IDH1-R132H acts as a tumor suppressor in glioma via epigenetic up-regulation of the DNA damage response

Abstract

Glioma patients whose tumors carry a mutation in Isocitrate Dehydrogenase 1 (IDH1R132H) are younger at diagnosis and live longer. IDH1 mutations co-occur with other molecular lesions, such as 1p/19q co-deletion, inactivating mutations in the tumor suppressor protein 53 (TP53) gene, and loss of function mutations in alpha thalassemia/mental retardation syndrome X-linked gene (ATRX). All adult low-grade gliomas (LGGs) harboring ATRX loss also express the IDH1R132H mutation. The current molecular classification of LGGs is based, in part, on the distribution of these mutations. We modelled the molecular glioma subtype which harbors IDH1R132H, and TP53 and ATRX inactivating mutations. Previously, we established that ATRX deficiency, in the context of wt-IDH1, induces genomic instability, impairs non homologous end joining DNA repair, and increases sensitivity to DNA damaging therapies. In this study, we investigated the function of IDH1R132H in the context of TP53 and ATRX loss. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 gene inactivation: (i) increases median survival (MS) in the absence of any treatment, (ii) enhances DNA damage response (DDR) via epigenetic upregulation of the Ataxia-telangiectasia mutated (ATM) signaling pathway, and (iii) elicits tumor radioresistance. Accordingly, pharmacological inhibition of ATM or checkpoint kinase 1 and 2 (CHK1/2), essential kinases in the DDR, restored the tumors’ radiosensitivity. Translation of these findings to IDH1132H glioma patients harboring TP53 and ATRX loss, could significantly improve the therapeutic efficacy of radiotherapy, and consequently patient survival.

Therapeutic efficacy of an immune stimulatory gene therapy strategy in a mouse model of high grade brainstem glioma

“…ACVR1 is frequently mutated to ACVR1 G328V in DIPG (4-7). The SB system efficiently and reproducibly integrates plasmid DNA into the neural progenitor cells' host chromosomal DNA of neonatal mice, allowing for the functional assessment of the role of candidate DIPG genes in promoting tumor progression (19)(20)(21)(22). Mutations in ACVR1 confer ligand independent activation of ACVR1, which is a mediator of the BMP signaling pathway (4,5,35,36).…”

“…The plasmids utilized were as follows: (i) SB Transposase and luciferase (pT2C-LucPGK-SB100X, henceforth referred to as SB/Luc) (ii) a short-hairpin against p53 (pT2-shp53-GFP4, henceforth referred to as shp53) or shp53-NO-GFP (iii) a constitutively active mutant of NRAS (pT2CAG-NRASV12, henceforth referred to as NRAS) with or without (4) mutant ACVR1 G328V (pkt-ACVR1-G328V-IRES-Katushka; henceforth referred to as mACVR1) (19,20,22). To create the mACVR1 plasmid we cloned pCMV5-ALK2-WT into pKT2-IRES-Katushka by blunt cloning.…”

“…Mouse neurospheres (NS) were generated from tumors that were developed using the SB system by injection of the following plasmid combinations (1) (i) shp53 and NRAS, or (ii) shp53, NRAS, and ACVR1m into the lateral ventricle (1.5 mm AP, 0.7 mm lateral, and 1.5 mm deep from the λsuture) following previously described protocols and detailed in the Supplementary Methods (20)(21)(22)25). Western blot analysis and inhibitor treatment with LDN-214117 is also detailed in the Supplementary Methods and antibody information is available in Supplementary Table S1.…”

“…To test the efficacy of this immune-mediated gene therapy approach in syngeneic mouse brainstem glioma models, we developed a genetically engineered mouse model encoding mutant ACVR1 using the Sleeping Beauty (SB) transposase system (19)(20)(21)(22). SB is a transposase that is able to recognize inverted repeats/direct repeats (IR/DR) sites on DNA transposons and carry out a cut and paste reaction to integrate transposon DNA into a host genome(23).…”

“…SB is a transposase that is able to recognize inverted repeats/direct repeats (IR/DR) sites on DNA transposons and carry out a cut and paste reaction to integrate transposon DNA into a host genome(23). The SB system can be utilized to generate endogenous tumors that resemble gliomas through delivery of DNA transposons that encode oncogenes and tumor suppressors (19)(20)(21)(22)24). Transcriptome analysis of ACVR1 mutant neurospheres identified an increase in the TGF-β signaling pathway and signaling pathways regulating the pluripotency of stem cells, and a decrease in the focal adhesion pathway.…”