Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30–50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.
Background Diffuse midline gliomas with histone H3 K27M (H3K27M) mutations occur in early childhood and are marked by an invasive phenotype and global decrease in H3K27me3, an epigenetic mark that regulates differentiation and development. H3K27M mutation timing and effect on early embryonic brain development are not fully characterized. Results We analyzed multiple publicly available RNA sequencing datasets to identify differentially expressed genes between H3K27M and non-K27M pediatric gliomas. We found that genes involved in the epithelial-mesenchymal transition (EMT) were significantly overrepresented among differentially expressed genes. Overall, the expression of pre-EMT genes was increased in the H3K27M tumors as compared to non-K27M tumors, while the expression of post-EMT genes was decreased. We hypothesized that H3K27M may contribute to gliomagenesis by stalling an EMT required for early brain development, and evaluated this hypothesis by using another publicly available dataset of single-cell and bulk RNA sequencing data from developing cerebral organoids. This analysis revealed similarities between H3K27M tumors and pre-EMT normal brain cells. Finally, a previously published single-cell RNA sequencing dataset of H3K27M and non-K27M gliomas revealed subgroups of cells at different stages of EMT. In particular, H3.1K27M tumors resemble a later EMT stage compared to H3.3K27M tumors. Conclusions Our data analyses indicate that this mutation may be associated with a differentiation stall evident from the failure to proceed through the EMT-like developmental processes, and that H3K27M cells preferentially exist in a pre-EMT cell phenotype. This study demonstrates how novel biological insights could be derived from combined analysis of several previously published datasets, highlighting the importance of making genomic data available to the community in a timely manner.
In the version of this article initially published, the Acknowledgements statement should have included the following: "S.C.M. is
Pediatric diffuse midline gliomas are lethal cancers, the majority of which harbor the H3 p.K27M mutations. Although it has potential implications on the treatment of diffuse midline glioma as a disease driver; the timing, cell type of origin, and effect of the H3 p.K27M mutation on early embryonic brain development is poorly understood. The purpose of our study is to elucidate the molecular mechanisms by which the histone H3 p.K27M mutation drives tumorigenesis of pediatric diffuse midline gliomas using the analysis of genomic datasets. Here, we performed differential RNA sequencing gene expression analysis of a cohort of H3K27M and H3 wild type (WT) pediatric diffuse midline gliomas, revealing that genes in the epithelial-mesenchymal transition (EMT) pathway were significantly differentially expressed between the mutant and WT tumors. Several EMTs are required for normal brain development. Overall, pre-EMT genes, including the master regulator of EMT SNAI1, were overexpressed in H3K27M tumors compared to the WT tumors, while post-EMT genes were underexpressed. We hypothesized that the H3 p.K27M mutation may lead to gliomagenesis by inducing a stall in the EMT in early brain development. To test this hypothesis, we examined published single-cell RNA sequencing data from pediatric diffuse midline gliomas alongside similar data from organoid models of neural development, collected from multiple developmental timepoints. This analysis revealed transcriptional similarities between H3K27M and pre-EMT neural stem cells. Currently, we are investigating the expression of EMT markers in H3K27M and WT pediatric glioma primary cell lines, using Western blotting, RT-PCR, and CRISPRi screening. In conclusion, we observed aberrant expression of genes involved in EMT in H3K27M pediatric gliomas. Our observations are consistent with a model in which the p.H3K27M mutation is associated with a pre-EMT cell phenotype, potentially due to an arrest in the EMT pathway or de-differentiation of mature astrocytes. Citation Format: ALLISON R. CHENEY, Lauren M. Sanders, Lucas Seninge, Holly C. Beale, Ellen Towle Kephart, Jacob Pfeil, Katrina Learned, A. Geoffrey Lyle, Isabel Bjork, David Haussler, Sofie R. Salama, Olena M. Vaske. H3K27M gliomas are characterized by a stall in the epithelial-mesenchymal transition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6154.
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