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. 2024 Oct 15;14(10):4747–4759. doi: 10.62347/LQIJ5334

A patient-derived xenograft mouse platform from epithelioid glioblastoma provides possible druggable screening and translational study

Chiao-Yun Lin 1,*, Chen-Yang Huang 2,3,*, Cheng-Chi Lee 3,4, Lien-Min Li 4, Ya-Fang Lee 4, Shi-Ming Jung 2,5, Hsien-Chi Fan 3, An-Chi Lin 1, Cheng-Lung Hsu 2,3, Yin-Cheng Huang 2,4
PMCID: PMC11560820  PMID: 39553226

Abstract

Despite advancements in targeted therapy, glioblastoma remains a challenging condition with limited treatment options. While surgical techniques and external radiation therapy have improved, the median survival for glioblastoma stands at around 12-18 months, with a 5-year survival rate of only 6.8%. Epithelioid glioblastoma (eGBM) represents a rare subtype within the glioma spectrum. Utilizing patient-derived xenograft (PDX) models in mice offers a promising avenue for drug screening and translational research, particularly for this specific glioblastoma subtype. Establishing a stable PDX model for eGBM revealed consistent genetic abnormalities, including BRAF V600E mutation and CDKN2A deletion, in both primary and PDX tumors. Leveraging a curated drug database, compounds potentially targeting these aberrations were identified. By using the novel PDX platform, the results presented in this study demonstrate that the treatments with Palbociclib or Dabrafenib/Trametinib significantly reduced tumor size. RNA sequencing analysis further validated the responsiveness of the tumors to these targeted therapies. In conclusion, PDX models offer a deeper understanding of eGBM at the genomic level and facilitate the identification of potential therapeutic targets. Further translational studies of this novel PDX model hold promise for advancing the diagnosis and treatment of this specific subtype of glioblastoma.

Keywords: Glioblastoma multiform, patient derived xenograft, BRAF V600E, CDK4/6 inhibitor

Introduction

Glioblastoma multiforme (GBM) is a formidable challenge disease with little significant improvement for overall survival (OS) during the past few decades. Despite exhaustive efforts involving maximal surgical debulking, radiation, and chemotherapy, the median OS remains distressingly brief, typically less than 18 months [1,2]. In addition, more than 257 clinical trials target therapy against EGFR/VEGFR, tyrosine kinase receptors and other pathways for glioblastomas were launched in the past 20 years and the promising results are yielding less-than-optimistic results [3]. As for immune checkpoint inhibitors therapy, unlike other cancers, glioblastomas are not responding in the phase 3 trial [4].

The application of patient-derived xenograft (PDX) is a powerful model for pre-clinical study [5]. The PDX model recapitulates the tumor micro-environment and maintains the heterogeneity within the xenografted tumor. Accordingly, the PDX model provides an excellent platform for drug screening in glioblastoma, which is notorious for the intra-tumor heterogeneity with low mutation burdens [6].

In glioblastomas, the IDH1/2 (isocitrate dehydrogenase 1/2) mutation, ATRX (α-thalassemia/mental retardation syndrome x-linked mutation) mutation, 1p/19q co-deletion and TERT (telomerase reverse transcriptase) promoter mutation are commonly discussed in the molecular classification of gliomas [7]. In addition to the common markers, TP53, BRAF (v-raf murine sarcoma viral oncogene homolog B1), CDKN2A, PTEN, PIK3R1, H3F3A, EGFR, PDGFRA and MET, are all important detectable gene aberrations for glioblastomas [8].

The miscellaneous genetic aberrations are critical for personalized medicine. The next generation sequencing (NGS) may screen the gene aberrations in one test. The applications of NGS are widely exploited in the field of lung cancer, melanoma, breast cancer and colorectal cancers [9]. However, in the field of gliomas, NGS has just started in clinical applications, this is due to the increasing availability of target therapy drugs are available, such as Rindopepimut, Imatinib, Dasatinib, Voxtalisib, AMG232 and Ribociclib [10,11].

In this study, we conducted whole exome sequencing (WES) and effectively established the PDX model as a screening platform for validating the efficacy of targeted therapy drugs. Moreover, RNA sequencing analysis of treated animals’ expression profiles offers a thorough insight into the in vivo mechanisms of action for each drug.

Material and methods

Glioma PDX establishment

The PDX model was generated according to the protocol published by Hsu CL, 2018 [12]. In brief, the surgical resected tumor was obtained from a glioma patient with informed consent and approved by our IRB (Chang Gung Memorial Hospital, Linkou, IRB No. 201802110B0A3). Male NOD/SCID mice, 6-8 weeks old, came from BioLASCO (Taiwan) and were breed/maintained in specific pathogen-free room of Laboratory Animal Center, CGMH, Linkou (full accreditation of Association for Assessment and Accreditation of Laboratory Animal Care, International, in 2023).

After PDX tumors had been sub-implanted in NOD/SCID mice and xenograft had reached a volume of approximately 150 mm3, animals were randomized (6-8 mice with tumors on the flank per group) for candidate drug testing. The pain scale of the animals was assessed weekly (score: normal to severe, 0-4). Furthermore, the assessment also includes body weight, general appearance, general behavior, and response to the stimuli. For a score of 5-8, observation will be performed daily and environment enrichment will be supplied to the animal. For a score of 10-14, twice consecutive observation will be performed in 8 hours and administer Carprofen (5 mg/Kg, q12h). If there is no improvement, humane euthanasia will be performed. For a score of 15 to 20, humane euthanasia will be performed. Additionally, euthanasia will be carried out via cervical dislocation if the tumor burden exceeds 10% of the body weight, exceeds 2 grams, prevents the animal from moving, reaching food or water, causes severe ulceration or abscess.

The sample was cut into pieces and immersed into PBS (2% penicillin-streptomycin) and implanted subcutaneously in the flank of NOD/SCID mice (NOD.CB17-Prkdcscid/JNarl) under anesthesia (approved by IACUC2021071202, Chang Gung Memorial Hospital, Linkou). The xenograft was observed daily until reaching 0.5-1 cm in diameter. A serial passage was performed after tumor removed and cut into 2-3 mm diameter pieces. In this patient, the PDX, GBM-1, was considered stable until passage 5.

Whole exome sequencing (WES) analysis

The DNA was extracted both from formalin-fixed paraffin embedded tissue (FFPE) and PDX tumor with DNA extraction kit. The quantification and integrity of DNA was determined prior to sequencing. The WES was performed from both primary and PDX tumors. The fastq files of WES obtained from Macrogen were filtered and trimmed. The reads of PDX tumors sequencing from matched patients’ primary tumor was aligned to the human reference genome, hg19, separately using Burrows-Wheeler Aligner (BWA0.5.9). Variants from both primary and PDX tumors were identified using the Genome Analysis Toolkit (GATK) pipeline. GATK Unified Genotyper was used for identifying single nucleotide variants (SNVs), short inserts and deletions.

Target therapy drugs

All the target therapy drugs are available clinically. Bevacizumab (Roche, Switzerland), Regorafenib (Bayer, Germany), Palbociclib (Pfizer, USA) and Dabrafenib/Trametinib (Norvatis, Switzerland) are acquired from MedChemExpress (Monmouth Junction, NJ).

Transcriptome profiles post-treatment

With RNA-seq (RNA sequencing), we evaluated the transcription profiles of PDX tumors after selected drug treatment. In brief, total RNA was extracted from the PDX tumors samples with standard RNA extraction kit (TRIzol, Invitrogen, USA). After treated with DNase I, the mRNAs with PolyA tails were isolated with mRNA Magnetic kit and the mRNA libraries were constructed with NEBNext Ultra library prep kit according to the manufacture’s standard protocol (both from New England Biolabs, UK). The sequence analyzer platform was Illumina HiSeq X. The reads from Illumina HiSeq which passed the quality filters were included to further analysis. Since the PDX could be contaminated by rodent DNA, the reads mapped to mouse sequences were discarded. Gene differential expression profiles (DEG) were analyzed according to edgeR package (ver. 3.12.0).

Antibody

The primary antibodies used in Western blotting and IHC included IDH1 R132H (H09): Dianova, Germany (1:100), Rb: #9313, Cell Signaling Technology, USA (1:1000), p-JNK: #4668, Cell Signaling Technology, USA (1:1000), JNK: #9252, Cell Signaling Technology, USA (1:1000), pRb: #9307, Cell Signaling Technology, USA (1:1000), ERK: SC-1647, Santa Cruz Biotechnology, USA (1:500), p-ERK: SC-7383, Santa Cruz Biotechnology, USA (1:500), CCND1: GTX61306, GeneTex, USA (1:1000), CCNE2: 11935-AP, Proteintech (1:1000), CDK4: SC-23896, Santa Cruz Biotechnology, USA (1:1000), CDK6: SC-7961, Santa Cruz Biotechnology, USA (1:1000), β-actin: GT5512, GeneTex, USA (1:10000), BRAF V600E, VE1 (Ventana Medical Systems, automated Ventana Benchmark ULTRA platform).

Statistical analysis

The mutational analyses of WES and RNA-seq were performed with software (Variant Effect Predictor ver. 77 and edgeR). The tumor volume was presented and compared as mean and two-tailed ANOVA analysis. The overall survival was analyzed with Kaplan-Meier curves, and the immunohistochemistry H-score analysis was used for quantifying the expression of biomarkers in tissue samples (range 0-300). The p-values less than 0.05 is defined as statistically significant.

Results

Establishment of glioblastoma multiforme (GBM) PDX line

The PDX model, GBM-1, was developed from a 55-year-old male with a right temporal tumor, proven pathology of IDH1 wild type glioblastoma (WHO grade 4). The patient received standard concurrent chemo-radiotherapy and had a relapse 5 months later after first craniotomy. Small pieces of resected glioma freshly dissociated from resected tumor bed were embedded to the flank of immune-compromised mouse (NOD/SCID) and this study was based on the serial passages of PDX animals. The tumor in the PDX animals was examined with H&E staining for the diagnostic histopathology. As for the histopathology, both primary and PDX tumors demonstrated characteristic pattern of an epithelioid subtype of glioblastomas (eGBM), recognized in the 2021 WHO classification [13]. The immunohistochemistry of primary and PDX tumors also revealed identical wild type IDH1 expression and mutated BRAF V600E (Figures 1 and 2C).

Figure 1.

Figure 1

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Patient-derived xenograft (PDX), primary and animal, hematoxylin & eosin staining, immuno-histo-chemistry (IHC) staining of isocitrate dehydrogenase 1 (IDH1). A. Primay tumor hemotoxylin and eosin (H&E) staining, 100×, scale bar: 50 μm. Arrow head: hypercellularity large epithelioid cells with numerous giant glioma cells, mocrovascular proliferation and regions of necrosis. The glioma is IDH1 wild type. B. PDX (patient-derived xenograft) tumor H&E staining, 100×, scale bar: 50 μm. The PDX tumor exhibited similar epithelioid cells with clear cytoplasm and frequent mitosis, necrosis and also IDH1 wild type.

Figure 2.

Figure 2

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Whole-exome sequencing of primary and PDX, IHC of BRAF V600E and possible drugs according to database. A. Whole-exome sequencing (WES) reads of normal PBMC (peripheral blood mononuclear cell), primary tumors, PDX tumors and RNA-seq of PDX tumors. The BRAF V600E mutation is consistently present in the primary tumors and PDX tumors, but not in PBMC. B. Calculated by dividing singal intensities, b-allele frequency demonstrated imbalance of CDKN2A gene loci (chormosome 9p21) both in primary and PDX tumors. C. The immunohistochemistry (IHC) stainings of BRAF V600E mutation in primary and PDX tumors (100×). D. Utilizing OncoKB (https://www.oncokb.org), level 1 drugs against BRAF mutation and CDKN2A were selected.

WES analysis from primary and PDX tumor

WES analysis was conducted on genomic DNA from PBMC, primary tumors, and PDX tumors. The primary tumors demonstrated BRAF V600E mutation (exon 16, codon 600, chromosome 7, shown in Figure 2A), as well as subsequent PDX tumors. However, the BRAF V600E mutation is not in the PBMC. Accordingly, the BRAF V600E mutation might be somatic but not germline. In addition to the missense mutation, the CDKN2A loci (Chromosome 9p21) in both primary and PDX tumors demonstrated loss of heterozygosity (LOH), as shown in Figure 2B. The BRAF V600E mutation was confirmed with IHC staining both in primary and PDX tumors (Figure 2C).

Drug screening platform

We utilized OncoKB (https://www.oncokb.org) as our drug screening platform (Figure 2D). Specifically, Dabrafenib/Trametinib targeting the BRAFV600E mutation is recognized as a level 1 evidence treatment for all solid tumor (excluding colorectal cancer) with FDA approval [14]. However, only level 4 evidence based on a case-control study in solid tumors, has been reported with Palbociclib in the context of CDKN2A. Palbociclib was initially approved in breast cancer by FDA as a CDK4/6 inhibitor [15]. As control target therapy drugs, both Bevacizumab, a VEGF monoclonal antibody, and Regorafenib, a multi-targeted tyrosine kinase inhibitor, were used in this study.

Glioma PDX animal drug screening

Selected from drug screening platform, target therapy drugs were evaluated on the GBM PDX model to validate the WES analysis. As depicted in Figure 3A, Bevacizumab (5 mg/Kg/week) failed to impede PDX tumor progression, whereas Regorafenib (30 mg/Kg/day), effectively inhibited the PDX progression (P<0.01). Notably, CDK4/6 inhibitor Palbociclib (150 mg/Kg/day) and BRAF inhibitors, Dabrafenib/Trametinib (Dabrafenib 30 mg/Kg/day, Trametinib 0.15 mg/Kg/day, BRAFi) also effectively inhibited the growth of the PDX tumors compared to the control PDX animal (Figure 3C and 3D).

Figure 3.

Figure 3

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The PDX model was employed for drug screening, evaluating the efficacy of Bevacizumab, Regorafenib (for control) and Palbociclib, Dabrafenib/Trametinib combination therapy. A. Flank implantation was conducted with a smaple size from 4-8 animlas. B. Gross tumor size was assessed post-treatment with respective drugs. C. The estimated tumor size, compared to day 1 before treatment. D. Tumor weight was measured following drug administration, including Bevacizumab, Regorafenib, Palbociclib and BRAFi (BRAF inhibitors) combination.

Transcriptome analysis of post-drug treatment

To further demonstrate the mechanisms of targeting drugs, the transcriptome analysis of PDX tumors was performed. As shown in Figure 4A, the volcano plot diagram revealed differential expression in the Palbociclib-treated PDX tumors (Figure 4A). CHI3L1 and DDIT4L are upregulated while CDK1, PBK, MELK, ZWINT, ESCO2, CLSPN are downregulated by Palbociclib treatment. Further analysis with GO enrichment analysis (https://geneontology.org/docs/go-enrichment-analysis/), revealed pathways in association with Palbociclib treatment including: increased cell mitosis, DNA metabolic and cell cycle (Figure 4B).

Figure 4.

Figure 4

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RNA-seq of treated PDX tumors, volcano plot diagram, gene ontology enrichment analysis and heat-map illustrations. A. Differential expression genes (DEGs) observed in Palbociclib-treated PDX tumors compared to control samples. DEGs with log2 fold changes >1 and adjusted p values <0.05 were considered significant. B. Gene ontology enrichment analysis based on DEGs of Palbociclib-treated PDX tumors compared to control samples. C. Differential gene expressions (DEGs) of Dabrafenib/Trametinib-treated PDX tumors compared to control samples. D. Heatmap of depicting pathway activities of treated-PDX tumors. Each column stands for one PDX animal and each row is the Z score of pathway activities in the Reactome database.

As for BRAFi treated PDX tumors, TENM1, PDE1C, EFEMP1, etc. are up-regulated, while CD55, KRTAP2901, SPATA18 are down-regulated (volcano plot, Supplementary Figure 1). Pathway analysis demonstrated that BRAFi inhibited AMPA receptor trafficking and other microRNA synthesis/processing (Figure 4C). Figure 4D illustrated the heatmap of pathway analysis according various treatment. In this heatmap of pathway analysis, BRAFi and Palbociclib treated PDX tumors demonstrated distinctly different responses. Palbociclib treatment mainly inhibited cell cycle-related pathways, while the treatment of BRAF inhibitors additionally upregulated RNA polymerase-associated signal transduction, JNK/β-catenin phosphorylation cascade and AMPA receptor activation. Additionally, Bevacizumab and Regorafenib exhibited similar pathway profiling, which indicated similar receptors targeting effects.

Validation of targeted drug therapy using cell lines

To validate the efficacy of targeted drug therapy at protein level, we employ human glioma cell line, T98 and LN229, along with tumor lysates from PDX samples. Treatment of PDX tumors with Palbociclib resulted in decreased Rb phosphorylation, whereas this effect was not observed with Regorafenib treatment. Notably, the expression level of cell cycle-associated molecules, CDK4, CDK6 and CCNE2 remained unchanged in both Palbociclib and Regorafenib treatment group, except for a slightly decreased in CCND1 expression with Regorafenib treatment.

In T98 and LN229 glioma cell lines, Palbociclib treatment led to decreased Rb phosphorylation and down-regulation of ERK phosphorylation, suggesting heterogenicity between cell lines and PDX tumors (Figure 5A). In addition, variations were observed in the expressions of CCND1 and CCNE2 in T98 and LN229 cell lines. While Palbociclib treatment did not inhibit CCND1 in either cell line, Regorafenib treatment resulted in decreased CCND1 expression in both. Additionally, CCNE2 was down-regulated with both drug treatments.

Figure 5.

Figure 5

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Western blot analysis was conducted on PDX tumors and glioma cell lines, T98, LN229 and DBTRG-05MG. Tumor lysate of PDX were obtained from animals utilized in the previous drug experiments. A. T98 and LN229 glioma cell lines exhibited similar responses to Palbociclib (10 μM, 24 h) and Regorafenib (10 μM, 24 h), compared to control PDX tumors. Notably, alterations in the expression of CCNE2, Rb phosphorylation 4 and ERK phosphorylation. B. DBTRG-05MG, a glioma cell line known for its BRAF V600E mutation, treatment with Dabrafenib (10 μM, 24 h) and Trametinib (10 μM, 24 h) resulted in a higher JNK phosphorylation. C. The endogenous protein levels of p-ERK, ERK, CCND2, p-JNK JNK and β-actin were detected with specific antibodies by western blotting in the PDX tumor treated with Dabrafenib and Trametinib. The intensity of western blotting of CCND2 was normalized for β-actin, whereas p-ERK and p-JNK were normalized by endogenous total ERK and JNK expression levels; respectively, following normalized by expression level of β-actin.

To further validate the BRAF inhibition mechanisms, we utilized DBTRG-05MG human glioma cell lines harboring BRAF V600E mutation for comparison with our PDX tumors. Treatment with Dabrafenib and Trametinib resulted in JNK phosphorylation in both PDX tumor and DBTRG-05MG, consistent with the RNA seq results (Figure 5B). In addition, Dabrafenib and Trametinib inhibited ERK phosphorylation potentially via CCND2 pathway [16] (Figure 5C).

Cyclin D1 expression and survival analysis in clinical patients

As evident from BRAFi treatment, cyclin D was consistently down-regulated either in PDX tumors and cell line (DBTRG-05MG), as shown in Figure 5B. Despite the relative infrequency of BRAF mutation in glioblastoma, cyclin D remains a common pathway in glioma. To investigate further, we examined 58 glioblastoma patients. Among whom 20 exhibited a high expression of cyclin D1, as determined by H-score analysis (Figure 6A, 6B). There was a negative correlation between the expression of cyclin D1 and OS of glioma patients (Figure 6C). We observed the expression of cyclin D1, depicted a worst OS in these glioma patients (Figure 6C). Subsequent Kaplan-Meier analysis suggested that nuclear cyclin D1 expression may be indicative of a worst prognosis (Figure 6D).

Figure 6.

Figure 6

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Cyclin D1 IHC staining of 56 glioblastoma patients and survival analysis. A, B. Cyclin D1 IHC staining of 56 glioblastoma patients, 200×. The inset was enlarged to highlight the pure nuclear staining/cytoplasm staining. C. High Cyclin D1 staining patients exhibited poor overall survival based on H-score analysis. D. Subgroup analysis revealed that nuclear cyclin D1 staining is the most significant predictor of poor overall survival.

Discussion

For GBM, aside from surgical excision, radiation and chemotherapy, the standard target therapy is limited, with Bevacizumab being the primary option. While Bevacizumab has been shown to be effective in extending progression-free survival, its impact on OS remained limited [17]. However, leveraging a comprehensive WES analysis, precision cancer medicine holds promise in devising treatment strategies tailored to target personalized cancer-related gene expressions.

WES is pivotal for pinpointing actionable targets in cancers. Both primary and PDX tumors (GBM-1) exhibited a somatic mutation of BRAF V600E, a finding reinforced by RNAseq analysis of the PDX tumors (refer to Figure 2A). While BRAF V600E mutations are prevalent in colorectal cancer, melanoma, myeloma and non-small cell lung cancer, they are less common in glioma [18]. In a systemic review, Andrews et al. summarized findings from 182 publications, revealing a heterogenous distribution of BRAF V600E mutations across all subtypes of gliomas: 69% in epithelioid glioblastoma, 56% in pleomorphic xanthoastrocytoma, 8% in subependymal giant cell astrocytoma and only 1% in glioblastoma [19]. While BRAF V600E mutation is regarded as a favorable factor in pediatric glioma, its role in adult glioblastoma is still under investigation [20].

The alteration of CDKN2A exhibited significant variabilities across multiple cancers as well, spanning from 6 to 85% in lung cancer, skin cancer, melanomas, esophageal carcinomas, pancreas adenocarcinoma, and prostate cancer [21]. In glioblastoma, the LOH involving CDKN2A is detected in 9-43% (median 22%) of cases, encompassing both low- and high-grade gliomas. In addition, the LOH of CDKN2A emerges as a crucial adverse prognostic factor for both low-grade and high-grade gliomas [22].

Considering the WES findings mentioned above, it is crucial to explore the potential drugs targeting these specific alterations. DisGENET (https://www.disgenet.org) and DrugBank (https://go.drugbank.com) are both online database for selecting target drugs. Additionally, National Cancer Institute offers comprehensive information on targeted therapy options based on the cancer type, accessible through their approved drug list (https://www.cancer.gov/about-cancer/treatment/types/targeted-therapies/approved-drug-list). Memorial Sloan Kettering Cancer center offers a user-friendly drug screening online platform called OncoKB (https://www.oncokb.org), developed by Suehnholz SP, 2024 [23]. In this database, the targeted drug Dabrafenib/Trametinib for BRAF V600E mutation is classified as a level 1 combination therapy for melanoma, lung cancer and colorectal cancer. However, for CDKN2A LOH, only level 4 evidence is available, supporting the use of Palbociclib, a CDK4/6. Inhibitor, against solid tumors. Despite lacking of clinical evidence against glioblastomas, we utilized these 2 targets to assay the feasibility according to the WES results.

In this PDX drug screening, Bevacizumab and regorafenib served as control drugs, commonly used in treating solid tumors. Their efficacy in controlling PDX tumor progression varied, with Bevacizumab showing inadequate control (Figure 3A-D). However, Dabrafenib/Trametinib (BRAFi) and Palbociclib (CDK4/6 inhibitor) resulted in notable tumor volume reduction among PDX animals, affirming the precision of prediction from the WES analysis. Presently, the BRAFi is neither regarded as a standard glioma therapy, nor is Palbociclib. Although BRAF V600E mutant glioma cells respond to the BRAFi in animal study, human GBM clinical trials are pending [16]. In a recent clinical trial, Palbociclib was proved to be effective in the CDKN2A mRNA-expressing advanced sarcoma in a phase II study [24]. Nevertheless, a phase 2 clinical trial indicated Palbociclib’s inefficacy in recurrent glioblastoma patients with 22 RB1-positive patients, with a median OS of 15.4 weeks [25]. Notably, in these clinical studies, the use of target therapy did not based on a detailed WES analysis.

To elucidate the specific impact of drugs, examining the expression profiles of tumors post-treatment via RNA-seq detailed insights into tumor responsiveness. As evidenced in our findings, Palbociclib predominantly influences cell-cycle related pathways, along with significantly downregulation of specific genes (ESCO2, CLSPN, ZWINT, PBK, MELK, and CDK1) (Figure 4A, 4B). GO enrichment analysis further indicates that suppressed genes primary cluster with cell cycle pathways, including DNA metabolic and mitotic cell pathways (depicted in Figure 4B). Additionally, pathway analysis of BRAFi treated PDX tumors demonstrated inhibition of AMPA receptors and microRNA synthesis/process, with potential implications for glioma invasion promotion through AMPA receptors overexpression [26]. JNK phosphorylation/activation plays complex roles in cancer microenvironment, including glioma [27]. The results shed light on potential effects of BRAFi on glioblastomas warranting further validation through clinical data to support the utilization of WES analysis and PDX models in pre-target therapy screening.

Human glioma cell lines, T98 and LN229 were utilized to investigate cell cycle markers via Western blot analysis in Figure 5. Our results reveal that treatment with Palbociclib, a CDK4/6 inhibitor, effectively inhibits downstream Rb phosphorylation without affecting ERK activation, in both PDX tumors and T98 or LN229 glioma cells. Conversely, treatment with Regorafenib, a multiple kinase inhibitor, suppresses ERK phosphorylation without affecting the RB pathway, suggesting the potential synergistic inhibition of the cell cycle machinery when combining Palbociclib and Regorafenib (illustrated in Figure 5A). However, this combination is currently in the pre-clinical investigation stage [28].

On the other hand, the BRAFi combination of Dabrafenib and Trametinib has emerged as a standard treatment in solid tumors, supported by level I evidence for patients harboring BRAF V600E mutations [29]. The DBTRG-05MG glioma cell line, characterized by the BRAFV600E mutation, was employed to validate the targets of Dabrafenib and Trametinib [30]. In Dabrafenib- and Trametinib-treated PDX tumor and DBTRG-05MG, JNK phosphorylation is significantly upregulated. Besides microenvironment control, JNK activation is also associated with both apoptosis and methuosis (non-apoptotic cell death) of glioblastoma [16]. In addition, our results also demonstrated that Dabrafenib and Trametinib effectively inhibited ERK phosphorylation potentially via CCND2 pathway showcasing synergistic effects (Figure 5). Notably, this effect was observed in the cell line but not replicated in the PDX model [16].

The prevalence of the BRAF V600E mutation varies significantly between different subtypes of gliomas. While approximately two-thirds of the epithelioid glioblastomas exhibit this mutation, only a small percentage, ranging from 1-2% is reported in glioblastomas multiforme [19,31]. Consequently, the BRAF V600E mutation is not a definitive marker for prognosis prediction, due to its relatively low incidence in glioblastoma. Alternatively, our observations in both PDX tumors and DBTRG-05MG cell line indicate a significantly decreased in CCND2 following BRAFi treatment (as shown in Figure 5B). Leveraging cyclin D, a target of BRAFi, as a prognostic marker, we conducted survival analysis. As expected, elevated cyclin D1 expression correlated with poot survival in glioblastoma (depicted in Figure 6C). Consistent findings in the literature support the role of cyclin D1 results in cancer and glioma progression, whereas nuclear accumulation of cyclin D1 leads to dysregulated cell cycle progression [32-35].

In conclusion, the elucidated WES results from glioblastoma underscore the potential of druggable targets for more effective tumor inhibition and suppression, particularly within this immune-privilege region. Utilizing a patient-derived xenograft (PDX) model enhances not only drug screening for efficacy but also facilitates a deeper comprehension of tumor responsiveness to targeted therapies.

In the context of glioblastomas, specific druggable targets such as the BRAF V600E mutation and CDKN2A LOH stand out. As depicted in Figure 7, targeting cell surface receptors by acting on the cell surface receptors like EGFR and VEGFR can effectively impede cancer progression. However, glioma harboring BRAF mutation and CDKN2A LOH may continue to receive signals from receptors unaffected by Regorafenib and Bevacizumab. Precision therapies employing Dabrafenib/Trametinib and Palbociclib targeting BRAF and Cyclin D-CDK4/6 complex, presenting a promising approach for inhibiting glioma progression through targeted WES interventions.

Figure 7.

Figure 7

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A schematic illustration depicting various target therapy in the glioma cells. Regorafenib and Bevacizumab target receptors, while BRAFi and Palbociclib target ERK and Cyclin D, respectively. Inhibition of various receptors may lead to subsequent RAS/RAF inhibition, and downstream targets such as BRAF and CDK4/6 could serve as adjuvant targets.

Acknowledgements

We thank the staff at the Laboratory Animal Center, Chang Gung Memorial Hospital, Linkou, Taiwan, for the animal husbandry and care. We also thank Genomic Medicine Core Laboratory, Gung Memorial Hospital, Linkou and Biobank, Chang Gung Memorial Hospital, Linkou for providing surgical specimen and genomic analysis. This work was supported by grants from National Science and Technology Council and Chang Gung Memorial Hospital, Taiwan (NMRPG3P0431, NMRPG3J6141~2, CMRPG3N1361, CMRPG3M1011, NMRPG3M0401, CORPG3L0491~3, NSTC 112-2629-B-182A-001).

Disclosure of conflict of interest

None.

Supporting Information

ajcr0014-4747-f8.pdf (316.9KB, pdf)

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