Dataset Information

Drosophila S2R+ / TSC1 / TSC2 mutant phosphoproteomics

ABSTRACT: The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of substantial clinical relevance to TSC and sporadic cancers. We developed a CRISPR-based method to generate homogenous mutant Drosophila cell lines. By combining TSC1 and TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Knockdown of three candidate genes (mRNA-cap, Pitslre and CycT; orthologs of RNGTT, CDK11 and CCNT1 in humans) reduced the population growth rate of both Drosophila TSC1 and TSC2 mutant cells but not that of wild-type cells. Moreover, knockdown of all three genes displayed similar selective effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross species screening strategy to identify potential drug targets.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Cell Culture, Cell Line Cell

DISEASE(S): Tuberous Sclerosis

SUBMITTER: Benjamin Housden

LAB HEAD: Norbert Perrimon

PROVIDER: PXD002670 | Pride | 2018-10-24

REPOSITORIES: Pride

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Publications

Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi.

Housden Benjamin E BE Valvezan Alexander J AJ Kelley Colleen C Sopko Richelle R Hu Yanhui Y Roesel Charles C Lin Shuailiang S Buckner Michael M Tao Rong R Yilmazel Bahar B Mohr Stephanie E SE Manning Brendan D BD Perrimon Norbert N

Science signaling 20150908 393

The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevan ...[more]

PMID: 26350902

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Project description:Tuberous Sclerosis Complex (TSC), an autosomal dominant condition, is engendered by heterozygous mutations in either TSC1 or TSC2 genes, manifesting in systemic growth of benign tumors. In addition to brain lesions, neurologic sequelae represent the greatest morbidity in TSC patients. Investigations utilizing TSC1/2-knockout animal or human stem cell models suggest that TSC deficiency-causing hyper-activation of mTOR signaling might precipitate anomalous neurodevelopmental processes. However, how the pathogenic variants of TSC1/2 genes identified in TSC patients affect the trajectory of human brain development and how they contribute to the neurological manifestations in TSC remain largely unexplored. Here, we employed 3-dimensional cortical organoids derived from induced pluripotent stem cells (iPSCs) from TSC patients harboring TSC2 mutations, alongside organoids from age- and sex-matched healthy individuals as controls. Through comprehensively longitudinal molecular and cellular analysis of TSC organoids, including transcriptomics and single cell transcriptomics, we found that TSC2 pathogenic variants led to dysregulated neurogenesis, synaptogenesis, and gliogenesis, particularly for reactive astrogliosis. The altered developmental trajectory of TSC organoids significantly resembles the molecular signatures of neuropsychiatric disorders, including autism spectrum disorders, epilepsy, and intellectual disability. Through cell-cell communication analysis at the single cell level, we identified that TSC2 pathogenic variants disrupted the cell-cell communications, in particular, neuron-astrocyte interactions within the NLGN-NRXN signaling network. Furthermore, cellular and electrophysiological assessments of TSC cortical organoids, along with proteomics and phosphoproteomics analyses of synaptosomes, revealed that pathogenic TSC2 variants precipitate perturbations in mitochondrial translational integrity, neurofilament formation, synaptic transmission, and neuronal network activity. Intriguingly, the increased neu

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