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
H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health Organization. DMGs are inoperable and resistant to chemo/radio therapies. Median survival ranges from 8-11 months, with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and transcriptional reprogramming driven by global loss of negative transcriptional regulator H3K27 trimethylation (H3K27me3). Loss of H3K27me3 is an initiating event in gliomagenesis. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach identified that H3K27M but not isogenic controls relied on the amino acid methionine and the enzyme Methionine Adenosyltransferase 2A (MAT2A). MAT2A is a central regulator of one-carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl-donor for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation, genetic/pharmacological, would alter DMG viability by disrupting the methylome. The current MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M cells are sensitive to MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1) overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein levels, conferring a sensitivity by inhibiting residual MAT2A. Genetic/pharmacological aberrations to MAT2A resulted in reduced proliferation. Parallel H3K36me3 ChIP and RNA-sequencing identified loss of oncogenic and developmental transcriptional programs associated with MAT2A loss. In vivo syngeneic and patient-derived xenograft models with both inducible MAT2A knockdown or methionine restricted diets showed extended survival. These results suggest novel interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence that MAT2A, presents exploitable therapeutic vulnerabilities in histone mutant gliomas.
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.
Introduction: Leukemia is the most common cancer in children, accounting for approximately one third of all malignancies that occur in the pediatric age group. Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML) account for most leukemia diagnosed in this age group. While known markers for poor prognosis include higher age, higher white blood cell count at diagnosis and certain translocations, innovative approaches in tumor RNA sequencing (RNA-Seq) data analysis can discover novel prognostic factors that could be exploited for future therapeutic development in fusion-negative ALL and AML. Methods: To reveal gene expression signatures among fusion-negative leukemias, we used a novel unsupervised analysis model called Hydra. Hydra uses a Dirichlet process mixture model to detect multimodally expressed genes to use in characterizing clusters within cancer cohorts. This approach can detect subtle yet robust differences in gene expression without the reliance on reference normal RNA-Seq datasets. The Hydra model reveals clusters of the cancer cohort, and differences among these clusters can be investigated by finding enriched pathways via Gene Set Enrichment Analysis (GSEA). The cluster-specific enriched pathways can be used in conjunction with survival data to determine how certain pathways are associated with outcome. This analysis used publicly available data from the National Cancer Institute (NCI) Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database that was uniformly processed by the Treehouse Childhood Cancer Initiative. Results: First, 202 fusion-negative AML and fusion-negative B-cell precursor ALL samples were run through Hydra and five clusters were identified. These clusters had different enriched pathways, such as high mitochondrial activity, high cell proliferation, and high cell signaling. Though these are characteristics of all cancer cells, each cluster demonstrated that one pathway was most distinctive of those samples. Most clusters were differentiated by disease, however, one cluster with enriched heme metabolism and immunoglobulin pathways contained almost equal amounts of AML and ALL samples, suggesting that specific cohorts of AML and ALL patients had increased inflammatory response. Another cluster contained 72 AML samples and 4 ALL samples. The four ALL samples in this cluster showed lowered expression of CD19, a B-cell lineage immune marker, and elevated expression of CD14, a myeloid lineage immune marker. These ALL patients exhibited genomic characteristics of AML, which may suggest a more specialized treatment regimen. Discussion: Despite extensive characterization of pediatric high-risk leukemias using genomic approaches, there is ample opportunity to study RNA-Seq-derived gene expression profiles to help accurately diagnose and treat pediatric patients. Citation Format: Sneha S. Jariwala, Alfred Geoffrey Lyle, Jacob Pfeil, Lauren Sanders, Holly C. Beale, Ellen T. Kephart, Katrina Learned, Allison Cheney, Olena M. Vaske. Molecular classification of pediatric high-risk leukemias using expression profiles of multimodally expressed genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3033.
Synovial sarcoma (SS) is an aggressive soft-tissue malignancy, accounting for 10% of all soft-tissue sarcomas. These tumors can occur at any age but most often affect young adults and adolescents, developing deep in the distal extremities. The prognosis of SS tumors is poor, with a 5-year survival rate of 36-76%, a high rate of metastasis and few treatments. The purpose of this study was to identify novel overexpressed oncogenes that could serve as druggable targets for treating synovial sarcoma patients. We compared the RNA-Seq expression profiles of a cohort of 36 synovial sarcomas to our compendium of RNA-Seq expression data from 12,236 tumor samples (treehousegenomics.ucsc.edu) from pediatric and adult cancer patients. In comparing gene expression in the synovial sarcoma cohort samples against the compendium samples, gene expression outliers were defined as having expression above the gene-specific outlier threshold as defined by the Tukey's outlier method. Among the overexpression outliers, pathway enrichment analysis was used to identify common and druggable pathways, with implications for potential therapeutics for patients with SS. Our analysis identified the overexpression of members of the Sonic Hedgehog pathway in the majority of synovial sarcoma samples. For example, GLI1 expression exceeded the outlier threshold in 35 out of 36 samples. This pathway can be targeted by available small molecule inhibitors. Ongoing work focuses on evaluating the role of Sonic Hedgehog signaling in the pathogenesis of SS using pharmacological inhibition, CRISPRi studies in cell line models of the disease and nanopore sequencing. We currently have 4 patient-derived synovial sarcoma cell lines (HSSY-II, SYO-1, YAMATO, and ASKA) that we can grow in both adherent conditions and in 3D cell culture as sarcospheres. We detected the expression of the SYT-SSX fusion transcript in each of the cell lines by RT-PCR to confirm the cell lines maintained expression of the pathogenic fusion. This work has implications for using comparative tumor RNA-seq derived gene expression data for nominating novel druggable targets specific to synovial sarcoma tumors. Citation Format: Yvonne A. Vasquez, Jacob Pfeil, Letitia Mueller, Holly Beale, Alfred G. Lyle, Lauren Sanders, Katrina Learned, Ellen Kephart, Anouk van den Bout, Allison Cheney, Sahar Hosseinzadeh, Isabel Bjork, Sofie R. Salama, Olena Vaske. Identifying potential druggable targets for synovial sarcoma using comparative RNA-seq analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3035.
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