An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.
Journal
Communications biology
ISSN: 2399-3642
Titre abrégé: Commun Biol
Pays: England
ID NLM: 101719179
Informations de publication
Date de publication:
10 Apr 2024
10 Apr 2024
Historique:
received:
22
08
2023
accepted:
29
03
2024
medline:
11
4
2024
pubmed:
11
4
2024
entrez:
10
4
2024
Statut:
epublish
Résumé
Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.
Identifiants
pubmed: 38600351
doi: 10.1038/s42003-024-06129-1
pii: 10.1038/s42003-024-06129-1
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
441Subventions
Organisme : American Cancer Society (American Cancer Society, Inc.)
ID : 131356-RSG-17-160-01-CSM
Informations de copyright
© 2024. The Author(s).
Références
Lennon, A. M. et al. Feasibility of blood testing combined with PET-CT to screen for cancer and guide intervention. Science 369, https://doi.org/10.1126/science.abb9601 (2020).
Wan, J. C. M. et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat. Rev. Cancer 17, 223–238 (2017).
pubmed: 28233803
doi: 10.1038/nrc.2017.7
Cescon, D. W., Bratman, S. V., Chan, S. M. & Siu, L. L. Circulating tumor DNA and liquid biopsy in oncology. Nat. Cancer 1, 276–290 (2020).
pubmed: 35122035
doi: 10.1038/s43018-020-0043-5
Parsons, H. A. et al. Sensitive detection of minimal residual disease in patients treated for early-stage breast cancer. Clin. Cancer Res. 26, 2556–2564 (2020).
pubmed: 32170028
pmcid: 7654718
doi: 10.1158/1078-0432.CCR-19-3005
Turner, N. C. et al. Circulating tumour DNA analysis to direct therapy in advanced breast cancer (plasmaMATCH): a multicentre, multicohort, phase 2a, platform trial. Lancet Oncol. 21, 1296–1308 (2020).
pubmed: 32919527
pmcid: 7599319
doi: 10.1016/S1470-2045(20)30444-7
Guo, Q. et al. Heterogeneous mutation pattern in tumor tissue and circulating tumor DNA warrants parallel NGS panel testing. Mol. Cancer 17, 131 (2018).
pubmed: 30153823
pmcid: 6114875
doi: 10.1186/s12943-018-0875-0
Chae, Y. K. et al. Concordance between genomic alterations assessed by next-generation sequencing in tumor tissue or circulating cell-free DNA. Oncotarget 7, 65364–65373 (2016).
pubmed: 27588476
pmcid: 5323161
doi: 10.18632/oncotarget.11692
Chae, Y. K. et al. Concordance of genomic alterations by next-generation sequencing in tumor tissue versus circulating tumor DNA in breast cancer. Mol. Cancer Ther. 16, 1412–1420 (2017).
pubmed: 28446639
doi: 10.1158/1535-7163.MCT-17-0061
Said, R., Guibert, N., Oxnard, G. R. & Tsimberidou, A. M. Circulating tumor DNA analysis in the era of precision oncology. Oncotarget 11, 188–211 (2020).
pubmed: 32010431
pmcid: 6968778
doi: 10.18632/oncotarget.27418
Zhang, Y. et al. Pan-cancer circulating tumor DNA detection in over 10,000 Chinese patients. Nat. Commun. 12, 11 (2021).
pubmed: 33397889
pmcid: 7782482
doi: 10.1038/s41467-020-20162-8
Zviran, A. et al. Genome-wide cell-free DNA mutational integration enables ultra-sensitive cancer monitoring. Nat. Med 26, 1114–1124 (2020).
pubmed: 32483360
pmcid: 8108131
doi: 10.1038/s41591-020-0915-3
Newman, A. M. et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat. Biotechnol. 34, 547–555 (2016).
pubmed: 27018799
pmcid: 4907374
doi: 10.1038/nbt.3520
Cohen, J. D. et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 359, 926–930 (2018).
pubmed: 29348365
pmcid: 6080308
doi: 10.1126/science.aar3247
Gydush, G. et al. Massively parallel enrichment of low-frequency alleles enables duplex sequencing at low depth. Nat. Biomed. Eng. 6, 257–266 (2022).
pubmed: 35301450
pmcid: 9089460
doi: 10.1038/s41551-022-00855-9
Kato, S. et al. Analysis of circulating tumor DNA and clinical correlates in patients with esophageal, gastroesophageal junction, and gastric adenocarcinoma. Clin. Cancer Res 24, 6248–6256 (2018).
pubmed: 30348637
pmcid: 6384095
doi: 10.1158/1078-0432.CCR-18-1128
Phallen, J. et al. Direct detection of early-stage cancers using circulating tumor DNA. Sci. Transl. Med. 9, https://doi.org/10.1126/scitranslmed.aan2415 (2017).
Kustanovich, A., Schwartz, R., Peretz, T. & Grinshpun, A. Life and death of circulating cell-free DNA. Cancer Biol. Ther. 20, 1057–1067 (2019).
pubmed: 30990132
pmcid: 6606043
doi: 10.1080/15384047.2019.1598759
Grabuschnig, S. et al. Putative origins of cell-free DNA in humans: a review of active and passive nucleic acid release mechanisms. Int. J. Mol. Sci. 21, https://doi.org/10.3390/ijms21218062 (2020).
Bronkhorst, A. J., Ungerer, V. & Holdenrieder, S. The emerging role of cell-free DNA as a molecular marker for cancer management. Biomol. Detect Quantif. 17, 100087 (2019).
pubmed: 30923679
pmcid: 6425120
doi: 10.1016/j.bdq.2019.100087
Jahr, S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61, 1659–1665 (2001).
pubmed: 11245480
Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M. & Shendure, J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164, 57–68 (2016).
pubmed: 26771485
pmcid: 4715266
doi: 10.1016/j.cell.2015.11.050
Lo, Y. M. et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci. Transl. Med. 2, 61ra91 (2010).
pubmed: 21148127
doi: 10.1126/scitranslmed.3001720
Thierry, A. R. et al. Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts. Nucleic Acids Res. 38, 6159–6175 (2010).
pubmed: 20494973
pmcid: 2952865
doi: 10.1093/nar/gkq421
Ungerer, V., Bronkhorst, A. J., Van den Ackerveken, P., Herzog, M. & Holdenrieder, S. Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures. Sci. Rep. 11, 9460 (2021).
pubmed: 33947882
pmcid: 8096822
doi: 10.1038/s41598-021-88866-5
Rostami, A. et al. Senescence, necrosis, and apoptosis govern circulating cell-free DNA release kinetics. Cell Rep. 31, 107830 (2020).
pubmed: 32610131
doi: 10.1016/j.celrep.2020.107830
Bronkhorst, A. J. et al. Characterization of the cell-free DNA released by cultured cancer cells. Biochim. Biophys. Acta 1863, 157–165 (2016).
pubmed: 26529550
doi: 10.1016/j.bbamcr.2015.10.022
Wang, W. et al. Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines. Oncotarget 8, 43180–43191 (2017).
pubmed: 28574818
pmcid: 5522137
doi: 10.18632/oncotarget.17858
Jiang, P. et al. Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc. Natl Acad. Sci. USA 112, E1317–E1325 (2015).
pubmed: 25646427
pmcid: 4372002
doi: 10.1073/pnas.1500076112
Markus, H. et al. Refined characterization of circulating tumor DNA through biological feature integration. Sci. Rep. 12, 1928 (2022).
pubmed: 35121756
pmcid: 8816939
doi: 10.1038/s41598-022-05606-z
Mouliere, F. et al. High fragmentation characterizes tumour-derived circulating DNA. PLoS One 6, e23418 (2011).
pubmed: 21909401
pmcid: 3167805
doi: 10.1371/journal.pone.0023418
Abbosh, C. et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature 545, 446–451 (2017).
pubmed: 28445469
pmcid: 5812436
doi: 10.1038/nature22364
Kahlert, C. et al. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J. Biol. Chem. 289, 3869–3875 (2014).
pubmed: 24398677
pmcid: 3924256
doi: 10.1074/jbc.C113.532267
Thakur, B. K. et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 24, 766–769 (2014).
pubmed: 24710597
pmcid: 4042169
doi: 10.1038/cr.2014.44
Lazaro-Ibanez, E. et al. DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology. J. Extracell. Vesicles 8, 1656993 (2019).
pubmed: 31497265
pmcid: 6719264
doi: 10.1080/20013078.2019.1656993
Vagner, T. et al. Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma. J. Extracell. Vesicles 7, 1505403 (2018).
pubmed: 30108686
pmcid: 6084494
doi: 10.1080/20013078.2018.1505403
Jin, Y. et al. DNA in serum extracellular vesicles is stable under different storage conditions. BMC Cancer 16, 753 (2016).
pubmed: 27662833
pmcid: 5035490
doi: 10.1186/s12885-016-2783-2
Fernando, M. R., Jiang, C., Krzyzanowski, G. D. & Ryan, W. L. New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes. PLoS One 12, e0183915 (2017).
pubmed: 28850588
pmcid: 5574584
doi: 10.1371/journal.pone.0183915
Yokoi, A. et al. Mechanisms of nuclear content loading to exosomes. Sci. Adv. 5, eaax8849 (2019).
pubmed: 31799396
pmcid: 6867874
doi: 10.1126/sciadv.aax8849
Takahashi, A. et al. Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nat. Commun. 8, 15287 (2017).
pubmed: 28508895
pmcid: 5440838
doi: 10.1038/ncomms15287
Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428–445 e418 (2019).
pubmed: 30951670
pmcid: 6664447
doi: 10.1016/j.cell.2019.02.029
Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).
pubmed: 22460905
pmcid: 3320027
doi: 10.1038/nature11003
Klijn, C. et al. A comprehensive transcriptional portrait of human cancer cell lines. Nat. Biotechnol. 33, 306–312 (2015).
pubmed: 25485619
doi: 10.1038/nbt.3080
Lapin, M. et al. Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer. J. Transl. Med. 16, 300 (2018).
pubmed: 30400802
pmcid: 6218961
doi: 10.1186/s12967-018-1677-2
Zabransky, D. J. et al. HER2 missense mutations have distinct effects on oncogenic signaling and migration. Proc. Natl Acad. Sci. USA 112, E6205–E6214 (2015).
pubmed: 26508629
pmcid: 4653184
doi: 10.1073/pnas.1516853112
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).
pubmed: 26780180
pmcid: 4744125
doi: 10.1038/nbt.3437
Sanson, K. R. et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416 (2018).
pubmed: 30575746
pmcid: 6303322
doi: 10.1038/s41467-018-07901-8
Li, W. et al. Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol. 16, 281 (2015).
pubmed: 26673418
pmcid: 4699372
doi: 10.1186/s13059-015-0843-6
Mi, H., Muruganujan, A., Casagrande, J. T. & Thomas, P. D. Large-scale gene function analysis with the PANTHER classification system. Nat. Protoc. 8, 1551–1566 (2013).
pubmed: 23868073
pmcid: 6519453
doi: 10.1038/nprot.2013.092
Schneider, P. et al. TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kappaB. Immunity 7, 831–836 (1997).
pubmed: 9430228
doi: 10.1016/S1074-7613(00)80401-X
Fu, K. et al. Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-kappaB activation. Elife 5, https://doi.org/10.7554/eLife.15018 (2016).
Sun, X. et al. Sam68 is required for DNA damage responses via regulating poly(ADP-ribosyl)ation. PLoS Biol. 14, e1002543 (2016).
pubmed: 27635653
pmcid: 5026359
doi: 10.1371/journal.pbio.1002543
Frisone, P. et al. SAM68: signal transduction and RNA metabolism in human cancer. Biomed. Res. Int. 2015, 528954 (2015).
pubmed: 26273626
pmcid: 4529925
doi: 10.1155/2015/528954
Ramakrishnan, P. & Baltimore, D. Sam68 is required for both NF-kappaB activation and apoptosis signaling by the TNF receptor. Mol. Cell 43, 167–179 (2011).
pubmed: 21620750
pmcid: 3142289
doi: 10.1016/j.molcel.2011.05.007
Lafont, E. et al. The linear ubiquitin chain assembly complex regulates TRAIL-induced gene activation and cell death. EMBO J. 36, 1147–1166 (2017).
pubmed: 28258062
pmcid: 5412822
doi: 10.15252/embj.201695699
Walczak, H. Death receptor-ligand systems in cancer, cell death, and inflammation. Cold Spring Harb. Perspect. Biol. 5, a008698 (2013).
pubmed: 23637280
pmcid: 3632055
doi: 10.1101/cshperspect.a008698
Jin, Z. & El-Deiry, W. S. Distinct signaling pathways in TRAIL- versus tumor necrosis factor-induced apoptosis. Mol. Cell Biol. 26, 8136–8148 (2006).
pubmed: 16940186
pmcid: 1636728
doi: 10.1128/MCB.00257-06
Holoch, P. A. & Griffith, T. S. TNF-related apoptosis-inducing ligand (TRAIL): a new path to anti-cancer therapies. Eur. J. Pharm. 625, 63–72 (2009).
doi: 10.1016/j.ejphar.2009.06.066
Sanchez-Margalet, V. & Najib, S. Sam68 is a docking protein linking GAP and PI3K in insulin receptor signaling. Mol. Cell Endocrinol. 183, 113–121 (2001).
pubmed: 11604231
doi: 10.1016/S0303-7207(01)00587-1
Li, Z. et al. Sam68 expression and cytoplasmic localization is correlated with lymph node metastasis as well as prognosis in patients with early-stage cervical cancer. Ann. Oncol. 23, 638–646 (2012).
pubmed: 21700735
doi: 10.1093/annonc/mdr290
Bielli, P. et al. The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis. EMBO Rep. 15, 419–427 (2014).
pubmed: 24514149
pmcid: 3989673
doi: 10.1002/embr.201338241
Paronetto, M. P., Achsel, T., Massiello, A., Chalfant, C. E. & Sette, C. The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x. J. Cell Biol. 176, 929–939 (2007).
pubmed: 17371836
pmcid: 2064079
doi: 10.1083/jcb.200701005
Popgeorgiev, N., Jabbour, L. & Gillet, G. Subcellular localization and dynamics of the Bcl-2 family of proteins. Front. Cell Dev. Biol. 6, 13 (2018).
pubmed: 29497611
pmcid: 5819560
doi: 10.3389/fcell.2018.00013
Dang, D. K. & Park, B. H. Circulating tumor DNA: current challenges for clinical utility. J. Clin. Investig. 132, https://doi.org/10.1172/JCI154941 (2022).
Cristiano, S. et al. Genome-wide cell-free DNA fragmentation in patients with cancer. Nature 570, 385–389 (2019).
pubmed: 31142840
pmcid: 6774252
doi: 10.1038/s41586-019-1272-6
Foda, Z. H. et al. Detecting Liver Cancer Using Cell-Free DNA Fragmentomes. Cancer Discov. 13, 616–631 (2023).
pubmed: 36399356
doi: 10.1158/2159-8290.CD-22-0659
Klein, E. A. et al. Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set. Ann. Oncol. 32, 1167–1177 (2021).
pubmed: 34176681
doi: 10.1016/j.annonc.2021.05.806
Martin-Alonso, C. et al. Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies. Science 383, eadf2341 (2024).
pubmed: 38236959
doi: 10.1126/science.adf2341
Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 20, 332–343 (2018).
pubmed: 29459780
pmcid: 5931706
doi: 10.1038/s41556-018-0040-4
Zhuang, H. et al. Down-regulation of HSP27 sensitizes TRAIL-resistant tumor cell to TRAIL-induced apoptosis. Lung Cancer 68, 27–38 (2010).
pubmed: 19540014
doi: 10.1016/j.lungcan.2009.05.014
Li, X. et al. Reversal of the apoptotic resistance of non-small-cell lung carcinoma towards TRAIL by natural product toosendanin. Sci. Rep. 7, 42748 (2017).
pubmed: 28209994
pmcid: 5314365
doi: 10.1038/srep42748
Carne Trecesson, S. et al. BCL-X(L) directly modulates RAS signalling to favour cancer cell stemness. Nat. Commun. 8, 1123 (2017).
pubmed: 29066722
pmcid: 5654832
doi: 10.1038/s41467-017-01079-1
Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).
pubmed: 26627737
doi: 10.1016/j.cell.2015.11.015
Tiedt, R. et al. Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors. Cell Rep. 42, 112297 (2023).
pubmed: 36961816
doi: 10.1016/j.celrep.2023.112297
Cohen, J. D. et al. Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands. Nat. Biotechnol. 39, 1220–1227 (2021).
pubmed: 33941929
pmcid: 8627329
doi: 10.1038/s41587-021-00900-z