Glioblastoma Patient-derived Xenografts Research Articles

Bystander effects, pharmacokinetics, and linker-payload stability of EGFR-targeting antibody-drug conjugates Losatuxizumab vedotin and Depatux-M in glioblastoma models.

Antibody-drug conjugates (ADCs) are targeted therapies with robust efficacy in solid cancers, and there is intense interest in using EGFR-specific ADCs to target EGFR-amplified glioblastoma (GBM). Given the molecular heterogeneity of GBM, bystander activity of ADCs may be important for determining treatment efficacy. In this study, the activity and toxicity of two EGFR-targeted ADCs, Losatuxizumab vedotin (ABBV-221) and Depatuxizumab mafodotin (Depatux-M), with similar auristatin toxins, were compared in GBM patient-derived xenografts (PDXs) and normal murine brain following direct infusion by convection enhanced delivery (CED). EGFRviii-amplified and non-amplified GBM PDXs were used to determine in vitro cytotoxicity, in vivo efficacy, and bystander activities of ABBV-221 and Depatux-M. Non-tumor bearing mice were used to evaluate pharmacokinetics and toxicity of ADCs using LC-MS/MS and immunohistochemistry. CED improved intracranial efficacy of Depatux-M and ABBV-221 in three EGFRviii-amplified GBM PDX models (Median survival: 125 to >300 days vs 20-49 days with isotype-control AB095). Both ADCs had comparable in vitro and in vivo efficacy. However, neuronal toxicity and CD68+ microglia/macrophage infiltration were significantly higher in brains infused with ABBV-221, with the cell-permeable MMAE, as compared to Depatux-M, with the cell-impermeant MMAF. CED infusion of ABBV-221 into brain or incubation of ABBV-221 with normal brain homogenate resulted in significant release of MMAE, which is consistent with linker instability in the brain microenvironment. EGFR-targeting ADCs are promising therapeutic options for GBM when delivered intra-tumorally by CED. However, the linker and payload for the ADC must be carefully considered to maximize the therapeutic window.

Deuterium metabolic imaging differentiates glioblastoma metabolic subtypes and detects early response to chemoradiotherapy.

Metabolic subtypes of glioblastoma have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. Here, we used 2H magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) measurements of [6,6'-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived glioblastoma xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial TCA cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6'-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H MRS and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6'-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for TCA cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response.

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 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 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.

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.

Abstract B050: Metabolic rewiring in human brain cancer

Abstract Metabolic adaptation can promote oncogenic phenotypes in glioblastoma (GBM), but the metabolic pathways utilized by GBM and how they differ from the pathways utilized in normal cortex are poorly understood. Here, we utilize in vivo stable isotope tracing in mice and patients with glioblastoma to define carbon fate and metabolic rewiring in cancer. We infused uniformly labeled 13C glucose into mice bearing orthotopic GBM patient-derived xenografts or into patients undergoing surgical resection for likely GBM. Tumor and cortex were physically separated in mice by fluorescence-guided microdissection. In humans, samples of enhancing tumor, non-enhancing tumor and normal cortex were separated intraoperatively using MRI guidance. Samples were subsequently analyzed by liquid chromatography mass spectrometry and MALDI imaging mass spectrometry to determine the downstream metabolic fates of infused 13C glucose. Of eight patients infused with 13C6-glucose, six were confirmed to have GBM while the others had non-GBM high grade gliomas. In mice and in patients, glucose-derived carbon effectively entered glycolysis and labeled glycolytic intermediates equivalently in tumor and cortex. Compared to GBM tissue, cortical tissue preferentially oxidized glucose-derived carbon in the TCA cycle and used glucose-derived carbon to synthesize the neurotransmitter GABA. Cortical tissue utilized glucose-derived carbon to synthesize the amino acid and neurotransmitter precursor serine, while GBM tissue preferentially derived serine from extracellular sources. By contrast, GBM preferentially utilized glucose-derived carbon for the synthesis of metabolites required for growth: purines, pyrimidines and NAD+/NADH. These studies are the first measurements of numerous metabolic pathways in human brain cancer and cortex and suggest that GBMs suppress the physiologic utilization of glucose carbons for ATP and neurotransmitter generation and instead use these carbons to synthesize the biomolecules they need to proliferate. Consistent with this hypothesis, eliminating dietary serine in mice slowed GBM PDX growth and forced tumors to synthesize serine from glucose, which in turn lowered tumor nucleotide and NAD+/NADH levels. Dietary serine depletion had minimal effects on cortical metabolism, consistent with glucose being the primary serine source in the normal brain. These direct measurements of metabolic pathway activity in human and mouse brain cancer reveal adaptive metabolic rewiring and new therapeutic targets. Citation Format: Andrew Scott, Anjali Mittal, Sravya Palavalasa, Baharan Meghdadi, Nathan Qi, Alexandra O'Brien, Ayesha Kothari, Nathalie Agar, Ethan Yang, Joshua Fischer, Vijay Tarnal, Wajd Al-Holou, Costas Lyssiotis, Deepak Nagrath, Daniel Wahl. Metabolic rewiring in human brain cancer [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B050.

Abstract A002: The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies

Abstract Large PDX collections provide broad representation of tumor genetics and are commonly used to evaluate the clinical potential of novel therapeutic strategies. In many solid malignancies, profound benefit in PDX preclinical studies has foreshadowed eventual FDA approval of clinically impactful therapeutics. With a lack of corresponding progress in glioblastoma (GBM), the value of PDX testing has been questioned. Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients, and over the past 23 years, we have used this collection for extensive in vivo preclinical testing of 145 drugs in both orthotopic and heterotopic settings. As part of this work, the impact of standard of care therapy with temozolomide (TMZ) and bevacizumab was evaluated in orthotopic models using clinically relevant dosing regimens. For each animal study, the ratio of median survival for treated vs. control animals were used to define a ‘survival ratio’ as a normalized metric of benefit. TMZ efficacy was evaluated in 26 newly diagnosed GBM PDXs, with equal representation of MGMT methylated and unmethylated tumors. As expected, MGMT methylated PDXs were much more sensitive to TMZ as compared to unmethylated PDXs (median survival ratio 4.47 vs 1.48, respectively), and similar to clinical experience, 2 MGMT unmethylated PDXs were markedly sensitive to TMZ, while 1 MGMT methylated PDX was relatively resistant. In a more limited study, dose-dense vs. standard TMZ dosing was compared in 7 PDXs (4 MGMT methylated, 3 MGMT unmethylated). In a pooled analysis of all lines, both standard (HR=0.09; 95% CI: 0.06–0.14) and dose-dense TMZ therapy (HR=0.09; 95% CI: 0.06–0.14) were superior to vehicle controls (p&amp;lt;0.001). Similar to the clinical results in the Phase III randomized RTOG 0525 trial, survival with the dose-dense TMZ was no different than standard dosing (p=0.88, HR=0.98; 95% CI: 0.72-1.32). In an analysis of bevacizumab across 32 PDXs, mice were dosed once a week until moribund, and a minimum overall median survival benefit of 20% was used to define ‘responders’. Thirteen PDXs met this criterion, although the overall survival ratio among this subset was limited (range 1.21-1.65). Only 2 models had a 50% or greater survival benefit. Stratification by disease state (24 newly diagnosed vs 8 recurrent) showed equivalent performance (median survival benefit 1.13 for both). These results are consistent with the limited survival benefit observed with bevacizumab and the RTOG 0825 clinical trial. In summary, the results from these relatively inexpensive PDX studies demonstrate the potential of pre-clinical PDX trials to evaluate the extent of benefit and fraction of responsive PDXs to a novel therapeutic. If analyzed across an ‘adequate’ number of PDXs, these results potentially can be used to identify predictive biomarkers or to compare various treatment regimens prior to clinical testing. 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. The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies [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 A002.

EPCO-14. IDENTIFYING EPIGENETIC VULNERABILITIES OF MINIMAL RESIDUAL DISEASE IN GLIOBLASTOMA USING IN VIVO CRISPR SCREENS

Abstract Therapy for glioblastoma (GBM) includes surgical resection, radiation, and chemotherapy, which functionally reduces tumor burden to a state of minimal residual disease (MRD). However, recurrence over time is universal. Large-scale longitudinal studies of GBM patient samples did not identify selection pressure for specific DNA mutations in recurrent disease, highlighting the importance of epigenetic mechanisms during relapse. In this study, we aim to model MRD and the functional and transcriptional effects of epigenetic perturbations in GBM patient derived xenografts (PDXs). We developed an in vivo inducible genome editing system by engineering 827 GBM stem cells to express doxycycline inducible Cas9 and a pool of 14 CRISPR sgRNAs targeting 5 epigenetic factors (readers/erasers of H3K27me3 and H4 acetyltransferase KAT5) and controls. Tumor cells are implanted orthotopically and mice are followed by serial MRIs until tumor volumes reach 2mm3, after which in vivo editing is induced by administration of doxycycline. Cells are harvested for scRNA-sequencing and targeted amplification of sgRNA libraries, allowing for assignment of sgRNAs to individual cells. Genome editing efficiencies ranged from 50-90% in vivo. Knockout of core Polycomb (PcG) complex members led to dysregulated expression of genes involved in therapeutic resistance and altered cell state distributions. Current directions include optimization of sgRNA targeting and recovery of CRISPR-edited cells from tumors for scaling up screens. In addition, we have developed a “standard of care” treatment regimen in GBM PDX models whereby temozolomide and radiation therapy is dose optimized to achieve a latent state that mimics MRD followed by eventual tumor recurrence. Future experiments will combine MRD modeling in PDXs with in vivo CRISPR screening to characterize functional dependencies of MRD in GBM. This data provides proof of concept for utilizing these tools for studying genotype to phenotype connections in a complex GBM model system.

DDDR-04. TRANSIENT SUPPRESSION OF XPO1 ACTIVITY IS SUFFICIENT FOR SELINEXOR-INDUCED CYTOTOXICITY IN GLIOBLASTOMA CELLS LINES

Abstract Inhibitors of the nuclear export protein exportin 1 (XPO1i) show promise in the treatment of leukemias and solid tumors, including brain tumors. XPO1i such as Selinexor reversibly bind and inhibit XPO1 activity, resulting in a lethal nuclear accumulation of a variety of XPO1 substrates. Although XPO1i activity has been suggested to require prolonged suppression of XPO1 activity, this point has not been directly addressed. To do so we generated glioblastoma and patient-derived glioblastoma xenograft cells that express either endogenous wild-type (WT) XPO1 or endogenous WT XPO1 plus a doxycycline-inducible point-mutant form of XPO1 (C528S) that exhibits full XPO1 activity but cannot bind or be inhibited by Selinexor. These cells were then incubated with doxycycline before, or at various time after, the initiation of continuous Selinexor exposure, after which the ability of C528S XPO1 to rescue cells from Selinexor-induced toxicity could be determined. All four glioblastoma cell lines examined were drug-sensitive (IC50 &amp;lt; 600nM) and exhibited a dose-dependent nuclear retention of the XPO1 substrate RanBP1 following Selinexor exposure. Induced expression of C528S led to a doubling of XPO1 protein levels (WT plus C528S), and variable increases in Selinexor IC50, from 3-fold in GBM43 cells to 12,000-fold in LN229 cells. While induction of C528S expression prior to, or up to 8 h after, Selinexor exposure completely prevented Selinexor-induced cytotoxicity, cells could not be rescued after a 24 hr Selinexor exposure. Furthermore, the resistance provided by prolonged expression of C528S, or by shRNA-mediated suppression of XPO1 levels, was nearly 10-fold less than that provided by complete CRISPR-based replacement of WT XPO1 with C528S. These results therefore show that a transient suppression of XPO1 activity is sufficient for Selinexor-induced cytotoxicity in glioblastoma cells, and suggest that the Selinexor-induced lethality results from a combination of loss of XPO1 activity and Selinexor-XPO1 complex formation.

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.

TMET-03. REPROGRAMMING OF GLIOBLASTOMA GLUCOSE METABOLISM FUELS TUMOR GROWTH

Abstract Metabolic adaptation can promote oncogenic phenotypes in glioblastoma (GBM), but the metabolic pathways utilized by GBM and how they differ from the pathways utilized in normal cortex are poorly understood. Here, we utilize in vivo stable isotope tracing in mice and patients with GBM to define carbon fate and metabolic rewiring in cancer. We infused uniformly-labeled 13C glucose into mice bearing orthotopic GBM patient-derived xenografts or into patients undergoing surgical resection for likely GBM. Tumor and cortex were physically separated in mice by fluorescence-guided microdissection. In humans, samples of enhancing tumor, non-enhancing tumor and normal cortex were separated intraoperatively using MRI guidance. Samples were subsequently analyzed by liquid chromatography mass spectrometry to determine the downstream metabolic fates of infused 13C glucose. Of eight patients infused with 13C6-glucose, six were confirmed to have GBM while the others had non-GBM high grade gliomas. In mice and in patients, glucose-derived carbon effectively entered glycolysis and labeled glycolytic intermediates equivalently in tumor and cortex. Compared to GBM tissue, cortical tissue preferentially oxidized glucose-derived carbon in the TCA cycle and used glucose-derived carbon to synthesize neurotransmitters. Cortical tissue utilized glucose-derived carbon to synthesize the amino acid and neurotransmitter precursor serine, while GBM tissue derived serine from extracellular sources. By contrast, GBM tissue preferentially utilized glucose-derived carbon for the synthesis of purines, pyrimidines and NAD+/NADH. Using time-course isotope tracing studies in mice with GBM and a novel metabolic flux analysis framework, we quantified absolute metabolic fluxes in vivo and confirmed elevated nucleotide synthesis and serine salvage in GBM. Consistent with these results, eliminating dietary serine in mice slowed GBM PDX growth. These studies are the first measurements of numerous metabolic pathways in humans and indicate that GBMs suppress the physiologic utilization of glucose carbons to fuel biosynthesis, which represents a targetable metabolic liability.

STEM-03. TARGETING ANDROGEN SIGNALING IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is a lethal brain cancer. Androgen receptor (AR) transcript levels and protein expression are upregulated in glioblastoma compared to normal brain. AR signaling has a role in cancer stem cell function in various cancers. Stem cell activity and cancer cell plasticity represent a key resistance mechanism in glioblastoma. Thus, potent brain-penetrant anti-androgen therapies have the potential to improve treatment outcomes in glioblastoma. METHODS Clinical samples and patient derived xenografts were assessed for AR expression by immunohistochemistry. Cytoplasmic AR+ and AR- glioblastoma patient-derived xenografts maintained under stem cell conditions were tested in vitro for their response to anti-androgen therapies (abiraterone, enzalutamide and seviteronel). Stem cell function was assessed using a tumoursphere assay. Changes in plasticity markers, including ZEB1, were measured with immunofluorescence. RESULTS ~55% of people with glioblastoma have detectable cytoplasmic AR based on immunohistochemistry. AR-positive patient-derived glioma stem cell line RN1 was inhibited by low concentrations of anti-androgen agents, with seviteronel having the lowest half maximal inhibitory concentration (IC50) (at 96 hours: enzalutamide 52µM, abiraterone 12µM, seviteronel 7µM). AR-negative line WK1 was also inhibited though with higher IC50s (at 96 hours: enzalutamide 63µM, seviteronel 21µM). Conventional cell lines U87 and U251 were also inhibited by anti-androgen monotherapy. Tumoursphere assays demonstrated that anti-androgen agents inhibit the tumour-forming ability of cells, with seviteronel and abiraterone showing more inhibition than enzalutamide. Immunofluorescence analysis showed downregulation of plasticity marker ZEB1 with anti-androgen treatment (normalised mean fluorescent intensity: abiraterone 0.84, enzalutamide 0.91, seviteronel 0.74). CONCLUSIONS Glioblastoma is a devastating disease, lacking effective or targeted treatments. Targeting AR with repurposed anti-androgen drugs may be a promising therapeutic strategy, with the potential to abrogate treatment resistance. We are now evaluating these anti-androgen regimens in animal experiments, which will provide a more replicative microenvironment and confirm blood-brain barrier penetrance.

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.

Structure-Based Design of a Lead Compound Derived from Natural Schweinfurthins with Antitumor Properties That Target Oxysterol-Binding Protein.

Schweinfurthins (SWs) are naturally occurring prenylated stilbenes with promising anticancer properties. They act through a novel mechanism of action similar to that of other families of natural compounds. Their known target, oxysterol-binding protein (OSBP), plays a crucial role in controlling the intracellular distribution of cholesterol. We synthesized 15 analogues of SWs and demonstrated for the first time that their cytotoxicity as well as that of natural derivatives correlates with their affinity for OSBP. Through this extensive SAR study, we selected one synthetic analogue obtained in one step from SW-G. Using its fluorescence properties, we showed that this compound recapitulates the effect of natural SW-G in cells and confirmed that it leads to cell death via the same mechanism. Finally, after pilot PK experiments, we provided the first evidence of its in vivo efficacy in combination with temozolomide in a patient-derived glioblastoma xenograft model.

Decreased eukaryotic initiation factors expression upon temozolomide treatment—potential novel implications for eIFs in glioma therapy

PurposeSince glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment.MethodsWe evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX.ResultsWe observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis.ConclusionOur study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy.

Hypoxia-Inducible Transgelin-2 Confers Treatment Resistance through Activation of PI3K/Akt/GSK3β Pathway in Glioblastoma

Glioblastoma (GBM) patients with wild-type IDH experience worse survival response to the standard treatment of surgery followed by radiation therapy (RT) and temozolomide (TMZ) chemotherapy compared to their mutant IDH counterparts. This treatment has remained relatively ineffective partly due to the highly invasive phenotype of GBM leading to therapeutic resistance and tumor recurrence. Hypoxia is one of the key characteristic features of GBM which results in cancer metastasis and confers treatment resistance. Therefore, it is paramount to identify targets to help overcome hypoxia-induced treatment resistance in glioblastoma. Our lab has identified transgelin-2 (TAGLN2) to be significantly upregulated in IDH-wt GBM through multiple molecular profiling studies. This study aims to understand the mechanisms by which TAGLN2 confers treatment resistance for developing additional treatments for GBM. Additionally, active drug development efforts are also underway to target TAGLN2 for circumventing these therapeutic resistance mechanisms for effective GBM therapy. RNAi-mediated TAGLN2 knockdown (KD) approach was employed to assess the functions of TAGLN2 in GBM patient-derived xenograft (PDX) cell lines. Series of in vitro functional assays were performed to assess the role of TAGLN2 in these cell lines. Cell proliferation, invasion ± RT and/or TMZ were assessed by MTS and Trans-well invasion assays. Subsequently, WB analysis of oncogenic signaling pathways was performed following Transgelin-2 KD. Co-IP assays and Biacore/SPR analyses were performed to study the binding affinity and kinetics for the interaction of PTEN with TAGLN2. Further, cells were intracranially implanted in nude mice to assess the role of TAGLN2 on tumor growth in vivo. Conditional KD of TAGLN2 reduces cell proliferation, survival and invasive potential of GBM PDX cell lines. TAGLN2 KD also improved the sensitivity of these cells to both TMZ and radiation in vitro, as assessed by proliferation, survival, clonal expansion, and invasion. Histopathological studies of human GBM tumors and mouse xenograft tumors showed elevated expression of TAGLN2 in the peri-necrotic region of the tumors indicating that TAGLN2 protein level was upregulated by hypoxia. We also show that TAGLN2 is induced in hypoxic microenvironments with GBM PDX cell lines and its overexpression may enhance cellular resistance towards conventional therapy. Subsequently, we also show that hypoxia-induced TAGLN2 activates the PI3K/Akt oncogenic pathway through binding and inhibition of PTEN. Finally, in vivo data using an orthotopic xenograft mouse model shows reduction of tumor growth with knockdown of TAGLN2. Our in vitro and in vivo xenograft studies suggest that TAGLN2 confers treatment resistance to GBM contributing to tumor recurrence. Altogether, TAGLN2 may serve as a potential therapeutically vulnerable target in GBM specifically through its role in cell survival and invasion.

Evaluating cancer cell line and patient-derived xenograft recapitulation of tumor and non-diseased tissue gene expression profiles in silico.

Preclinical models like cancer cell lines and patient-derived xenografts (PDXs) are vital for studying disease mechanisms and evaluating treatment options. It is essential that they accurately recapitulate the disease state of interest to generate results that will translate in the clinic. Prior studies have demonstrated that preclinical models do not recapitulate all biological aspects of human tissues, particularly with respect to the tissue of origin gene expression signatures. Therefore, it is critical to assess how well preclinical model gene expression profiles correlate with human cancer tissues to inform preclinical model selection and data analysis decisions. Here we evaluated how well preclinical models recapitulate human cancer and non-diseased tissue gene expression patterns in silico with respect to the full gene expression profile as well as subsetting by the most variable genes, genes significantly correlated with tumor purity, and tissue-specific genes. By using publicly available gene expression profiles across multiple sources, we evaluated cancer cell line and patient-derived xenograft recapitulation of tumor and non-diseased tissue gene expression profiles in silico. We found that using the full gene set improves correlations between preclinical model and tissue global gene expression profiles, confirmed that glioblastoma (GBM) PDX global gene expression correlation to GBM tumor global gene expression outperforms GBM cell line to GBM tumor global gene expression correlations, and demonstrated that preclinical models in our study often failed to reproduce tissue-specific expression. While including additional genes for global gene expression comparison between cell lines and tissues decreases the overall correlation, it improves the relative rank between a cell line and its tissue of origin compared to other tissues. Our findings underscore the importance of using the full gene expression set measured when comparing preclinical models and tissues and confirm that tissue-specific patterns are better preserved in GBM PDX models than in GBM cell lines. Future studies can build on these findings to determine the specific pathways and gene sets recapitulated by particular preclinical models to facilitate model selection for a given study design or goal.

A phase 0/Ia study of BI 907828 concentrations in brain tissue and a nonrandomized, open-label, dose escalation study of BI 907828 in combination with radiotherapy in patients with newly diagnosed glioblastoma (GBM).

TPS2081 Background: Murine double minute 2 (MDM2) directly regulates the stability of the tumor suppressor p53, and dysregulation of this pathway can critically disrupt cellular response to genomic stress and lead to cell death. BI 907828 is a highly potent inhibitor of the MDM2-p53 interface that triggers accumulation of p53 and subsequent apoptosis in multiple GBM patient-derived xenografts (PDXs) with wild-type p53. Moreover, the combination of BI 907828 and radiotherapy provides significant survival extension in multiple orthotopic GBM PDXs. Methods: An integrated Phase 0/1 trial design is being used to evaluate the potential for BI 907828 to achieve a therapeutic concentration in brain tumor tissue and to test the safety of the combination with radiation. The co-primary endpoints for the Phase 0 study are i) measured total concentration of BI 907828 and ii) calculated unbound concentration of BI 907828 in contrast and non-contrast enhancing regions of tumor. Key inclusion criteria for the Phase 0 study are patients at least 18 years of age, ECOG performance status of 0 or 1, histologic or radiologic new diagnosis of GBM, and planned surgical resection. The first approximately 6 patients enrolled in the Phase 0 will be treated at the starting dose of 30 mg, and the future patients enrolled will be dosed at higher levels (45 mg or 60 mg) provided there are no safety concerns. If dose-limiting toxicities (DLTs) are encountered, then a Bayesian Logistic Regression Model (BLRM) with overdose control will be used to guide the dose level decisions for the additional patients. Multiple, image-registered tumor samples and intra-operative plasma samples will be analyzed for BI 907828 by LC-MS/MS. Intra-tumoral variation in BI 907828 will be correlated with pharmacodynamic effects of MDM2 inhibition (elevation of p53, p21, MDM2, GDF15, PUMA). Those patients completing the Phase 0 study and who meet additional eligibility criteria can enroll on the Phase 1a portion of the study. The pathologic inclusion criteria are TP53 wild-type, IDH1/2 wild-type, MGMT unmethylated GBM. Co-primary endpoints for the Phase 1a trial are i) occurrence of DLTs during and within 21 days after completion of radiation therapy, and ii) occurrence of adverse events throughout the treatment period. Tolerability of inter-patient dose-escalation of BI 907828 (30, 45, 60 mg) combined with radiation therapy (60 Gy in 30 fractions) will be evaluated with dose-escalation guided by a two-parameter BLRM with overdose control. Patients will remain on BI 907828 monotherapy following completion of radiotherapy until progression. To date, two patients have enrolled on the Phase 0 study, and one patient went on to enroll on the Phase 1a portion of the study. Clinical trial information: NCT05376800 .

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.