Kimia Ghannad-Zadeh

More than one third of patients with glioblastoma experience tumour progression during adjuvant therapy. In this study, we performed a high-throughput drug repurposing screen of FDA-approved agents capable of crossing the blood-brain barrier that to find agents to counteract acquired or inherent glioma cell resistance to temozolomide-associated cytotoxicity. We identified the cholesterol processing inhibitor, lomitapide, as a potential chemosensitizer in glioblastoma. In vitro treatment of temozolomide-resistant glioblastoma cells with lomitapide resulted in decreased intracellular ubiquinone levels and sensitized cells to temozolomide-induced ferroptosis. Concomitant treatment with lomitapide and temozolomide (TMZ) prolonged survival and delayed tumour recurrence in a mouse glioblastoma model, compared to treatment with TMZ alone. Our data identified lomitapide as a potential adjunct for treatment of temozolomide-resistant glioblastoma.

Glioblastoma accounts for nearly half of all primary malignant brain tumors in adults, and despite an aggressive standard of care, including excisional surgery and adjuvant chemoradiation, recurrence remains universal, with an overall median survival of 14.6 months. Recent work has revealed the importance of passenger mutations as critical mediators of metabolic adaptation in cancer progression. In our previous work, we identified a role for the epigenetic modifier ID-1 in temozolomide resistance in glioblastoma. Here, we show that ID-1-mediated glioblastoma tumourigenesis is accompanied by upregulation of one-carbon (1-C) mediated de novo purine synthesis. ID-1 knockout results in a significant reduction in the expression of 1-C metabolism and purine synthesis enzymes. Analysis of glioblastoma surgical specimens at initial presentation and recurrence reveals that 1-C purine synthesis metabolic enzymes are enriched in recurrent glioblastoma and that their expression correlates with a shorter time to tumor recurrence. Further, we show that the 1-C metabolic phenotype underlies proliferative capacity and temozolomide resistance in glioblastoma cells. Supplementation with exogenous purines restores proliferation in ID-1-deficient cells, while inhibition of purine synthesis with AICAR sensitizes temozolomide-resistant glioblastoma cells to temozolomide chemotherapy. Our data suggest that the metabolic phenotype observed in treatment-resistant glioma cells is a potential therapeutic target in glioblastoma.

Glioblastoma is the most common primary brain tumor in adults. While the introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid tumor recurrence remains universal. The transcriptional regulatory protein, inhibitor of DNA-binding-1 (ID1), is a key regulator of cell phenotype in cancer. We show that CRISPR-mediated knockout of ID1 in glioblastoma cells, breast adenocarcinoma cells, and melanoma cells dramatically reduced tumor progression in all three cancer systems through transcriptional downregulation of EGF, which resulted in decreased EGFR phosphorylation. Moreover, ID1-positive cells were enriched by chemotherapy and drove tumor recurrence in glioblastoma. Addition of the neuroleptic drug pimozide to inhibit ID1 expression enhanced the cytotoxic effects of temozolomide therapy on glioma cells and significantly prolonged time to tumor recurrence. Conclusively, these data suggest ID1 could be a promising therapeutic target in patients with glioblastoma.Significance:These findings show that the transcriptional regulator ID1 is critical for glioblastoma initiation and chemoresistance and that inhibition of ID1 enhances the effect of temozolomide, delays tumor recurrence, and prolongs survival.

(A) From left: Lane 1-5 U251 CTR. Lane -. Negative control. Lane +. Positive control. Lane D+. Diluted positive control. Lane 7-10 ID1-/-U251.1. Lane 11-14 ID1-/-U251.2. (B) PCR method.

(A) Western blot analysis for the identification of two clones lacking ID1 protein expression, consistent with successful gene knockout by CRISPR. (B) DNA sequence analysis of chosen clones. Red boxes mark the sgRNA target sequence and denote predicted cut-site. (C) Cell viability was measured using Alamar Blue in a time-dependent manner. (D) Cells were cultured for 48h followed by staining with propidium iodide for flow cytometric analysis. Bar chart shows quantification of the cell cycle distribution.

Measurement of ID1 half-life by cycloheximide chase assays. U251 cells were pretreated with or without TMZ for 3 days, followed by treatment with cycloheximide for the indicated times. The cell lysates were then analyzed by Western blots.

(A) Spearman correlation of ID1 vs EGFR gene expression in all glioblastoma patients. (B) Spearman correlation of ID1 vs EGFR gene expression in the Classical subtype. (C) Spearman correlation of ID1 gene expression and EGFR_pY1173 RPPA in the Classical subtype. (D) Spearman correlation of ID1 gene expression and EGFR_pY1173 RPPA in all GBM patients with available RPPA data.

(A) TMZ IC50 curves for U251 control and U251.TR cells. (B) U251 and U251.TR cells colony forming assay at different concentrations of TMZ. (C) Western blot analysis for EGFR and p-EGFR-1068 from lysates of U251and U251.TR cells.

(A) Flow chart of patient sample analysis (B) Representative images of IHC staining for ID1 expression in a TMA cohort. Images of weak and strong intensity staining for tumor tissues are shown. (C) Representative qPCR analysis of ID1 expresssion in U251 cells. Cells were treated with 100uM TMZ for indicated timepoints. *P>0.1

(A) Representative H&E images of control and ID1 KO mouse brains. (B) ID1 KO mice demonstrated greater accumulation of TMEM within the brain tissues compared to control animals. TMEM-positive cells were diffusely distributed throughout the brain, while in the control group. The boxes indicate the areas shown at higher magnification. (C) Quantification of positive TMEM cells in the brain relative to area. **P<0.005 Western blot analysis shows successful gene knockout clone for ID1 protein expression in (D) MDA-MB231 and (E) A375 cells.