AB002. DNA methylation-regulated genes contribute to temozolomide (TMZ) resistance by scaffolding paraspeckle proteins

Background: Temozolomide (TMZ) resistance in glioblastoma (GBM) remains a challenge in clinical treatment and the mechanism is largely unknown. Emerging evidence shows that epigenetic modifications including DNA methylation and non-coding RNA were involved in diverse biological processes, including therapeutic resistance. However, the underlying mechanisms by which DNA methylation-mediated non-coding RNA regulates TMZ resistance remain poorly characterized.

Methods: RNA microarray and DNA methylation chips of TMZ-resistant and parental GBM cells were performed for the gain of unreported long non-coding RNA HSD52. Quantitative reverse transcription polymerase chain reaction (PCR) and fluorescence in situ hybridization assays were used to detect HSD52 levels in GBM cells and tissues. The investigation into HSD52’s impact on TMZ resistance was conducted utilizing both in vitro assays and intracranial xenograft mouse models. The mechanism of HSD52 expression and its relationships with paraspeckle proteins, non-POU domain-containing octamer-binding protein (NONO) and splicing factor proline/glutamine rich (SFPQ), as well as alpha-thalassemia mental retardation X-linked (ATRX) mRNA were determined by pyrosequencing assay, chromatin immunoprecipitation, chromatin isolation by RNA purification, RNA immunoprecipitation, RNA pulldown, immunofluorescence, and western blot assays.

Results: HSD52 was highly expressed in high-grade glioma and TMZ-resistant GBM cells. Phosphorylated p38 mitogen-activated protein kinase (p38 MAPK)/ubiquitin specific peptidase 7 (USP7) axis mediates H3 ubiquitination, impairs the interaction between H3K23ub and DNA methyltransferase 1 (DNMT1) and the recruitment of DNMT1 at the HSD52 promoter to attenuate DNA methylation, which makes the transcription factor 12 (TCF12) more accessible to the promoter region to regulate HSD52 expression. Further analysis showed that HSD52 can serve as a scaffold to promote the interaction between NONO and SFPQ, and then increase the paraspeckle assembly and activate the paraspeckle/ataxia telangiectasia mutated (ATM) kinase pathway in GBM cells. In addition, HSD52 forms an RNA-RNA duplex with ATRX mRNA, and facilitates the association of heteromer of SFPQ and NONO with RNA duplex, thus leading to the increase of ATRX mRNA stability and level. In clinical patients, HSD52 is required for TMZ resistance and GBM recurrence.

Conclusions: Our results reveal that HSD52 in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.

Keywords: Glioblastoma (GBM); HSD52; DNA methylation; temozolomide resistance (TMZ resistance); paraspeckle protein