18 research outputs found
Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication
Gain-of-function (GOF) p53 mutations are observed frequently in most intractable human cancers and establish dependency for tumor maintenance and progression. While some of the genes induced by GOF p53 have been implicated in more rapid cell proliferation compared with p53-null cancer cells, the mechanism for dependency of tumor growth on mutant p53 is unknown. This report reveals a therapeutically targetable mechanism for GOF p53 dependency. We have shown that GOF p53 increases DNA replication origin firing, stabilizes replication forks, and promotes micronuclei formation, thus facilitating the proliferation of cells with genomic abnormalities. In contrast, absence or depletion of GOF p53 leads to decreased origin firing and a higher frequency of fork collapse in isogenic cells, explaining their poorer proliferation rate. Following genome-wide analyses utilizing ChIP-Seq and RNA-Seq, GOF p53–induced origin firing, micronuclei formation, and fork protection were traced to the ability of GOF p53 to transactivate cyclin A and CHK1. Highlighting the therapeutic potential of CHK1’s role in GOF p53 dependency, experiments in cell culture and mouse xenografts demonstrated that inhibition of CHK1 selectively blocked proliferation of cells and tumors expressing GOF p53. Our data suggest the possibility that checkpoint inhibitors could efficiently and selectively target cancers expressing GOF p53 alleles
Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells
The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity
Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.
This is the final version. Available from the National Academy of Sciences via the DOI in this record. SI Appendix contains detailed descriptions of the cloning of bacterial expression constructs for the 64 shuffled BRC peptide variants, cloning of mammalian expression constructs, and notes on the soluble expression of the shuffled BRC peptide variants. Also included is a description of ITC used to cross-validate the microfluidic measurements, single concentration point measurements carried out with microfluidics, and exemplary titrations carried out by microfluidics. The fluorescence anisotropy data obtained for the 64 separate titrations as well as the Matlab script used in the analysis have been uploaded as separate files. The supplementary data also contain an analysis on the effect of shuffling of BRC peptides and in particular on the effect of the exact shuffle cutoff point placement. X-ray crystallography electron density map images, data collection, and refinement statistics are also to be found in SI Appendix. Additional cell images highlighting the pan-nuclear signal of RAD51 are also included in SI Appendix. The coordinates and corresponding structure factors for the monomeric RAD51:BRC8-2 complex have been deposited to the PDB under accession code 6HQU. As described previously (49), the transformation from intensity maps into anisotropy values from image data was carried out with a custom Matlab code available on GitHub (https://github.com/quantitativeimaging/icetropy). A custom Matlab script used to fit Kd values for the unlabeled competitive GB1-BRC peptides can be found in SI Appendix, Datasets S1–S4. All other study data are included in the article and/or supporting information.Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.Biotechnology and Biological Sciences Research CouncilEuropean Research CouncilMarie Curie Research GrantCancer Research UKEngineering and Physical Sciences Research CouncilEngineering and Physical Sciences Research CouncilWellcome TrustWellcome TrustMedical Research CouncilMedical Research CouncilSchweizerischer Nationalfond
Microtubule interfering agents and KSP inhibitors induce the phosphorylation of the nuclear protein p54(nrb), an event linked to G2/M arrest
Microtubule interfering agents (MIAs) are anti-tumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause G2/M arrest and cell death. Using 2D-PAGE followed by Nano-LC-ESI-Q-ToF analysis, we found that MIAs such as vincristine (Oncovin) or paclitaxel (Taxol) and KSP inhibitors such as S-tritil-l-cysteine induce the phosphorylation of the nuclear protein p54(nrb) in HeLa cells. Furthermore, we demonstrate that cisplatin (Platinol), an anti-tumor drug that does not cause M arrest, does not induce this modification. We show that the G2/M arrest induced by MIAs is required for p54(nrb) phosphorylation. Finally, we demonstrate that CDK activity is required for MIA-induced phosphorylation of p54(nrb)
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Fosforilación del EF1B-GAMMA de eucariotas durante la parada en mitosis producida por los mias y los inhibidoresde KSP
Los MIAs o agentes que interfi eren con los microtúbulos incluyen compuestos como el nocodazol y fármacos antitumorales como paclitaxel y docetaxel. Estos agentes y los inhibidores de KSP (STLC, etc.) son compuestos capaces de inhibir la dinámica de los microtúbulos, parar el ciclo celular en mitosis e inducir muerte celular. En nuestro laboratorio se ha descrito que el paclitaxel induce la fosforilación del eEF1B-gamma (Prado, M.A. y cols., Proteomics, 2007, Vol.7, 3299-3304), siendo esta proteína parte fundamental del complejo eEF1B. Este complejo, de gran importancia en el proceso de elongación proteica, se encarga de activar el eEF1A (transportador del aminoaciltRNA al ribosoma) gracias a la actividad intercambiadora de nucleótidos de guanina que posee. Utilizando 2D-PAGE del proteoma de células HeLa demostramos que el cisplatino, un fármaco antitumoral independiente de fase, no induce la fosforilación del eEF1B-gamma, mientras que otros compuestos antimitóticos, como docetaxel, nocodazol y STLC, sí inducen la fosforilación de esta proteína. El posterior análisis del proteoma de células HeLa tratadas con paclitaxel, pero paradas previamente al comienzo de fase S por acción de la afi dicolina, revela que la parada en mitosis es necesaria para la fosforilación del eEF1B-gamma. Una 2D-PAGE de extractos tratados con roscovitina, un inhibidor de CDK1, mostró que esta vía de fosforilación del eEF1B-gamma es dependiente de esta quinasa. Finalmente, un análisis por espectrometría de masas MALDI-ToF del eEF1B-gamma digerido con la endoproteinasa GluC, mostró que el péptido comprendido entre los residuos 224 y 234 se fosforila tras el tratamiento con paclitaxel, aunque no es posible determinar si se produce en el residuo T224 o T230
Aneuploidy tolerance caused by BRG1 loss allows chromosome gains and recovery of fitness.
Aneuploidy results in decreased cellular fitness in many species and model systems. However, aneuploidy is commonly found in cancer cells and often correlates with aggressive growth, suggesting that the impact of aneuploidy on cellular fitness is context dependent. The BRG1 (SMARCA4) subunit of the SWI/SNF chromatin remodelling complex is frequently lost in cancer. Here, we use a chromosomally stable cell line to test the effect of BRG1 loss on the evolution of aneuploidy. BRG1 deletion leads to an initial loss of fitness in this cell line that improves over time. Notably, we find increased tolerance to aneuploidy immediately upon loss of BRG1, and the fitness recovery over time correlates with chromosome gain. These data show that BRG1 loss creates an environment where karyotype changes can be explored without a fitness penalty. At least in some genetic backgrounds, therefore, BRG1 loss can affect the progression of tumourigenesis through tolerance of aneuploidy
Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.
Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.BBSRC (BB/K013629/1), Cancer Research UK (C7905/A25715), EPSRC (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z), MRC (grants MR/K015850/1 and MR/K02292X/1), ERC, H2020 Marie-Curi
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A recombinant approach for stapled peptide discovery yields inhibitors of the RAD51 recombinase
Acknowledgements: We are grateful to Dr Laurens Lindenburg for the gift of BRC4-fluorescein. We thank the Biophysical and X-ray crystallographic facilities (Department of Biochemistry, University of Cambridge) for access to instrumentation and support. We thank Diamond Light Source for access to beamlines i03 and i04 (proposals mx18548, mx25402). We thank Dr Peter Martin and the Institute of Cancer Research Light Microscopy Facility for support. TP was supported by MRC DTP. The Spring Laboratory acknowledges support from the EPSRC, BBSRC and MRC. PZ-V and JAD were supported by CRUK (C7905/A25715).An approach for stapled peptide preparation in small scale using recombinant expression of peptide–protein fusions in bacteria. We use this approach to design binders of RAD51, characterise their interaction and demonstrate activity in cells.</jats:p