Transposon mutagenesis identifies chromatin modifiers cooperating with Ras in thyroid tumorigenesis and detects ATXN7 as a cancer gene

Abstract

Oncogenic mutations are present in 15-30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with Hras in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway., a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with or mutations. Expression of mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to as a previously unrecognized cancer gene.

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

“…Mutagenic SB transposons were developed to modify expression of endogenous genes in specific ways depending on their relative position and orientation, as previously described [8]. This feature, combined with our experience analyzing insertion site data from 15 SB-induced models of cancer [2, 3, 20, 29, 30, 33, 34, 37, 40, 41, 45–47, 52, 55], led us to develop an algorithm to predict the functional impact that recurrent transposon insertion has on a given gene. This approach evaluates clustered transposon insertions within each candidate locus to determine if there is a bias for insertion in the same orientation as the gene, indicative of an over-expression mechanism, or if the transposons are randomly orientated, suggesting a gene disruption mechanism (see Additional file 2: Supplemental Methods).…”

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

“…Mutagenic SB transposons were developed to modify expression of endogenous genes in specific ways depending on their relative position and orientation, as previously described (Dupuy 2010). This feature, combined with our experience analyzing insertion site data from 15 SB-induced models of cancer Mann et al 2012;Wu et al 2012;Keng et al 2013;Quintana et al 2013;Rogers et al 2013;Zanesi et al 2013;Bard-Chapeau et al 2014;Mann et al 2015;Takeda et al 2015;Montero-Conde et al 2017;Morris et al 2017;Suarez-Cabrera et al 2017;Tschida et al 2017;Riordan et al 2018), led us to develop an algorithm to predict the functional impact that recurrent transposon insertion has on a given gene. This approach evaluates clustered transposon insertions within each candidate locus to determine if there is a bias for insertion in the same orientation as the gene, indicative of an over-expression mechanism, or if the transposons are randomly orientated, suggesting a gene disruption mechanism (see Supplemental Methods).…”

Deleterious Mutations in the TPO Gene Associated with Familial Thyroid Follicular Cell Carcinoma in Dutch German Longhaired Pointers

“…RAS somatic mutations are the second most common type of mutations found in fine needle aspiration of thyroid nodules [ 19 ]. Somatic RAS mutations are present in 15–30% of TC [ 20 ]. In addition, PAX8/PPARG was also identified to be an oncogenic driver for TC [ 19 , 21 ].…”

“…20 (p-value 0.09) Note: A represents alternative allele, and R represents reference allele. Numbers in the cell are the counts of affected and unaffected GLPs.…”