Abstracts

Rationale: BRAF mutations are major driver mutations in various types of brain tumors and associated with poor prognosis. Pediatric glioma with BRAF mutations is frequently associated with intractable epilepsy. Recently, our group showed that BRAF mutations arise from neural stem cells (NSCs) during the brain development and cause pediatric glioma with intractable epilepsy in human and mouse models (Koh et al, Nat Med 2018). However, current chemical BRAF inhibitors have limited effectiveness and cause significant systemic side effects because they poorly penetrate the blood-brain barrier. Here, we show that antisense oligonucleotide (ASO) therapy targeting BRAF induces long-lasting anti-tumor and anti-epiletic effects by alternating the fate of BRAF mutant tumor cells into normal oligodendrocytes.

Methods: We developed the mouse model mimicking Braf-mutant pediatric brain tumors with intractable epilepsy by introducing Braf p.V600E mutation into neural stem cells during embryonic brain development. Next, we screened to find the most effective ASO inhibiting mouse Braf RNA. ASO was delivered by single bolus intracerebroventricular (ICV) injection at 10 weeks after pre-monitoring of behavior seizure and tumor size. We observed the symptoms for 3 months to 1 year. Braf mutated cells in the tumor lesion of the model mouse were collected and analyzed their fate changes by single-cell RNA sequencing.

Results: The Braf p.V600E mutated mice show a gradual increase in tumor size and epileptic seizures and a decrease in survival rate over time. Single-cell analysis of tumor cells show dual characteristics of OPC and NSC with dysregulation of glutamatergic synaptic transmission and synaptic localization, potentially contribute to the epileptogenicity. While oral administration of existing BRAF inhibitors could not significantly reduce seizures and tumor size, single ICV injection of ASO, which sufficiently knock-down the Braf for at least 3 months without any significant toxicity, resulted in the robust decrease of seizures and tumors with enlarged dysmorphic neurons as well as the increase of survival rate. Surprisingly, the therapeutic effect maximally lasts up to 1 year. We found that the near-complete elimination of tumor cells and emergence of a normal oligodendrocyte cluster carrying a Braf mutation after the ASO treatment, suggesting the reprograming of tumor cells into normal oligodendrocytes.

Conclusions: ASO targeting Braf shows strong therapeutic effects on various aspects of BRAF-derived pediatric brain tumors, including the epileptic seizures, tumor size, survival rate as well as dysmorphic neurons. Braf ASO profoundly altered the fate by inducing differentiation of tumor cells into oligodendrocytes with normal function. Therefore, ASO therapy will be a promising alternative in BRAF-mutant brain tumors with intractable epilepsy.

Funding: National Research Foundation of Korea (NRF), Korea government, Ministry of Science and ICT. Grant Number: 2019R1A3B2066619 / 2020R1A6A3A13076897

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