achieves pharmacologically relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Ongoing studies are evaluating in vivo survival benefits from combining VT03989 inhibition with radiation therapy Citation Format: Tigran Margaryan, Yu-Wei Chang, Jennifer Molloy, Barbara Hopkins, Mackenna Elliott, Ernesto Luna Melendez, Connor White, Nader Sanai, An-Chi Tien, Artak Tovmasyan, Shwetal Mehta. Tumor pharmacokinetics and pharmacodynamics assessment of TEAD inhibitor VT03989 in patient-derived xenograft 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 1660.

Abstract 6620: PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease

Abstract Chemotherapy, along with the PD1 inhibitor pembrolizumab, is the recommended standard of care for patients with primary triple negative breast cancer (TNBC). Nearly half of TNBC patients treated with standard neoadjuvant chemotherapy (NACT) have excellent responses. However, those patients with significant residual cancer burden (RCB) after NACT are at high risk of recurrence or metastatic relapse within two years. Due to the challenges posed by the inter- and intratumoral heterogeneity of TNBC, it is critical to develop appropriate models for predicting and overcoming therapy resistance. We established a collection of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from 84 patients before, during, and after NACT while they were enrolled in the ARTEMIS trial (NCT02276443) at MD Anderson Cancer Center. Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), after a 3-month course of additional chemotherapy and/or different experimental therapies in cases of AC resistance (post-NACT), and from the metastatic lesions of two patients. The collection includes 12 longitudinal sets. Models were established from chemo-sensitive and -resistant tumors, but engraftment success was higher from those cancers that proved to be treatment resistant (RCB II/III). The PDX collection includes models encompassing a broad heterogeneity of chemotherapy responses, histologic features, and molecular TNBC subtypes. In addition, the majority of models develop spontaneous lung metastases. Whole exome sequencing demonstrated conservation of mutations between patient tumors and corresponding PDX models, with TP53 being the most commonly mutated gene. A similar comparison of patient and PDX transcriptomes revealed conservation of signaling pathway signatures, with those related to immune and stromal interactions being the most variable, as anticipated when adapting human tumors to growth in mice. The subclonal architecture of the tumors exhibited little overall change throughout NACT, suggesting that selection for resistant subclones is not likely to be a significant contributor to resistance. Tumor cells from the longitudinal PDX collections were subjected to high throughput drug susceptibility profiling to identify targetable vulnerabilities associated with resistance to NACT. We found that post-NACT tumors exhibited enhanced sensitivity to drugs targeting protein homeostasis pathways. Preclinical studies with pevonedistat validated the neddylation pathway as an effective target in chemoresistant TNBC. Citation Format: Amanda L. Rinkenbaugh, Yuan Qi, Reid T. Powell, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Lei Guo, Xiaomei Zhang, Sabrina Jeter-Jones, Chunxiao Fu, Rebekah Gould, Jason B. White, Clifford Stephan, Gloria V. Echeverria, Peter J. Davies, Stacy Moulder, W. Fraser Symmans, Jeffrey T. Chang, Helen Piwnica-Worms. PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease [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 6620.

Abstract 6867: TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis

Abstract Glioblastoma remains difficult to treat with immunotherapy due to the immunosuppressive tumor microenvironment and the ability of glioma cells to invade diffusely into normal brain parenchyma. While it is known that glioblastoma-associated myeloid cells (GAMs) comprise a large proportion of the tumor microenvironment and can suppress adaptive and innate immune responses through various mechanisms, detailed mapping of myeloid lineages in glioblastoma is still missing. Further characterization of pro-tumoral myeloid-lineages in glioblastoma is important for identifying myeloid-based drug targets that may synergize with T cell-based therapies and produce a more meaningful anti-tumor immune response. Towards this end, we examined numerous scRNAseq datasets derived from glioblastoma patients and identified expression of TNFRSF12A, the gene encoding the TNF- family receptor, Fn14, in a subset of GAMs enriched for expression of cytokines such as CCL3, CCL5, IL-6, and IL-8. This result is intriguing since TWEAK (TNFSF12), the ligand for Fn14, is abundantly expressed within numerous patient-derived glioblastoma cell lines, namely GBM6, GBM8, GBM22, and GBM39. To validate Fn14 protein expression in GAMs, we isolated murine bone-marrow derived macrophages and treated them with glioma-conditioned media, which induced Fn14 gene expression. We then conducted in vitro experiments utilizing the human microglia line HMC3, the murine microglia line SIM-A9, and primary murine bone-marrow-derived macrophages to assess the effects of Fn14 activation in myeloid cells. We found that treatment of Fn14+ myeloid cells with TWEAK ligand upregulates CCL3, CCL5, IL-6, and IL-8 expression. We tested the conditioned media generated from TWEAK-treated Fn14+ myeloid cell lines and found that it promotes glioblastoma invasion in a CCL5-dependent manner. This aligns with previous reports demonstrating the CCL5-CCR5 signaling in glioma cells promotes invasion through calcium-dependent matrix metalloproteinase 2. To assess the spatial distribution of this myeloid subpopulation, we performed RNAscope hybridization against AIF1, TMEM119, CCR5, CCL5, and Fn14 transcripts on sections of murine gliomas generated using the RCAS/tv-a model of human glioblastoma. We found an abundance of Fn14+ plus AIF1+ as well as Fn14+plus TMEM119+ myeloid cells throughout the tumor core and rim of murine tumors and identified CCL5 expression in pseudo-palisades bordering necrotic regions. Our results to date suggest that Fn14 expression in GAMs can promote the invasion of tumor cells into normal brain parenchyma by release of CCL5. Currently, we are utilizing orthotopic patient-derived xenograft models co-implanted with Fn14+/- myeloid cells to test the hypothesis that TWEAK-Fn14 signaling in GAMs provides strong pro-tumoral signals in the glioblastoma microenvironment. Citation Format: Angad Beniwal, Matthew Dufault, Pranjali Kanvinde, David Nascari, Ryan Eghlimi, Adarsha Malla, Dolores Hambardzumyan, Henrique Borges da Silva, Graeme Woodworth, Jeffrey Winkles, Nhan Tran. TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis [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 6867.

Regional delivery of mesothelin-targeted chimeric antigen receptor T-cell effectively and safely targets colorectal cancer liver metastases in mice.

Liver metastasis is the major cause of colorectal cancer related death. Mesothelin (MSLN)-targeted chimeric antigen receptor (CAR) T-cell therapy has been illustrated effective and safe through regional delivery of breast cancer, ovarian cancer and malignant mesothelioma tumors. Herein, we investigated the safety, efficacy, and immune microenvironment of regional delivery of MSLN (CAR) T-cell in the treatment of colorectal carcinoma liver metastases (CRLM). Second-generation MSLN CAR T-cells were administered by portal vein (PV) or caudal vein (CV, systemic administration) delivery in an orthotopic MSLN+ CRLM nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/γc-/- (NSG) mouse model. A total of 20 mice were randomly divided into control group, non-transduced T cell (NT)-CV group, NT-PV group, MSLN CAR T-cell CV (MSLN-CV) group, and MSLN CAR T-cell PV (MSLN-PV) group, with each group containing four mice to examine the safety and efficacy. The bioluminescence intensity (BLI) of tumor burden, tumor tissue macroscopic and microscopic observation were used to evaluate treatment efficacy. The safety was examined by body weight, survival time, and vital organ damage of mice. CAR T-cell infiltration and cytokine concentration were analyzed by flow cytometry, and immunostaining. The change of immune microenvironment between regional delivery and systemic delivery was investigated on an immune reconstructed CRLM patient-derived xenograft (PDX) model. Additionally, T cell subsets and immunosuppressive markers were examined. PV administration of 1×107/100 μL MSLN CAR T-cells in 20 NSG mice was well tolerated, and no overt toxicity was observed. The tumor burden in the PV group was obviously alleviated. The BLI was (0.73±0.52)×109 in PV group and (1.97±0.11)×109 in CV group (P<0.05), CD8+ granzyme B (GB)+ T cell percentage (MSLN-CV 4.42%±0.47% vs. MSLN-PV 13.5%±4.67%, P<0.01) and cytokine concentration were obviously increased in the MSLN-PV group. In the immune reconstituted CRLM PDX model, intratumor (IT) delivery of MSLN CAR T-cells exhibited much more infiltration of CD4+ and CD8+ T cells accompanied with elevated expression levels of PD-1, LAG-3, and TIM-3. Regional delivery of MSLN-targeted CAR T-cell therapy has encouraging results in the orthotopic CRLM NSG mouse model and PDX model, and converts the tumor microenvironment from cold to hot. This study may provide a new therapeutic approach for CRLM. Further clinical study is needed.

Abstract B031: Evaluating the consequence of homologous recombination deficiency in models of pediatric solid tumor

Abstract Homologous recombination deficiency (HRD) has been extensively studied in adult cancers where its association with sensitivity to Poly (ADP-ribose) polymerases (PARP) inhibitor sensitivity is well established. In these cancers, HRD is most frequently caused by biallelic inactivation of BRCA1/2 and tumors often exhibit characteristic genomic scars such as elevated HRD.sum score and specific mutational signatures derived from single nucleotide variants, insertions and deletions, and structural variants. Despite its clinical relevance, the therapeutic predictive value of HRD has not been systematically studied in pediatric cancers. Consequently, our team devised a strategy to identify and validate targetable deficiencies in DNA damage response (DDR) genes and assess genomic scars in pediatric tumors. Here, we report evaluation of HRD in a cohort of &amp;gt;200 patient-derived orthotopic xenograft models from the Childhood Solid Tumor Network (CSTN), a freely sharing resource established by our institution to accelerate the development of therapies for children with cancer. We provide an overview of our pipeline and discuss the landscape of genomic scars within our cohort. We profiled somatic and germline variants from over 500 DNA damage response genes and identified TP53 as the most frequently mutated gene with biallelic loss, whereas bi-allelic loss of BRCA1/2 was not found. Overall, our study suggests that HRD exists within solid tumors, but is driven by inactivation of genes other than BRCA1/2, resulting in a different set of genomic scars compared to those in adult cancers where HRD is common. Importantly, the CSTN contains models of pediatric solid tumors that are amenable to in vitro and in vivo interrogation, and therefore, will facilitate examination of the functional consequences of inactivated DDR genes, characterization of the unique genomic scar profiles underlying these inactivation events, and evaluation of novel therapeutic strategies. Citation Format: Evan Savage, Ke Xu, Jason Myer, Evadnie Rampersaud, Yawei Hui, Ti-Cheng Chang, Gang Wu, Christopher Tinkle, Anang Shelat. Evaluating the consequence of homologous recombination deficiency in models of pediatric solid tumor [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B031.

KCNA1 promotes the growth and invasion of glioblastoma cells through ferroptosis inhibition via upregulating SLC7A11

BackgroundThe high invasiveness and infiltrative nature of Glioblastoma (GBM) pose significant challenges for surgical removal. This study aimed to investigate the role of KCNA1 in GBM progression.MethodsCCK8, colony formation assay, scratch assay, transwell assay, and 3D tumor spheroid invasion assays were to determine how KCNA1 affects the growth and invasion of GBM cells. Subsequently, to confirm the impact of KCNA1 in ferroptosis, western blot, transmission electron microscopy and flow cytometry were conducted. To ascertain the impact of KCNA1 in vivo, patient-derived orthotopic xenograft models were established.ResultsIn functional assays, KCNA1 promotes the growth and invasion of GBM cells. Besides, KCNA1 can increase the expression of SLC7A11 and protect cells from ferroptosis. The vivo experiments demonstrated that knocking down KCNA1 inhibited the growth and infiltration of primary tumors in mice and extended survival time.ConclusionTherefore, our research suggests that KCNA1 may promote tumor growth and invasion by upregulating the expression of SLC7A11 and inhibiting ferroptosis, making it a promising therapeutic target for GBM.

Magnetic resonance imaging-guided intracranial resection of glioblastoma tumors in patient-derived orthotopic xenografts leads to clinically relevant tumor recurrence

BackgroundPreclinical in vivo cancer models are essential tools for investigating tumor progression and response to treatment prior to clinical trials. Although treatment modalities are regularly assessed in mice upon tumor growth in vivo, surgical resection remains challenging, particularly in the orthotopic site. Here, we report a successful surgical resection of glioblastoma (GBM) in patient-derived orthotopic xenografts (PDOXs).MethodsWe derived a cohort of 46 GBM PDOX models that faithfully recapitulate human disease in mice. We assessed the detection and quantification of intracranial tumors using magnetic resonance imaging (MRI).To evaluate feasibility of surgical resection in PDOXs, we selected two models representing histopathological features of GBM tumors, including diffuse growth into the mouse brain. Surgical resection in the mouse brains was performed based on MRI-guided coordinates. Survival study followed by MRI and immunohistochemistry-based evaluation of recurrent tumors allowed for assessment of clinically relevant parameters.ResultsWe demonstrate the utility of MRI for the noninvasive assessment of in vivo tumor growth, preoperative programming of resection coordinates and follow-up of tumor recurrence. We report tumor detection by MRI in 90% of GBM PDOX models (36/40), of which 55% (22/40) can be reliably quantified during tumor growth. We show that a surgical resection protocol in mice carrying diffuse primary GBM tumors in the brain leads to clinically relevant outcomes. Similar to neurosurgery in patients, we achieved a near total to complete extent of tumor resection, and mice with resected tumors presented significantly increased survival. The remaining unresected GBM cells that invaded the normal mouse brain prior to surgery regrew tumors with similar histopathological features and tumor microenvironments to the primary tumors.ConclusionsOur data positions GBM PDOXs developed in mouse brains as a valuable preclinical model for conducting therapeutic studies that involve surgical tumor resection. The high detectability of tumors by MRI across a substantial number of PDOX models in mice will allow for scalability of our approach toward specific tumor types for efficacy studies in precision medicine-oriented approaches. Additionally, these models hold promise for the development of enhanced image-guided surgery protocols.

Development of Orthotopic Patient-Derived Xenograft Models of Pediatric Intracranial Tumors.

The development of clinically relevant and reliable models of central nervous system tumors has been instrumental in advancing the field of Neuro-Oncology. The orthotopic intracranial injection is widely used to study the growth, invasion, and spread of tumors in a controlled environment. Orthotopic models are performed to examine tumor cells isolated from a specific region in a patient in the same site or location in an animal model. Orthotopic brain tumor models are also utilized for preclinical testing of therapeutics as they closely recapitulate the behavior of such cancer and the brain environment of patients. Below, we describe our experiences in the development of murine models of pediatric brain tumors including diffuse midline glioma (DMG), glioblastoma (GBM), and medulloblastoma. The method provides an overview of intracranial stereotactic injections in mice.

Patient-Derived Orthotopic Xenograft Models for High-Grade Pediatric Brain Cancers.

Patient-derived orthotopic xenograft (PDOX) mouse models are considered the gold standard for evidence-based preclinical research in pediatric neuro-oncology. This protocol describes the generation of PDOX models by intracranial implantation of human pediatric brain cancer cells into immune-deficient mice, and their continued propagation to establish cohorts of animals for preclinical research.

Abstract A003: A novel treatment strategy for high-risk and relapse/refractory hepatoblastoma

Abstract Background: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To better understand these aggressive phenotypes, our lab has developed a preclinical testing pipeline consisting of patient-derived spheroids (PDSp) and orthotopic xenograft models for chemotherapy and targeted therapy testing. We hypothesized that with this preclinical pipeline we could find an effective combination treatment strategy utilizing pan-histone deacetylase (HDAC) inhibition. Methods: RNA sequencing, microarray, NanoString, and immunohistochemistry (IHC) data of patient HB samples were analyzed for expression of all HDAC classes. HB cell lines were used to screen HDAC inhibitors. PDSp were used to screen which combination of standard chemotherapy regimens with a HDAC inhibitor. HB patient derived xenograft (PDX) mice models were developed and treated based off the lowest viability of PDSp drug screen. Mice were started on study when tumor volume reached 0.1-0.4 cm3 and were sacked when tumors reached diameter of 1.5 cm or at the end of the 6-week study period. Tumors were sectioned and evaluated for Ki67% and necrosis. Results: HDAC RNA and protein expression was elevated in HB tumors compared to normal livers. Four unique HB PDX models, including high risk, relapse and treatment refractory cases, were developed and validated from treatment-refractory patient tumors. Panobinostat (IC50 of 0.013-0.059 mM) showed strong in vitro effects with the lowest cell viability compared to other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). After 6 week of treatment[SV1] , all four models had a limited response to VI therapy; however, showed a much improved response with VIP. All four models responded to therapy with a decrease in tumor size compared to placebo and survival to 6 weeks. Two of our four models that had showed some initial response to VI therapy demonstrated necrotic cell death, lower Ki67%, decreased serum alpha fetoprotein (AFP) and reduced tumor burden compared to paired vincristine/irinotecan (VI) and placebo. Conclusions: Utilizing a preclinical pipeline for HB, we have shown that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from high risk, relapse, and treatment refractory HB patients. This clinically relevant methodology can be used to screen novel targeted combination therapies with the VI backbone in order to create novel treatment strategies for future clinical trials. Citation Format: Andres F Espinoza, Roma Patel, Sai Govindu, Pavan Kureti, Bryan Armbruster, Sarah Woodfield, Sanjeev A Vasudevan. A novel treatment strategy for high-risk and relapse/refractory hepatoblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A003.

Functional Dissection of Cellular Programs to Uncover Novel Gene Dependencies in AML

Genome-scale CRISPR-Cas9 screens have the power to unveil the Achilles' heel of neoplastic cells. Typically, analyses of such large-scale data sets focus on single gene dependencies. An alternative strategy is to evaluate functional networks enriched in a disease or molecular subset of interest. We applied this strategy to the Broad Institute's Cancer Dependency Map (DepMap) data set consisting of genome-scale CRISPR-Cas9 screens in over 1000 cancer cell line models. We hypothesized that interrogating enriched pathways with context-specific dependencies leads to the discovery of functionally informed gene dependencies. Single sample gene set enrichment analysis (ssGSEA) of over 1000 cancer cell lines in the DepMap identified hematologic malignancies to be highly enriched for signatures associated with elevated transcriptional activity. Notably, within hematologic malignancies, acute myeloid leukemia (AML) with a rearrangement in the KMT2A gene ( KMT2Ar-AML) was the most significantly enriched for dependency on transcriptional activity-associated gene signatures. We next interrogated KMT2Ar-AML gene dependencies (excluding common essential genes) with the human kinase database KinMap (http://www.kinhub.org/kinmap/) to identify readily druggable genes involved in transcriptional activity. Cyclin-dependent kinase 13 ( CDK13) was the only gene meeting these criteria. Further DepMap data analysis confirmed that KMT2Ar-AML cell lines were indeed more strongly dependent on CDK13 than all other cancer cell lines screened. Moreover, when we performed GSEA on the gene dependencies observed in CDK13-dependent versus non-dependent cell lines, we found transcriptional activity and KMT2A-target gene sets as top hits. We next validated AML cell dependency on CDK13 with orthogonal genetic approaches (CRISPR-Cas9 knock-out and shRNA) in vitro in human AML cell lines and cells from patient-derived xenograft (PDX) models of KMT2Ar-AML. Perturbation of CDK13 induced cell death with hallmarks of apoptosis. Doxycycline-inducible shRNA directed against CDK13 also impaired AML progression in the peripheral blood and bone marrow in an MV4-11- KMT2Ar xenograft. Moreover, in an orthotopic PDX model, doxycycline inducible knock-out of CDK13 in mice with established disease significantly reduced leukemia burden in the peripheral blood and bone marrow. CDK13 is reported to regulate transcriptional elongation and the clearance of prematurely terminated RNAs. To decipher why AML cells, particularly KMT2Ar, need CDK13 to survive, we identified the CDK13 binding sites and histone marks involved in transcriptional regulation in AML using CUT and RUN and ChIP-seq: We determined that CDK13 co-localizes with H3K4me3 and H3K27ac at promoter sites. Global gene expression studies following the knock-out of CDK13 revealed that the majority of CDK13 bound genes were also differentially expressed, with significantly more genes decreased than increased in expression after CDK13 knock-out. GSEA of CDK13-bound genes with decreased expression upon CDK13 knock-out identified gene signatures associated with transcriptional activation and KMT2A-targets. We next studied the characteristics of CDK13 target genes and observed a predominance of long genes (total length of gene exons &amp;gt; 10 kb, GRCh38.p12). GSEA of genes ranked by size, confirmed that longer genes were enriched for CDK13 target genes, CDK13 differential AML dependencies, and for KMT2A-target transcriptional signatures. Additionally, following CDK13 perturbation in KMT2Ar-AML we observed a decrease of Pol2/pPol2Ser2 occupancy at the transcription end site of long genes. In summary, using functional pathway analyses, we identified CDK13 as a candidate gene dependency enriched in KMT2Ar-AML, a target not immediately prioritized with single gene analysis. Mechanistically, our data support that CDK13 controls Pol2 processivity, with CDK13 perturbation leading to a decreased occupancy at the 3' end of long genes. KMT2Ar, CDK13 dependent cells are also enriched for dependency on long genes, thereby suggesting a molecular basis for the CDK13 dependency in KMT2Ar-AML.

Combining Menin and MEK Inhibition to Target Poor Prognostic KMT2A-Rearranged RAS Pathway-Mutant Acute Leukemia

Approximately 10% of all acute leukemias harbor a chromosomal translocation involving the Mixed Lineage Leukemia 1 ( MLL1/ KMT2A) gene locus with over 90 fusion partners. These fusions are especially prevalent in pediatric acute leukemias. The resulting KMT2A fusion proteins (KMT2A-r) drive leukemogenic gene expression through an interaction with a chromatin complex that includes the scaffold protein menin, giving rise to aggressive acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In infant ALL, frequently characterized by KMT2A-r, the presence of RAS pathway mutations has been associated with a poor prognosis. The prognostic impact of RAS pathway mutations in pediatric KMT2A-r AML is less established. We thus analyzed data from 1605 pediatric patients with AML treated on the Children's Oncology Group (COG) AAML0531 and AAML1031 studies, with 336 of these having KMT2A-r. 51% (170/336) of these patients had mutations in NRAS, KRAS or PTPN11. The combination of KMT2A-r with RAS pathway mutations was associated with worse EFS and OS, HR=1.71 (p&amp;lt;0.001) for EFS and OS multivariable models. Given the prevalence of RAS pathway mutations co-occurring with KMTA-r, and the high-risk leukemia associated with this mutational profile, we sought to identify efficacious targeted drug combinations for this subset of childhood leukemia. Early clinical trial data for the menin inhibitor revumenib (SNDX-5613) demonstrated efficacy in KMT2A-r leukemias. Responding patients, however, typically develop progressive disease with a subset having mutations in MEN1 the gene encoding menin. Effective combination treatments for full efficacy of this targeted therapy are needed. We evaluated the combination of RAS/MAPK targeting using the MEK1/2 inhibitor selumetinib with VTP-50469, a close analog of revumenib, in pediatric leukemias harboring both KMT2A-r and RAS mutations. Treatment of AML and ALL cell lines and cells from an AML patient-derived xenograft (PDX) model in vitro with this drug combination resulted in a synergistic decrease in viability, a G0/G1 cell cycle arrest, and an increase in apoptosis. The combination also resulted in decreased AML colony formation. We next explored potential mechanisms by which the combination treatment had a synergistic anti-leukemic effect. Both selumetinib and VTP-50469 treatment individually reduced phosphorylated ERK levels, but the combination had the most complete inhibitory effect. The combination treatment also resulted in loss of stem-like gene expression signatures, an increase in myeloid differentiation programs, and most strikingly, downregulation of targets of MYC signaling. Both MYC transcript and protein levels were reduced by each drug alone and even more effectively with the combination. Next, we evaluated the in vivo efficacy of this drug combination in three orthotopic pediatric AML and ALL pediatric PDX models harboring different KMT2A-r and RAS mutations (AML: KMT2A- AF10 with KRAS, KMT2A- PICALM with NRAS, ALL: MLL- AF4 with NRAS). NSG mice engrafted with PDX models via tail vein injection were assigned to four treatment groups: vehicle, chow containing revumenib (0.03%), selumetinib (50 mg/kg/day, 5 days a week), or the combination. After treatment for 18-28 days leukemia burden in bone marrow, spleen and peripheral blood was markedly decreased with the combination compared to the single drug-treated cohorts in 2 models with RAS mutations. In one of the AML models, single agent treatment with revumenib or selumetinib was highly efficacious at eradicating disease at the measured time point. We also found a striking decrease in levels of MYC, P-ERK and MEIS1 by western immunoblotting, highlighting on-target activity of the drugs and supporting our in vitro data for a role for MYC in the synergistic effect observed with the combination. In conclusion, new clinical data from a large cohort of COG patients demonstrates that KMT2A-r and co-occurring RAS pathway mutations portend a distinctively worse outcome for children with AML. The findings in our preclinical study suggest a promising, readily translatable targeted treatment for this patient population. Mechanistically, enhanced downregulation of both MYC signaling and the RAS/MAPK pathway with the combinatorial therapy is likely a strong contributor to the observed efficacy.

TMET-13. DNA DAMAGE SIGNALING ACTIVATES GTP SYNTHESIS TO PROMOTE GLIOBLASTOMA TREATMENT RESISTANCE

Abstract Glioblastoma (GBM) is the most common type of invasive brain tumor in adults and is uniformly fatal due to inherent resistance to radiation therapy (RT) and chemotherapy. Our group and others have found that metabolites can regulate DNA repair and therapy resistance in brain tumors, but little is known about how DNA damage regulates metabolic pathway activity in cancer. Here we show that following treatment with RT, GBMs increase rates of de novo guanylate synthesis in vitro and in orthotopic patient-derived xenograft models via signaling through the DNA repair protein DNA-PK. To determine if disrupting this regulation can augment GBM treatment efficacy, we combined an FDA-approved inhibitor of purine synthesis (mycophenolate mofetil, MMF) with chemoradiation in a variety of mouse models of GBM. Critically, targeting GTP synthesis improved the efficacy of both RT alone and chemoradiation in multiple patient-derived and syngeneic intracranial models. Our translational studies have shown that mycophenolic acid, the active metabolite of MMF, penetrates the blood-brain barrier at concentrations sufficient to inhibit GTP synthesis in GBMs of human patients. The phase 1 arms of our study, which are ongoing, are testing the safety and efficacy of combining MMF with standard of care chemoradiation for patients with primary and recurrent GBM.

EXTH-74. TUMOR PHARMACOKINETICS AND PHARMACODYNAMICS IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE PRMT5 INHIBITOR SKL27969

Abstract Epigenetic regulation plays a key role in the development of several cancers, including glioblastoma (GBM). Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising therapeutic target for GBM. PRMT5 is the primary type II methyltransferase that catalyzes symmetric arginine dimethylation in multiple proteins. In this study, we evaluated the pharmacokinetics, pharmacodynamics, and radiation sensitization properties of SKL27969, a highly selective PRMT5 inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 50 mg/kg (oral) SKL27969 vs. placebo for two cycles either alone or in combination with radiation. SKL27969 levels in plasma and contrast-enhancing tumor tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unbound fractions were determined by equilibrium dialysis. Immunohistochemistry was performed to assess levels of SDMA, phospho-Histone H3, pH2AX, and cleaved caspase 3 (CC3). For survival studies, mice with intracranial tumors were randomized into four cohorts – vehicle, radiotherapy, SKL27969 monotherapy, and SKL27969 plus radiotherapy. The median brain-to-plasma and tumor-to-plasma partition coefficients of unbound SKL27969 were 1.8 and 2.9, respectively – indicating favorable brain and tumor penetration ability of SKL27969. A decrease in SDMA (+) cells (54% vs. 0.1%, p &amp;lt; 0.001) indicated PRMT5 inhibition and was accompanied by an increase in CC3 (+) cells (0.7% vs. 1.2%, p = 0.01). SKL27969 is well tolerated, achieves pharmacologically relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Ongoing studies are evaluating in vivo survival benefit from combining PRMT5 inhibition with radiation therapy.