The evolution of alternative splicing in glioblastoma under therapy.

Alternative splicing is a rich source of tumor-specific neoantigen targets for immunotherapy. This holds promise for glioblastomas (GBMs), the most common primary tumors of the adult brain, which are resistant to standard-of-care therapy. Although most clinical trials enroll patients at recurrence, most preclinical studies have been done with specimens from primary disease. There are limited expression data from GBMs at recurrence and surprisingly little is known about the evolution of splicing patterns under therapy. We profiled 29 primary-recurrent paired human GBM specimens via RNA sequencing. We describe the landscape of alternative splicing in GBM at recurrence and contrast that to primary and non-malignant brain-tissue specimens. By screening single-cell atlases, we identify cell-type specific splicing patterns and novel splicing events in cell-surface proteins that are suitable targets for engineered T-cell therapies. We identify recurrent-specific isoforms of mitogen-activated kinase pathway genes that enhance invasiveness and are preferentially expressed by stem-like cells. These studies shed light on gene expression in recurrent GBM and identify novel targets for therapeutic development. We combined de novo RNA sequencing (RNA-seq) of paired GBM clinical specimens with public RNA-seq of non-malignant adult and fetal brain tissues to construct an integrated model of AS during GBM malignant progression. RNA-seq of non-malignant human brain tissues were obtained from the GTEx portal (https://www.gtexportal.org/home/datasets); see Supplementary file thirdparty-reanalysis_of_GTExSamples.txt. We screened the AS events we detected in suitable single-cell RNA-seq (scRNA-seq) data from human GBM specimens to determine their cell-type specificity. We identified an exon-retention event up-regulated in recurrent GBM in mitogen-activated protein 4 kinase 4 (MAP4K4). *** Raw data not available due to privacy concerns.