scholarly journals Interaction between DNA damage response, translation and apoptosome determines cancer susceptibility to TOP2 poisons

2019 ◽  
Author(s):  
Chidiebere U Awah ◽  
Li Chen ◽  
Mukesh Bansal ◽  
Aayushi Mahajan ◽  
Jan Winter ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Cancer Susceptibility ◽  
Protein Subunit ◽  
Ribosomal Subunits ◽  
Topoisomerase Ii Poisons ◽  
Etoposide Treatment ◽  
A Genome ◽  
Genome Scale

AbstractTopoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We show that glioblastoma (GBM), the most malignant of all primary brain tumors in adults is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 impaired the induction of pro-apoptotic gene APAF1 following etoposide treatment, and led to resistance to this drug and doxorubicin. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from multiple cancers.Graphical Abstract

scholarly journals LEDGF/p75 is Required for an Efficient DNA Damage Response

2020 ◽  
Author(s):  
Victoria Liedtke ◽  
Christian Schröder ◽  
Dirk Roggenbuck ◽  
Romano Weiss ◽  
Ralf Stohwasser ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Protein A ◽  
Signalling Pathways ◽  
Cancer Type ◽  
Knock Out ◽  
Signalling Molecules ◽  
Protein Levels

Abstract BackgroundLens epithelium derived growth factor splice variant of 75 kDa (LEDGF/p75), is overexpressed in different solid cancers and cancer cell lines and various autoinflammatory diseases. Due to its ability to bind chromatin, it acts as a transcriptional co-activator and promotes anti-apoptotic signalling pathways that lead to increased tumour aggressiveness and resistance to chemotherapy. The role of LEDGF/p75 in DNA-damage repair (DDR) is still not completely elucidated particularly regarding the ubiquitin-dependent regulation and degradation of DDR signalling molecules.MethodsDifferent LEDGF model cell lines were generated, a complete knock-out of LEDGF (KO) as well as the re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Then, various assays were performed to determine their proliferation and migration capacity as well as their chemosensitivity. Moreover, DDR signalling pathways were investigated by western blot and immunofluorescence.ResultsLEDGF-deficient cells exhibited a decreased proliferation (dt (WT) = 21 h, dt (KO) = 26 h) , 60 % decreased migration, as well as an 30-50 % increased sensitivity towards the topoisomerase II inhibitor etoposide. Moreover, LEDGF depleted cells showed a significant reduction by 65 % in the recruitment of downstream DDR-related proteins like replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. Re-expression of LEDGF/p75 rescued all knock-out effects, while re-expression of LEDGF/p52 had no effect.Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and Breast cancer type 1 susceptibility protein (BRCA1). In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. ConclusionsThis study provides evidence that LEDGF is not only an important player in the DDR after chemotherapeutic treatments but is also involved in the maintenance of the general genome integrity. Moreover, this study provides for the first time an insight into the possible role of LEDGF in the ubiquitin-dependent regulation of DDR signalling molecules and highlights the involvement of LEDGF/p75 in homology-directed DNA repair.

scholarly journals Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons

2012 ◽  
Vol 287 (46) ◽  
pp. 38590-38599 ◽  
Author(s):  
Stefanie C. Huelsenbeck ◽  
Anne Schorr ◽  
Wynand P. Roos ◽  
Johannes Huelsenbeck ◽  
Christian Henninger ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Topoisomerase Ii ◽  
Protein Signaling ◽  
Topoisomerase Ii Poisons ◽  
Damage Response

scholarly journals Genome-wide screening identifies cell cycle control as a synthetic lethal pathway with SRSF2P95H mutation

2021 ◽  
Author(s):  
Jane Jialu Xu ◽  
Alistair M Chalk ◽  
Iva Nikolic ◽  
Kaylene Simpson ◽  
Monique F Smeets ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Functional Screening ◽  
Wild Type ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Damage Response

Current strategies to target RNA splicing mutant myeloid cancers proposes targeting the remaining splicing apparatus. This approach has only been modestly sensitizing and is also toxic to non-mutant bearing wild-type cells. To explore potentially exploitable genetic interactions with spliceosome mutations, we combined data mining and functional screening for synthetic lethal interactions with an Srsf2P95H/+ mutation. Analysis of mis-splicing events in a series of both human and murine SRSF2P95H mutant samples across multiple myeloid diseases (AML, MDS, CMML) was performed to identify conserved mis-splicing events. From this analysis, we identified that the cell cycle and DNA repair pathways were overrepresented within the conserved mis-spliced transcript sets. In parallel, to functionally define pathways essential for survival and proliferation of Srsf2P95H/+ cells, we performed a genome-wide CRISPR loss of function screen using Hoxb8 immortalized R26-CreERki/+ Srsf2P95H/+ and R26-CreERki/+ Srsf2+/+ cell lines. We assessed loss of sgRNA representation at three timepoints: immediately after Srsf2P95H/+ activation, and at one week and two weeks post Srsf2P95H/+ mutation. Pathway analysis demonstrated that the cell cycle and DNA damage response pathways were amongst the top synthetic lethal pathways with Srsf2P95H/+ mutation. Based on the loss of guide RNAs targeting Cdk6, we identified that Palbociclib, a CDK6 inhibitor, showed preferential sensitivity in Srsf2P95H/+ cell lines and in primary non-immortalized lin-cKIT+Sca-1+ cells compared to wild type controls. Our data strongly suggest that the cell cycle and DNA damage response pathways are required for Srsf2P95H/+ cell survival, and that Palbociclib could be an alternative therapeutic option for targeting SRSF2 mutant cancers.

scholarly journals Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia with a Genome-Scale Crispr Knock-out Screen

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4233-4233 ◽  
Author(s):  
Matthieu Lewis ◽  
Valerie Prouzet-Mauleon ◽  
Elodie Richard ◽  
Beatrice Turcq ◽  
Richard Iggo ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Knock Out ◽  
Individual Gene ◽  
Oncogenic Pathways ◽  
A Genome ◽  
Tki Resistance ◽  
Genome Scale

Abstract Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system. Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells. Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS). Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance. Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases. Disclosures Mahon: ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.

scholarly journals Genetic Screens in Human Cells Using the CRISPR-Cas9 System

Science ◽  
2013 ◽  
Vol 343 (6166) ◽  
pp. 80-84 ◽  
Author(s):  
Tim Wang ◽  
Jenny J. Wei ◽  
David M. Sabatini ◽  
Eric S. Lander
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Negative Selection ◽  
Topoisomerase Ii ◽  
Massively Parallel Sequencing ◽  
Sequence Motifs ◽  
Loss Of Function ◽  
Specific Sequence ◽  
Guide Rna ◽  
A Genome

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

scholarly journals Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition

2021 ◽  
Vol 11 ◽  
Author(s):  
Arafat Siddiqui ◽  
Manuela Tumiati ◽  
Alia Joko ◽  
Jouko Sandholm ◽  
Pia Roering ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cell Lines ◽  
Combination Treatment ◽  
Topoisomerase Ii ◽  
Protein Tyrosine Kinase ◽  
Chemotherapy Resistance ◽  
Treatment Resistance ◽  
Clinical Problem ◽  
Repair Process

Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50–85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.

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