LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity.
Enhancement of regulatory T cell (Treg cell) frequency and function is the goal of many therapeutic strategies aimed at treating type 1 diabetes (T1D). The interleukin-2 (IL-2) pathway, which has been strongly implicated in T1D susceptibility in both humans and mice, is a master regulator of Treg cell homeostasis and function. We investigated how IL-2 pathway defects impact Treg cells in T1D-susceptible nonobese diabetic (NOD) mice in comparison with protected C57BL/6 and NOD congenic mice. NOD Treg cells were reduced in frequency specifically in the lymph nodes and expressed lower levels of CD25 and CD39/CD73 immunosuppressive molecules. In the spleen and blood, Treg cell frequency was preserved through expansion of CD25(low), effector phenotype Treg cells. Reduced CD25 expression led to decreased IL-2 signaling in NOD Treg cells. In vivo, treatment with IL-2-anti-IL-2 antibody complexes led to effective upregulation of suppressive molecules on NOD Treg cells in the spleen and blood, but had reduced efficacy on lymph node Treg cells. In contrast, NOD CD8(+) and CD4(+) effector T cells were not impaired in their response to IL-2 therapy. We conclude that NOD Treg cells have an impaired responsiveness to IL-2 that reduces their ability to compete for a limited supply of IL-2.
Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.
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