Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence

Abstract

Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A. We developed an innovative approach that integrates singlecell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16 INK4a , p21 CIP1 , and p15 INK4b , preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.

PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells

“…To generate IMR90 cells expressing ER:RAS G12V , miR30-based shRNAs (pGIPz) or κB-GFP (pTR-mCMV-copGFP), and MEFs expressing miRE-based shRNAs, retroviral and lentiviral infections were carried out as previously described ( Aarts et al., 2017 ). To generate IMR90 ER:RAS G12V cells expressing two miR30-based shRNAs simultaneously, viruses were mixed at a 1:1 ratio.…”

Single-cell analyses of aging, inflammation and senescence

“…setup: 10x Genomics Chromium; Illumina HiSeq 4000 Remarks: Investigation of senescence (beta-galactosidase, reduced proliferation, 6 specific CpGs, HMGB2 and CTCF). mTOR shRNA Atlas ( Aarts et al, 2017 ) Tissues: primary human fibroblasts (IMR90) expressing reprogramming factors Animals/Cells: in-vitro shRNA screen Heterogeneity: Not investigated Exp. setup: Illumina Nextera XT; Illumina HiSeq 2500 Remarks: Investigation of senescence (p16, p21, p19, SASP) & reprogramming and of the role of mTOR.…”

Galactose‐modified duocarmycin prodrugs as senolytics

“…To generate IMR90 ER:RAS‐expressing shRNAs against GLB1 , lentiviral infections were carried out as described before (Aarts et al, 2017). Briefly, HEK293T cells were transfected with the lentiviral and packaging vectors using PEI (PEI 2500, Polysciences).…”