Developmental Programs as Vulnerabilities in H3.3G34-Mutant High-Grade Glioma - Abstract/Project Summary Brain tumors are the leading cause of cancer-related deaths among children. Recent studies have identified new subtypes of brain tumors, including high-grade glioma with G34R/V mutations in the histone variant H3.3 (HGG-G34). This subtype primarily affects the cerebral hemispheres of adolescents and young adults. In addition to the histone mutation, HGG-G34 often exhibits mutations in both ATRX and TP53. However, the precise pathogenic mechanism of HGG-G34 remains poorly understood. Currently, there are no established therapeutic approaches tailored to this subtype, and patients continue to face a dismal prognosis. Therefore, there is an urgent clinical need to elucidate the cellular and molecular mechanisms underlying the development of HGG-G34 and to identify new therapeutic targets. To address these knowledge gaps, we have developed a new human embryonic stem cell-based model that allows us to introduce various combinations of mutations into a defined cell population. Using this mode, we have demonstrated that the three core mutations (H3.3G34R, ATRX, and TP53) specifically transform interneuronal progenitors of the ventral forebrain, shedding light on the cellular origin of HGG-G34. We also discovered that H3.3G34R and ATRX mutations cooperatively enhance the expression of DMRTA2, a forebrain-specific transcription factor, which is crucial for the high proliferation of HGG-G34 cells. Additionally, our data indicate that the majority of HGG-G34 cells exhibits characteristics of radial glial (RG) cell, a type of neural/glial progenitor cell that only exists in the developing brain. These findings collectively indicate a dysregulation in developmental programs in HGG-G34. However, the precise molecular mechanisms underlying tumorigenesis, including the transcriptional targets of DMRTA2, the exact role of ATRX mutation, and the maintenance of RG-like state, are still unclear. Additionally, the clinical relevance of RG-like cells in malignant brain tumors has not been fully studied. In this proposal, we aim to unravel the transcriptional targets of DMRTA2 by employing ChIP-seq and assess their involvement in tumorigenesis by loss-of-function experiments (Aim 1). Additionally, we will investigate the interplay between H3.3G34R and ATRX mutations by examining the impact of ATRX mutation on the expression, function, and distribution of DMRTA2 (Aim 2). Furthermore, we will examine the maintenance mechanisms and clinical relevance of RG-like cells through mouse xenograft models and analysis of clinical samples obtained from human patients (Aim 3). Our innovative HGG-G34 model, scientific expertise, and strong local collaborations uniquely position us to achieve these aims. The outcomes of this project are expected to provide novel insights into the tumor biology of HGG-G34 and serve as a foundation for our long-term goal of developing personalized treatment and diagnostic approaches for patients suffering from this devastating disease.
