NF2 Loss-of-Function and Hypoxia Drive Radiation Resistance in Grade 2 Meningiomas

Abstract

ABSTRACTBackgroundWorld Health Organization Grade 2 meningiomas (G2Ms) exhibit an aggressive natural history characterized by recurrence and therapy resistance. G2Ms with histopathological necrosis have been associated with worse local control (LC) following radiation therapy, but drivers and biomarkers of radiation resistance in these G2Ms remain unknown.MethodsWe performed genetic sequencing and histopathological analysis of 113 G2Ms and investigated the role of intratumoral hypoxia as well as genes of interest through knockdown and clonogenic survival following ionizing radiation. Lastly, we performed transcriptional profiling of ourin vitromodel and 18 G2M tumors using RNA sequencing.ResultsNF2loss-of-function (LOF) mutations were associated with necrosis in G2Ms (p=0.0127). Tumors withNF2mutation and necrosis had worse post-radiation LC compared toNF2wildtype tumors without necrosis (p=0.035). Under hypoxic conditions,NF2knockdown increased radiation resistancein vitro(p<0.001). Bulk RNA sequencing of ourin vitromodel revealedNF2- and hypoxia-specific changes and a 50-gene set signature specific to radiation resistant,NF2knockdown and hypoxic cells, which could distinguishNF2mutant and necrotic patient G2Ms by unsupervised clustering. Gene set enrichment analysis of patient tumor andin vitrodata revealed downregulation of apoptosis and upregulation of proliferation inNF2-deficient and hypoxic cells, which we validated with functional assays.ConclusionsNF2LOF in the setting of hypoxia confers radiation resistance through transcriptional programs that reduce apoptosis and promote proliferation. These pathways may identify tumors resistant to radiation and represent therapeutic targets that in the future could improve LC in patients with radiation resistant G2Ms.KEY POINTS1. Spontaneous necrosis withNF2mutations is associated with radio-resistance in WHO G2Ms.2.NF2knockdown in the setting of hypoxia confers radio-resistance to meningioma cellsin vitroand is driven by increased cell proliferation and decreased apoptosis.IMPORTANCE OF THE STUDYWorld Health Organization Grade 2 meningiomas (G2M) are often treated with surgical resection followed by radiation, especially in the case of recurrence. However, the mechanisms underlying radiation resistance in G2Ms remain to be identified, and moreover, we lack biomarkers to distinguish G2Ms that will respond to radiotherapy from those that are refractory. In this study we perform histological and molecular analysis of a large cohort of G2Ms to identify predictors of radiation resistance. Using these data and anin vitromodel of radiation therapy, we demonstrate that radiation resistance in G2Ms is likely driven by the combination ofNF2gene mutations and the hypoxia that accompanies tumor necrosis. Patients whose tumors bear these two features may therefore benefit from alternative treatments that target specific pathways implicated in radiation resistance.