scholarly journals Compromised CDK12 activity causes dependency on the non-essential spliceosome components

2021 ◽  
Author(s):  
Satu Pallasaho ◽  
Aishwarya Gondane ◽  
Damien Duveau ◽  
Craig Thomas ◽  
Massimo Loda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Treatment Options ◽  
Growth Defect ◽  
Small Scale ◽  
Cyclin Dependent Kinase ◽  
Synthetic Lethal ◽  
Castration Resistant ◽  
Synthetic Lethal Interactions ◽  
Glcnac Transferase ◽  
Mutant Cells

Prostate cancer (PC) is the most common cancer in men and after development of the castration-resistant PC (CRPC), there are no curative treatment options. Inactivating mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive sub-type of CRPC. We hypothesized that compromised CDK12 activity leads to a significant rewiring of the CRPC cells, and that this rewiring results in actionable synthetic lethal interactions. Methods: We used combinatorial lethal screening, ChIP-seq data, RNA-seq data, global alternative splicing analysis, and comprehensive mass spectrometry (MS) profiling to understand how the compromised CDK12 activity rewires the CRPC cells. In addition, we used DepMap-, PC- and CRPC-datasets as a strategy to identify factors that are selectively required by the CDK12-mutant cells. Results: We show that inhibition of O-GlcNAc transferase (OGT) and CDK12 induces cancer cell-selective growth-defect. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation, and we use unbiased MS-profiling to show that the short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in PC and CRPC cells. Integration of DepMap- and a small scale-drug screen data reveled that depletion of CDK12 activity causes addiction to non-essential spliceosome components (CLK1/4 and SRPK1). CDK12-mutant tumors overexpress CLK1/4 and SRPK1. Finally, we show that the genomes of the CDK12-mutant tumors have lowered DNA methylation, and that CDK12 inhibition induces the expression of the genes marked by DNA methylation. Conclusions: Compromised CDK12 activity rewires DNA methylation, transcription and splicing, and this rewiring renders the affected cells addicted on the non-essential spliceosome components. We propose that inactivation of CDK12 is a biomarker for sensitivity against inhibitors of the non-essential spliceosome components just entering the clinical trials.

scholarly journals A Late Mitotic Regulatory Network Controlling Cyclin Destruction in Saccharomyces cerevisiae

1998 ◽  
Vol 9 (10) ◽  
pp. 2803-2817 ◽  
Author(s):  
Sue L. Jaspersen ◽  
Julia F. Charles ◽  
Rachel L. Tinker-Kulberg ◽  
David O. Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Network ◽  
Growth Defect ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitotic Cyclin ◽  
Low Levels ◽  
Late Mitosis ◽  
Mutant Cells

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15,cdc5, cdc14, dbf2, andtem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, andSIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.

scholarly journals Synthetic Lethal Phenotypes Caused by Mutations Affecting Chromosome Partitioning in Bacillus subtilis

1999 ◽  
Vol 181 (18) ◽  
pp. 5860-5864 ◽  
Author(s):  
Robert A. Britton ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Double Mutant ◽  
Chromosome Structure ◽  
Growth Defect ◽  
Loss Of Function ◽  
Synthetic Lethal ◽  
Chromosome Partitioning ◽  
Nucleoid Structure ◽  
Mutant Cells ◽  
Dna Translocase

ABSTRACT We investigated the genetic interactions between mutations affecting chromosome structure and partitioning in Bacillus subtilis. Loss-of-function mutations in spoIIIE(encoding a putative DNA translocase) and smc (involved in chromosome structure and partitioning) caused a synthetic lethal phenotype. We constructed a conditional mutation in smc and found that many of the spoIIIE smc double-mutant cells had a chromosome bisected by a division septum. The growth defect of the double mutant was exacerbated by a null mutation in the chromosome partitioning gene spo0J. These results suggest that mutants defective in nucleoid structure are unable to move chromosomes out of the way of the invaginating septum and that SpoIIIE is involved in repositioning these bisected chromosomes during vegetative growth.

scholarly journals CDK12: an emerging therapeutic target for cancer

2018 ◽  
Vol 71 (11) ◽  
pp. 957-962 ◽  
Author(s):  
Goldie Y L Lui ◽  
Carla Grandori ◽  
Christopher J Kemp
Keyword(s):  
Rna Polymerase Ii ◽  
Therapeutic Target ◽  
Cell Function ◽  
Human Cancer ◽  
Cyclin Dependent Kinase ◽  
Cellular Functions ◽  
Synthetic Lethal ◽  
Pancreatic Cancers ◽  
Expression Of Genes ◽  
Synthetic Lethal Interactions

Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases that regulate transcriptional and post-transcriptional processes, thereby modulating multiple cellular functions. Early studies characterised CDK12 as a transcriptional CDK that complexes with cyclin K to mediate gene transcription by phosphorylating RNA polymerase II. CDK12 has been demonstrated to specifically upregulate the expression of genes involved in response to DNA damage, stress and heat shock. More recent studies have implicated CDK12 in regulating mRNA splicing, 3’ end processing, pre-replication complex assembly and genomic stability during embryonic development. Genomic alterations in CDK12 have been detected in oesophageal, stomach, breast, endometrial, uterine, ovarian, bladder, colorectal and pancreatic cancers, ranging from 5% to 15% of sequenced cases. An increasing number of studies point to CDK12 inhibition as an effective strategy to inhibit tumour growth, and synthetic lethal interactions have been described with MYC, EWS/FLI and PARP/CHK1 inhibition. Herein, we discuss the present literature on CDK12 in cell function and human cancer, highlighting important roles for CDK12 as a clinical biomarker for treatment response and potential as an effective therapeutic target.

Synthetic Lethal Interactions in Cancer Therapy

2017 ◽  
Vol 17 (4) ◽  
pp. 304-310 ◽  
Author(s):  
Xinwei Geng ◽  
Xiaohui Wang ◽  
Dan Zhu ◽  
Songmin Ying
Keyword(s):  
Cancer Therapy ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals Combinatorial CRISPR screen identifies fitness effects of gene paralogues

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicola A. Thompson ◽  
Marco Ranzani ◽  
Louise van der Weyden ◽  
Vivek Iyer ◽  
Victoria Offord ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Unknown Function ◽  
Single Copy ◽  
Genetic Redundancy ◽  
Synthetic Lethal ◽  
Fitness Effects ◽  
Synthetic Lethal Interactions ◽  
Crispr Screen ◽  
Multiple Cell

AbstractGenetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.

scholarly journals Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

NAR Cancer ◽  
2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Amrita Sule ◽  
Jinny Van Doorn ◽  
Ranjini K Sundaram ◽  
Sachita Ganesa ◽  
Juan C Vasquez ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Parp Inhibitor ◽  
Parp Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Isocitrate Dehydrogenase 1 ◽  
Normal Product ◽  
Synthetic Lethal Interactions ◽  
Mutant Cells

Abstract Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as ‘BRCAness’, which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.

scholarly journals The Putative Eukaryote-LikeO-GlcNAc Transferase of the Cyanobacterium Synechococcus elongatus PCC 7942 Hydrolyzes UDP-GlcNAc and Is Involved in Multiple Cellular Processes

2014 ◽  
Vol 197 (2) ◽  
pp. 354-361 ◽  
Author(s):  
Kerry A. Sokol ◽  
Neil E. Olszewski
Keyword(s):  
Deletion Mutant ◽  
Bacterial Species ◽  
Synechococcus Elongatus ◽  
Single Amino Acid ◽  
Content Type ◽  
Cellular Processes ◽  
Mutant Phenotypes ◽  
Glcnac Transferase ◽  
Pcc 7942 ◽  
Mutant Cells

The posttranslational addition of a single O-linked β-N-acetylglucosamine (O-GlcNAc) to serine or threonine residues regulates numerous metazoan cellular processes. The enzyme responsible for this modification,O-GlcNAc transferase (OGT), is conserved among a wide variety of organisms and is critical for the viability of many eukaryotes. Although OGTs with domain structures similar to those of eukaryotic OGTs are predicted for many bacterial species, the cellular roles of these OGTs are unknown. We have identified a putative OGT in the cyanobacteriumSynechococcus elongatusPCC 7942 that shows active-site homology and similar domain structure to eukaryotic OGTs. An OGT deletion mutant was created and found to exhibit several phenotypes. Without agitation, mutant cells aggregate and settle out of the medium. The mutant cells have higher free inorganic phosphate levels, wider thylakoid lumen, and differential accumulation of electron-dense inclusion bodies. These phenotypes are rescued by reintroduction of the wild-type OGT but are not fully rescued by OGTs with single amino acid substitutions corresponding to mutations that reduce eukaryotic OGT activity.S. elongatusOGT purified fromEscherichia colihydrolyzed the sugar donor, UDP-GlcNAc, while the mutant OGTs that did not fully rescue the deletion mutant phenotypes had reduced or no activity. These results suggest that bacterial eukaryote-like OGTs, like their eukaryotic counterparts, influence multiple processes.

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