Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence

Belk, Julia A.; Yao, Winnie; Ly, Nghi; Freitas, Katherine A.; Chen, Yanting; Shi, Quanming; Valencia, Alfredo M.; Shifrut, Eric; Kale, Nupura; Yost, Kathryn E.; Duffy, Connor V.; Dániel, Bence; Hwee, Madeline A.; Miao, Zhuang; Ashworth, Alan; Mackall, Crystal L.; Marson, Alexander; Carnevale, Julia; Vardhana, Santosh; Satpathy, Ansuman T.

doi:10.1016/j.ccell.2022.06.001

Cited by 186 publications

(132 citation statements)

References 79 publications

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“…Single cell CRISPR screens are contingent on successfully and accurately associating a cell’s transcriptome with the gRNA it expresses. Similar to what we observed here however, in every study published thus far, no gRNA is detected in a subset of cells (6–8,11,12). This is typically attributed to a lack of complete selection for gRNA-transformed cells, leading to inclusion of non-transformed cells in the analysis, or to an insufficiently high gRNA expression or an insufficient sequencing depth, implying that a gRNA is expressed but not detected.…”

Section: Resultssupporting

confidence: 92%

GiRAFR improves gRNA detection and annotation in single cell CRISPR screens

Minsel

Galle

et al. 2022

Preprint

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Novel single cell RNA-seq analysis combined with CRISPR screens enables the high- throughput characterization of transcriptional changes caused by genetic perturbations. Dedicated software to annotate CRISPR guide RNA (gRNA) libraries and associate them with single cell transcriptomes are however lacking. Here, we generated a CRISPR droplet sequencing dataset. We demonstrate that the current default tool fails to detect mutant gRNAs. We therefore developed GiRAFR, a pysam-based software tool to characterize intact and mutant gRNAs. We show that mutant gRNAs are dysfunctional, and failure to detect and annotate them leads to an inflated estimate of the number of untransformed cells as well as an underestimated multiplet frequency. These findings are mirrored in publicly available datasets, where we find that up to 34 % of cells are transduced with a mutant gRNA. Applying GiRAFR hence stands to improve the annotation and quality of single cell CRISPR screens.

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Section: Resultssupporting

confidence: 92%

GiRAFR improves gRNA detection and annotation in single cell CRISPR screens

Minsel

Galle

et al. 2022

Preprint

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“…Previous research has revealed that ARID1A mutation or ARID1A expression loss contributes to the dMMR subtype, higher PD-L1 expression and remodeling of the TIME, which leads to enhanced sensitivity to ICIs in ovarian cancer [ 31 ] and gastric cancer [ 32 ]. In addition, it was reported that ARID1A depletion limits acquisition of exhaustion-associated chromatin accessibility and leads to improved anticancer immunity [ 33 ]. The results of the current study offer clear support for the role of low ARID1A expression as a positive biomarker for ICI treatment in LUAD patients with or without EGFR mutation.…”

Section: Discussionmentioning

confidence: 99%

“…Two mechanisms, including inhibition of autophagy and promotion of type I IFN production, were detected and confirmed in ARID1A-KD EGFR -mutant LUAD cells, and we sought to explore whether there is a connection between these two mechanisms in EGFR -mutant LUAD cells. A previous review suggested that production of type I IFN is stimulated in autophagy-deficient cancer cells [ 33 ]. For EGFR -mutant LUAD cells, we found that type I IFN production was inhibited by autophagy and that knockdown of ARID1A-KD could reverse production of type I IFN via inhibition of autophagy.…”

Section: Discussionmentioning

confidence: 99%

ARID1A deficiency reverses the response to anti-PD(L)1 therapy in EGFR-mutant lung adenocarcinoma by enhancing autophagy-inhibited type I interferon production

et al. 2022

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Introduction EGFR mutations in non-small cell lung cancer (NSCLC) are associated with a poor response to immune checkpoint inhibitors (ICIs), and only 20% of NSCLC patients harboring EGFR mutations benefit from immunotherapy. Novel biomarkers or therapeutics are needed to predict NSCLC prognosis and enhance the efficacy of ICIs in NSCLC patients harboring EGFR mutations, especially lung adenocarcinoma (LUAD) patients, who account for approximately 40–50% of all NSCLC cases. Methods An ARID1A-knockdown (ARID1A-KD) EGFR-mutant LUAD cell line was constructed using lentivirus. RNA-seq and mass spectrometry were performed. Western blotting and IHC were used for protein expression evaluation. Effects of 3-MA and rapamycin on cells were explored. Immunofluorescence assays were used for immune cell infiltration examination. Results ARID1A expression was negatively associated with immune cell infiltration and immune scores for ICIs in LUAD with EGFR mutations. In vitro experiments suggested that ARID1A-KD activates the EGFR/PI3K/Akt/mTOR pathway and inhibits autophagy, which attenuates the inhibition of Rig-I-like receptor pathway activity and type I interferon production in EGFR-mutant LUAD cells. In addition, 3-MA upregulated production of type I interferon in EGFR-mutant LUAD cells, with an similar effect to ARID1A-KD. On the other hand, rapamycin attenuated the enhanced production of type I interferon in ARID1A-KD EGFR-mutant LUAD cells. ARID1A function appears to influence the tumor immune microenvironment and response to ICIs. Conclusion ARID1A deficiency reverses response to ICIs in EGFR-mutant LUAD by enhancing autophagy-inhibited type I interferon production.

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“…Ex vivo CRISPR has been used safely to deliver gene-edited tumorspecific T cells to humans with cancer (17), and the CRISPR platform has been optimized to conduct forward genetic screens in primary human T cells to identify novel targets to augment T cell function. These approaches have identified genes that regulate programmed cell death protein 1 (PD-1) expression, T cell proliferation and persistence, and resistance to adenosine-mediated immunosuppression (18)(19)(20), but work is ongoing to define genes for which editing will most potently augment antitumor responses in humans. In this study, we used CRISPR screening to identify genes that regulate effector function in primary human T cells expressing chimeric antigen receptors (CARs) and discovered that MED12 (Mediator complex subunit 12) and CCNC (cyclin C), genes encoding proteins in the kinase module of the Mediator complex, negatively regulate T cell effector activity.…”

Section: Rationalementioning

confidence: 99%

Enhanced T cell effector activity by targeting the Mediator kinase module

Freitas

Sotillo

Quinn

et al. 2022

Science

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T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC , components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor–engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12 -deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers—most notably for STAT and AP-1 transcription factors—and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

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Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence

Cited by 186 publications

References 79 publications

GiRAFR improves gRNA detection and annotation in single cell CRISPR screens

GiRAFR improves gRNA detection and annotation in single cell CRISPR screens

ARID1A deficiency reverses the response to anti-PD(L)1 therapy in EGFR-mutant lung adenocarcinoma by enhancing autophagy-inhibited type I interferon production

Enhanced T cell effector activity by targeting the Mediator kinase module

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