The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers

Abstract

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis.

Keywords: Glioblastoma; Immunosuppression; OAS2; Proteomics.

Fig. 1:. pGBM and rGBM have distinct genomic and protein landscapes.

a, Schematic representation of sample collection and preparation, MS proteomics, target identification and functional analysis workflow. b, Distribution of protein quantitation measured as median intensity by the number of samples they are detected in. Bar plot on top represents the total counts of proteins quantified by the number of samples they are present in. Missing values were omitted when calculating the median.

Fig. 2:. Proteomic subtypes of GBM across disease states

a, Proteomic subtypes were identified in 40 matched pGBM-rGBM pairs. Clinical covariates indicating tumour classification, age at treatment (years), transcriptomic subtype, sex and pathway enrichment for proteomic subtypes are shown. b, pGBM and rGBM samples from the same patient classify as different proteomic (n = 40; replicate pGBM or rGBM samples were removed for simplicity) and c, transcriptomic subtypes (n = 22).

Fig. 3:. Differential expression analysis between pGBM-rGBM matched pairs introduces OAS2, an essential gene for rGBM with significant upregulation at the recurrent stage.

a, Volcano plot depicting differential abundance analysis between pGBM and rGBM in matched sample pairs. Proteins significantly enriched in rGBM patients are indicated in red, while proteins enriched in pGBM are indicated in blue (Q value < 0.1; log₂ fold change > 1). b, Gene set enrichment analysis (GSEA) showing the top significantly enriched pathways in pGBM vs. rGBM. The dot plot shows the normalized enrichment scores (NES) with the background color gradient representing the Q values. c, Immunohistochemical analysis of OAS2 on TMA consisting of both matched and unmatched pGBM-rGBM samples indicated a significantly higher level of OAS2 at the recurrent stage. (The representative image shows pGBM (PM35)-rGBM (RM35) matched samples) (Scale bar: 200 um, P value: *** 0.0002, **** < 0.0001). d, Overexpression of OAS2 in rGBM was confirmed by immunohistochemical analysis on a TMA construct consisting of an independent cohort of 20 pGBM-rGBM matched samples.

Fig. 4:. OAS2, an essential gene for rGBM, has significant upregulation at the recurrent stage.

a, Western blotting analysis on the protein lysate of GBM BTIC (pGBM-rGBM matched and unmatched samples) confirmed a two-fold increase in OAS2 expression. b, BT972, an OAS2 high expressing recurrent GBM BTIC line, was used for functional analysis. OAS2 was knocked out using CRISPR knockout gene editing. Following confirmation of gene knockout (construct A, B and C) by western blotting, the effect of OAS2 on self-renewal and proliferation capacity of OAS2 KO BT972 vs OAS2 WT BT972 (BT972 AAVS1) were measured using secondary sphere formation assay and PrestoBlue proliferation assay, respectively (P value: *** p < 0.001, **** p < 0.0001) (One way ANOVA). c-d, OAS2 KO BT972 and BT972 control (BT972 AAVS1) (100,000) were intracranially implanted into the right frontal lobe of NSG mice (n=6). The tumor size was tracked weekly using MRI imaging. e, IHC analysis of IBA1 on GBM xenografts from OAS2 KO or WT GBM cell engrafted mice revealed higher levels of IBA1 expression in OAS2 WT vs OAS2 KO engrafted brains. Scale bars represent 100 μm.