Patient-derived Xenograft Models Of Glioblastoma Research Articles

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.

OGDH and Bcl-xL loss causes synthetic lethality in glioblastoma.

Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the α-ketoglutarate dehydrogenase (OGDH) gene, which encodes an enzyme that is part of the tricarboxylic acid (TCA) cycle, as essential for GBM growth. Moreover, by combining transcriptome and metabolite screening analyses, we discovered that loss of function of OGDH by the clinically validated drug compound CPI-613 was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3 mimetic, ABT263) in patient-derived xenografts as well neurosphere GBM cultures. CPI-613-mediated energy deprivation drove an integrated stress response with an upregulation of the BH3-only domain protein, Noxa, in an ATF4-dependent manner, as demonstrated by genetic loss-of-function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with disruption of the TCA cycle might be a treatment strategy for 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 < 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.

EXTH-38. VELCRIN MOLECULAR GLUES INDUCE CELL DEATH IN GLIOBLASTOMAS WITH HIGH PDE3A AND SLFN12 EXPRESSION

Abstract Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase, SLFN12, and the phosphodiesterase, PDE3A. Formation of the complex activates SLFN12, which cleaves tRNALeu(TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumor cell lines from the Cancer Cell Line Encyclopedia (CCLE), including glioblastoma cell lines. We therefore aimed to characterize velcrins as novel therapeutic agents in glioblastoma. We measured PDE3A and SLFN12 expression levels in commercially available glioblastoma cells as well as glioblastoma neurospheres from the Center for Patient Derived Models at Dana Farber Cancer Institute. We identified velcrin-sensitive glioblastoma cell lines and neurospheres, and determined that velcrin response is highly correlated with PDE3A expression. We also determined that sensitive cells exhibit a reduction in nascent polypeptide formation upon velcrin treatment, which is rescued by CRISPR-induced knockout of PDE3A. To assess velcrin efficacy in vivo, we measured central nervous system and plasma levels of the velcrin, BAY 2666605, and determined that it freely crosses the blood brain barrier. We also determined that twice daily dosing of BAY 2666605 elicits full tumor regression in an orthotopic glioblastoma xenograft model, with sustained complete responses after treatment withdrawal. Lastly, we demonstrated that the velcrins BAY 2666605 and BAY 2501293 induce tumor regression in biomarker-positive subcutaneous glioblastoma patient-derived xenograft models. In summary, our data demonstrates that velcrins have anti-tumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents.

Clinically relevant glioblastoma patient-derived xenograft models to guide drug development and identify molecular signatures.

Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.

Abstract 2796: Tumor pharmacokinetics, pharmacodynamics and efficacy analysis of WEE1 inhibitor, zn-c3 in patient-derived xenograft models of glioblastoma

Abstract WEE1 kinase regulates G2/M checkpoint transition via phosphorylation of CDK1 and prevents entry into mitosis. WEE1 inhibition increases genomic instability due to G2/M checkpoint inactivation and can result in mitotic catastrophe as monotherapy or when combined with DNA damaging agents. In this study, we evaluated the efficacy as well as pharmacokinetic and pharmacodynamic properties of ZN-c3, a best-in-class WEE1 inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 80 mg/kg ZN-c3 PO QD x 2 weeks vs. placebo. ZN-c3 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 pCDK1, phospho-Histone H3 (pHH3), and cleaved caspase 3 (CC3). The median unbound concentrations of ZN-c3 were 315 nmol/L and 15 nmol/kg in plasma and tumor tissue, respectively - higher than the biochemical IC50 of ZN-c3 for inhibition of WEE1 (3.8nM). An increase in pHH3(+) cells (0.40% vs. 6.57%, p=0.03) indicated drug-induced mitotic entry upon ZN-c3 treatment. ZN-c3 was potently cytotoxic across multiple patient-derived xenograft (PDX)-derived cell lines (IC50- 150-10000nM). Time and dose-escalation experiments exhibited target engagement at concentrations lower than IC50 values within one hour of treatment. Basal levels of WEE1 and pCDK1 were correlated with cell viability in response to ZN-c3 across multiple PDX cell lines. Overall, ZN-c3 is well tolerated, achieves pharmacologically-relevant unbound concentrations in GBM PDX models, and is associated with significant checkpoint modulation. Preclinical evaluation of ZN-c3 in combination with radiation therapy and DNA damage response pathway inhibitors is currently underway, which will support future combinatorial strategy for glioblastoma treatment. Citation Format: Shwetal Mehta, An-Chi Tien, Sonam Patel, Tigran Margaryan, Anilkumar Thaghalli Shivanna, Leonel Elena, Jennifer Molloy, William Knight, Ruiheng Hon, Mackenna Elliott, Mariya Stavnichuk, Zorana Opachich, Yu-Wei Chang, Chelsea Montgomery, Sarah Himes, Barbara Hopkins, Artak Tovmasyan, Nader Sanai. Tumor pharmacokinetics, pharmacodynamics and efficacy analysis of WEE1 inhibitor, zn-c3 in patient-derived xenograft models of glioblastoma [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 2796.

TMET-38. LOSS OF FUNCTION OF CDK7 IS SYNTHETICALLY LETHAL WITH FATTY ACID OXIDATION INHIBITION IN GLIOBLASTOMA

Abstract CDK7 has been identified as a potential drug target for glioblastoma (GBM), a highly lethal primary brain tumor. However, resistance to therapy develops quickly, which may be facilitated by drug-induced reprogramming of metabolism. By combination of a transcriptome and metabolite screening analyses followed by carbon tracing (U-13C-Glucose, U-13C-Glutamine and U-13C-Palmitic acid) and extracellular flux analysis we demonstrated that both genetic and pharmacological (YKL-5-124 and THZ1) CDK7 inhibition elicited substantial metabolic reprogramming. Specifically, CDK7i elicited an increase of oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO) manifested by enhanced labeling of citric acid cycle intermediates from palmitic acid. Consistently, the combination treatment of CDK7i inhibitors with blockers of FAO (etomoxir) or cellular respiration (gamitrinib) exerted substantial synergistic growth inhibition in patient derived xenograft as well as neurosphere GBM cultures, which was mainly driven by a collapse of oxidative energy metabolism. In turn, exogenous administration of adenosine triphosphate partially rescued from the cell death induced by the combination treatment. Moreover, the combination treatment activated intrinsic apoptosis through a reduction of both Mcl-1 and Bcl-xL as demonstrated by rescue experiments. Finally, the combined administration of YKL-5-124 and etomoxir extended overall in an orthotopic patient-derived xenograft model of GBM. In summary, these data support that simultaneous targeting of CDK7 and FAO might be a potential novel therapy against GBM.

59. Inferring copy number events in Glioblastoma using spatial gene expression data

<h3>Background</h3> Glioblastoma (GBM) is the most common malignant brain tumour in adults, with inevitable treatment resistance and poor outcomes. Genomic instability of GBM gives rise to large-scale chromosomal copy number variants (CNVs) that drive resistance. Genetically distinct clones in GBM are conventionally investigated using whole-genome sequencing (WGS), but recent bioinformatic approaches can infer CNVs using transcriptomics data only. <h3>Purpose</h3> To investigate whether patient-derived xenograft (PDX) mouse models of GBM exhibit clonal diversity as a function of space (across tumour regions) and time (early to late time points). <h3>Methods</h3> PDX data from 3 cell lines representing the tumour core, vascular regions, and infiltrating edge of one GBM patient's tumour were profiled at early and late time points in disease progression, using the 10X Genomics Visium platform for spatial transcriptomics. The computational tool inferCNV was used to identify CNVs. <h3>Results</h3> Successfully identified large-scale CNVs were concordant with events detected in matched WGS data. Additionally, we identified a minor subclone not detectable by WGS that expands in multiple PDX mice indicating it is pre-existing in the patient-derived cell lines. Further analysis revealed localization of this subclone to the tumour core region suggesting a microenvironment-specific adaptation. <h3>Conclusions</h3> We show that spatial gene expression data can be leveraged to infer CNVs in PDX models of GBM that correspond to known events from bulk WGS data. Subclones present below the limit of WGS detection can also be inferred. Charting the spatially resolved position of these clones in vivo supports the use of PDX models in GBM.

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma.

Recurrence of solid tumors renders patients vulnerable to advanced, treatment-refractory disease state with mutational and oncogenic landscape distinctive from initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profile barcoded tumor stem cell populations through therapy at tumor initiation, MRD, and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors show distinct patterns of recurrence in which clonal populations exhibit either a pre-existing fitness advantage or an equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing reveals a tumor-intrinsic immunomodulatory signature with prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from patients with GBM. Our results provide insight into the innate and therapy-driven dynamics of human GBM and the prognostic value of interrogating the MRD state in solid cancers.

Abstract 1086: PEGylated talazoparib enhances therapeutic window of its combination with temozolomide in Ewing sarcoma

Abstract Introduction: The Ewing family of sarcomas is the fourth most common highly malignant childhood cancer. Current standard therapy is relatively ineffective for relapsed and metastatic Ewing sarcoma predominantly due to development of resistance to chemotherapeutics. Nanoparticle-formulated drugs are reported to increase uptake into tumor tissue, while reducing drug access to normal tissues, because of reduced permeability of normal vasculature. This potentially leads to lower dose exposure in normal tissues and reduced toxicity. The purpose of this study was to evaluate the antitumor activity of the PEGylated poly(ADP) ribose polymerase 1/2 (PARP1/2) inhibitor talazoparib combined with the DNA alkylating agent temozolomide (TMZ) in Ewing sarcoma xenograft models and other pediatric cancers. Method:. We evaluated the preclinical efficacy of PEGylated talazoparib (PEG~TLZ), alone or in combination with TMZ, in Ewing sarcoma and glioblastoma patient derived xenograft (PDX) models. Additional solid tumor models, such as rhabdomyosarcoma, Wilms tumor, malignant rhabdoid tumor, osteosarcoma, and synovial sarcoma were evaluated using a novel single mouse testing approach. PEG~TLZ was administered as a single administration intraperitoneally to mice bearing subcutaneous tumors at a single dose of 5 - 20 µmol/kg alone, or at 10 µmol/kg on day 1 combined with TMZ administered orally at 40 mg/kg starting on day 3 or 4 for 5 consecutive days. Results: Mice bearing Ewing sarcoma and MGMT-deficient glioblastoma xenografts tolerated PEG~TLZ+TMZ with minimal toxicity and achieved maintained complete response (MCR). The time-separated drug administration schedule of the single dose of PEG~TLZ followed by the 5-day TMZ treatment demonstrated objective responses in Ewing sarcoma, rhabdoid tumor, rhabdomyosarcoma, and glioblastoma PDX models. Conclusion: We evaluated the range of PEG~TLZ+TMZ activity among pediatric solid tumor panels using conventional and single-mouse testing approaches, and demonstrated that PEG~TLZ combined with delayed TMZ administration enhances the therapeutic window of the treatment compared to free TLZ. From the clinical standpoint, the single intravenous administration of PEG~TLZ could be advantageous for treating infants and young children. Citation Format: Vanessa Del Pozo, Andrew J. Robles, Shaun Fontaine, Qianqian Liu, Joel E. Michalek, Peter J. Houghton, Raushan Kurmasheva. PEGylated talazoparib enhances therapeutic window of its combination with temozolomide in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1086.

Abstract 4001: Evaluation of novel therapeutics using the Mayo Clinic GBM Patient-Derived Xenograft (PDX) collection

Abstract The Mayo Clinic collection of patient-derived xenograft (PDXs) has been developed from the direct implantation of glioblastoma (GBM) samples into immunodeficient mice. Over 105 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated infiltration of tumor cells into the brain parenchymal in 90% of models with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed the PDX collection captures the genetic heterogeneity of GBM, with the majority of typical molecular alterations seen at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations (86%) and homozygous deletion of CDKN2A (70%) were the most frequent. Additional alterations in the p53 pathway included TP53 mutations (36%) and MDM2/4 amplification (10%/2%). IDH mutations were found in three models while MGMT promoter methylation was observed in 45% of PDXs. The available molecular sequencing results are viewable on cBioPortal. These genetically diverse models are extremely useful for biomarker discovery and efficacy evaluations. Using an innovative 1x1x1 PDX preclinical trial design, we are establishing a workflow to evaluate control and drug treatment in an individual animal across PDX lines in the flank. As our first use of this strategy, a small molecule MDM2 inhibitor was evaluated in 17 PDX lines with variable MDM2 and TP53 statuses. Tumors harboring MDM2 amplification without TP53 mutation (n=5) showed profound response, with 4 PDXs showing a complete response that lasted &amp;gt;150 days. Response in TP53 wild-type tumors lacking MDM2 amplification was variable, with a 1.9 to 4.8 fold longer time to endpoint (mean 3; median 2.7). A more traditionally powered intracranial efficacy study performed with one MDM2 amplified (GBM108) and two non-amplified (GBM14, GBM10) PDX lines confirmed these findings of reduced and more variable response to therapy in MDM2 non-amplified TP53 wild-type tumors. Future studies will aim to examine novel biomarkers indicative of therapeutic response to better understand molecular features associated with MDM2 inhibitor efficacy. With over 20 years of experience, our group has extensive expertise in development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Our diverse collection of clinically relevant GBM PDX models provides a highly sought-after resource which has been utilized by the pharmaceutical community with 58 working relationships to date (26 contracts, 32 collaborations). Through these collaborations, we aim to expand the use of our highly characterized GBM PDX panel for biomarker discovery using similar strategies. Citation Format: Danielle M. Burgenske, Ann C. Mladek, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Rachael Vaubel, Jann N. Sarkaria. Evaluation of novel therapeutics using the Mayo Clinic GBM Patient-Derived Xenograft (PDX) collection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4001.

Induction of Synthetic Lethality by Activation of Mitochondrial ClpP and Inhibition of HDAC1/2 in Glioblastoma.

Novel therapeutic targets are critical to unravel for the most common primary brain tumor in adults, glioblastoma (GBM). We have identified a novel synthetic lethal interaction between ClpP activation and HDAC1/2 inhibition that converges on GBM energy metabolism. Transcriptome, metabolite, and U-13C-glucose tracing analyses were utilized in patient-derived xenograft (PDX) models of GBM. Orthotopic GBM models were used for in vivo studies. We showed that activation of the mitochondrial ClpP protease by mutant ClpP (Y118A) or through utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (panobinostat) or selective (romidepsin) HDAC inhibitors caused synergistic reduction of viability in GBM model systems, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. This effect was partially mediated by activation of apoptosis along with activation of caspases regulated chiefly by Bcl-xL and Mcl-1. Knockdown of the ClpP protease or ectopic expression of a ClpP D190A mutant substantially rescued from the inhibition of oxidative energy metabolism as well as from the reduction of cellular viability by ClpP activators and the combination treatment, respectively. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC inhibitors and imipridones prolonged host survival more potently than single treatments or vehicle in vivo. Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

PTRF/Cavin-1 enhances chemo-resistance and promotes temozolomide efflux through extracellular vesicles in glioblastoma.

Background: The concentration and duration of intracellular drugs have always been the key factors for determining the efficacy of the treatment. Efflux of chemotherapeutic drugs or anticancer agents is a major reason for multidrug resistance generation in cancer cells. The high expression of polymerase I and transcript release factor (PTRF) is correlated with a worse prognosis in glioma patients. However, the importance of PTRF on temozolomide (TMZ) resistance in glioblastoma (GBM) is poorly understood.Methods: TCGA data analysis, CGGA data analysis, transmission electron microscopy (TEM), scanning electron microscopy (SEM), clone formation, cell counting kit-8 (cck-8), western blot (WB), immunofluorescence (IF), immunohistochemistry (IHC) and flow cytometry assays were performed to investigate the underlying mechanism and effect of PTRF on TMZ-resistance in a variety of GBM cell lines and GBM patient-derived xenograft (PDX) models. Clone formation, WB, IF, IHC and flow cytometry assays were performed to examine the efficacy of sequential therapy of TMZ followed by CQ in GBM cells and PDX models.Results: The prognosis of GBM patients treated with TMZ was negatively correlated with PTRF expression. Our results reveal that PTRF knockdown significantly decrease proliferation and increase apoptosis in GBM after TMZ treatment. Moreover, PTRF contribute to TMZ-resistance by increasing TMZ efflux through extracellular vesicles (EVs). Furthermore, our results demonstrate that sequential therapy of TMZ followed by CQ significantly promotes the TMZ efficacy against GBM by increasing intracellular TMZ concentration ([TMZ]i).Conclusion: This study highlights that PTRF can act as an independent biomarker to predict the prognosis of GBM patients after TMZ treatment and describes a new mechanism contributing to TMZ-resistance. In addition, this study may provide a novel idea for GBM therapy.

TMOD-15. CHARACTERIZATION OF THE MINIMAL RESIDUAL DISEASE STATE REVEALS DISTINCT EVOLUTIONARY TRAJECTORIES OF HUMAN GLIOBLASTOMA

Abstract Recurrence of solid tumors renders patients vulnerable to a distinctly advanced, highly treatment-refractory disease state that has an increased mutational burden and novel oncogenic drivers not detected at initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cancer cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profiled barcoded tumor cell populations through therapy at tumor initiation/engraftment, MRD and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors showed distinct patterns of recurrence in which clonal populations exhibited either a priori, pre-existing fitness, or equipotent fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing revealed a tumor-intrinsic immunomodulatory signature with strong prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from GBM patients at all stages of disease. Our results provide insight into the innate and therapy-driven dynamics of human glioblastoma, and the prognostic value of the interrogating of the MRD state in solid cancers.

EXTH-09. TUMOR PHARMACOKINETICS, PHARMACODYNAMICS AND RADIATION SENSITIZATION IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE AURORA KINASE A INHIBITOR LY3295668

Abstract Cell-cycle deregulation is at the crux of all malignancies, including glioblastoma (GBM). Aurora Kinase A (AURKA) plays a central role in G2/M transition and faithful chromosome segregation. In this study, we evaluated the pharmacokinetics, pharmacodynamics, and radiation sensitization properties of LY3295668, a highly specific AURKA inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 50 mg/kg LY3295668 PO BID x 4 days vs. placebo. LY3295668 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 pAURKA (T288), phospho-Histone H3 (pHH3), and cleaved caspase 3 (CC3). For survival studies, mice with intracranial tumors were randomized to four cohorts – vehicle, radiotherapy, LY3295668 monotherapy, and LY3295668 plus radiotherapy. The median unbound concentration of LY3295668 was 270.88 nmol/L and 22.33 nmol/kg in plasma and tumor tissue, respectively – significantly higher than the biochemical IC50 of LY3295668 for AURKA inhibition (0.8 nM). A decrease in pHH3(+) cells (0.8% vs. 6.4%, p=0.036) indicated drug-induced mitotic arrest and was accompanied by an increase in CC3(+) cells (6.4% vs. 8.0%, p=0.67). Combination of LY3295668 with radiotherapy prolonged survival compared to either therapy alone in orthotopic GBM PDX models. LY3295668 is well tolerated, achieves pharmacologically-relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Preclinical combination of LY3295668 with radiation therapy leads to synergistic effects and supports future clinical study of this multimodal strategy in glioblastoma patients.

EXTH-67. THERAPEUTIC EFFICACY OF GC1118, A NOVEL ANTI-EGFR ANTIBODY, AGAINST GLIOBLASTOMA WITH HIGH EGFR AMPLIFICATION IN PATIENT-DERIVED XENOGRAFTS

Abstract We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.

TAMI-17. INDUCTION OF SYNTHETIC LETHALITY BY ACTIVATION OF MITOCHONDRIAL CLPP AND INHIBITION OF HDAC1/2 IN GLIOBLASTOMA

Abstract Activation of the mitochondrial ClpP protease is an innovative therapeutic concept and the identification of synthetic lethal interactions may foster the development of novel therapies for glioblastoma (GBM). By integration of a transcriptome, metabolite and U-13C-glucose tracing analyses, we showed that activation of the mitochondrial ClpP protease through constitutively active ClpP (Y118A) or utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (Panobinostat) and selective (Romidepsin) HDAC inhibitors caused synergistic reduction of viability in established, neuro-sphere and patient-derived xenograft (PDX) cultures of human GBM, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. Notably, human astrocytes were significantly less susceptible to the combination treatment of HDAC-inhibitors and ClpP activators. The reduction of GBM viability occurred independent of TP53 status and was accompanied by activation of cell death with apoptotic features along with cleavage of caspases regulated chiefly by Bcl-xL and Mcl-1. Importantly, knockdown of the ClpP protease or ectopic expression of a ClpP D190A mutant almost completely rescued from the inhibition of oxidative energy metabolism as well as from the reduction of cellular viability by ClpP activators and the combination treatment, suggesting critical involvement of this protein. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC-inhibitors and imipridones reduced tumor growth and prolonged host survival more potently than single treatments or vehicle in vivo. Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

Antitumor efficiency of combination of bortezomib and themosolomide on subcutaneous PDX-model of human glioblastoma.

e15011 Background: Poor clinical effects of standard treatment for glioblastoma determine the need for the development of new therapeutic strategies. Aberrant functioning of the proteasome system, as well as activation of the HIF-1α signaling pathway, are characteristic of glial tumor cells; they can be considered as potential therapeutic targets in the treatment of malignant brain tumors. One of the possible options for improving the results of glioblastoma treatment may involve strategies for inhibiting the HIF-1α pathway. Bortezomib, a proteasome inhibitor, can block the biological effects of HIF-1α. Bortezomib showed a pronounced antitumor effect in in vitro testing on various models of solid malignant tumors, giving grounds for further studies of its effectiveness in vivo. Patient-derived xenograft (PDX) models are characterized by a variety of cell subclones and are therefore considered the most reliable tool for predicting therapeutic responses. Methods: A PDX model of glioblastoma was created in 20 Balb/c Nude mice implanted with a subcutaneously inoculated human glioblastoma. Temozolomide (0.5 mg/kg), bortezomib (0.25 mg/kg), or a combination of temozolomide and bortezomib were administered intraperitoneally daily for 21 days. The tumor histotype was confirmed by histological analysis (staining with hematoxylin and eosin). The antitumor effect was determined by the inhibition of tumor growth (ITG%), the volume of tumor nodes, and the index of tumor growth. Results: The highest value of the inhibition of tumor growth (ITG%) was registered in the group of animals receiving a combination of temozolomide and bortezomib – 85.38%. The values in the groups receiving temozolomide or bortezomib monotherapy were 57.32% and 63.11%, respectively. Conclusions: An analysis of the antitumor efficacy of bortezomib combined with temozolomide in human subcutaneous PDX-glioblastomas demonstrated their synergistic effect.

SPDR-01 Paired epithelioid glioblastoma patient-derived xenograft models to evaluate resistant mechanism for molecular target therapy

Epithelioid glioblastoma (E-GBM) arises at younger age, commonly disseminates to cerebrospinal fluid, and results in dismal prognosis. About half of E-GBM harbors BRAF V600E mutation, thus BRAF/MEK inhibitors are expected to be specifically sensitive to E-GBM like other BRAF V600E mutant carcinomas. However, therapeutic effect is limited by the emergence of drug resistance. To overcome this issue, it is crucial to elucidate the treatment resistance mechanisms by clinically representative models. Herein, we establish 2 paired E-GBM patient-derived xenograft (PDX) models from young adult patients (YMG62 and YMG89) with BRAF V600E, TERT promoter mutations and CDKN2A homozygous deletions. The YMG62 patient received dabrafenib with trametinib, while YMG89 patient received dabrafenib monotherapy after recurrence with standard treatment. The YMG62 patient was refractory to combination therapy. The YMG89 patient was initially responded to dabrafenib, but gradually became resistant and the 2 patients died due to CNS dissemination. Paired PDX models were established from tumors prior and after molecular target therapy. All PDXs were formed as CNS dissemination model, which were recapitulated to the patient characteristics. BRAF/MEK inhibitors strongly suppressed cell viability in primary tumor (YMG89P). However, BRAF/MEK inhibitors became resistant in recurrent tumor (YMG89R). YMG62 paired PDXs were resistant to molecular target therapy. Western blotting indicated retained MAPK signaling pathway and/or increased AKT phosphorylation after BRAF/MEK inhibitors treatment in refractory and recurrent cells, which indicates crucial role of re-activation in the MAPK signaling pathway and/or PI3 kinase pathway for tumor maintenance in BRAF V600E mutant E-GBM. We have done high throughput drug screening to identify compounds to overcome resistant to molecular target therapy. Our established E-GBM paired PDX models recapitulate patient characteristics, which may uncover treatment resistant mechanism and novel therapeutic target in E-GBM.

Therapeutic Efficacy of GC1118, a Novel Anti-EGFR Antibody, against Glioblastoma with High EGFR Amplification in Patient-Derived Xenografts.

Simple SummaryGC1118 is a novel anti-EGFR monoclonal antibody with a distinct mode of epitope binding. Its therapeutic efficacy has been validated in preclinical studies of several cancers. We evaluated the anti-tumor efficacy of GC1118 against glioblastoma (GBM) using patient-derived xenografts (PDXs). GC1118 exhibited anti-tumor efficacy comparable to that of cetuximab in a subset of PDXs, and EGFR amplification was a potential biomarker for predicting its therapeutic efficacy. Growth inhibitory and direct apoptotic effects on GBM tumor cells were confirmed in in vitro analyses. In intracranial PDXs, GC1118 significantly improved survival outcome, indicating its potential to cross the blood–brain barrier. These results support the clinical potential of GC1118 in treating GBM, further prompting the requirement of a clinical trial.We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.