Synthetic Lethality Triggered by Combining Olaparib with BRCA2–Rad51 DisruptorsClick to copy article linkArticle link copied!
- Federico Falchi
- Elisa Giacomini
- Tiziana Masini
- Nicolas Boutard
- Lorenza Di Ianni
- Marcella Manerba
- Fulvia Farabegoli
- Lara Rossini
- Janet Robertson
- Saverio Minucci
- Isabella Pallavicini
- Giuseppina Di Stefano
- Marinella Roberti
- Roberto Pellicciari
- Andrea Cavalli
Abstract
In BRCA2-defective cells, poly(adenosine diphosphate [ADP]-ribose) polymerase inhibitors can trigger synthetic lethality, as two independent DNA-repairing mechanisms are simultaneously impaired. Here, we have pharmacologically induced synthetic lethality, which was triggered by combining two different small organic molecules. When administered with a BRCA2–Rad51 disruptor in nonmutant cells, Olaparib showed anticancer activity comparable to that shown when administered alone in BRCA2-defective cells. This strategy could represent an innovative approach to anticancer drug discovery and could be extended to other synthetic lethality pathways.
Figure 1
Figure 1. (a) Rad51–BRCA2 BRC repeat complex. Rad51 is represented as a surface, BRCA as a cartoon. The two hot spots of the interaction between the proteins (Phe1524 and Phe1546) are highlighted in sticks. (b) SiteMap hydrophobic (green), hydrogen-bond donor (blue), and hydrogen-bond acceptor (red) maps for 1N0W in the proximity of Phe1524. (c) Schematic representation of the virtual screening protocol. The numbers on the left column represent the number of compounds in each step; the filters used to narrow down the compounds are reported on the right column.

(∗) All points were tested in triplicate with error bars indicating the standard deviation. (∗∗) Not active.
Figure 2
Figure 2. Experiments on cell cultures. (a) Effect of 7 days of Olaparib treatment on the viability of BxPC-3 and Capan-1 cells. (b, c) Dose–response curves assessed in BxPC-3 (b) and Capan-1 (c) cells exposed to 2. The compound doses used for combination experiments have been sketched with dark symbols. (d, e) Combination experiments of Olaparib with 2 in BxPC-3 (d) and Capan-1 (e). In the BxPC-3 cell line, the results obtained with Olaparib were compared with those obtained with Olaparib in combination with 2 using two-way ANOVA; for both 20 and 30 μM 2, p values < 0.001 were obtained at 3–6 days of treatment. (f) Combination experiment of Olaparib with 2, in BxPC-3 cells after stable Rad51 silencing, compared with scrambled controls. The effect was evaluated after a 96 h treatment. Results obtained in siRad51 cultures were statistically analyzed by ANOVA followed by Bonferroni post test. (g) Densitometric reading of immunoblots evaluating γH2AX expression (normalized on Actin levels) in cells treated with Cisplatin (12 μM) and 2 at the same doses used in the Olaparib combination experiments. For a detailed explanation, see the text. Vertical lines indicate the samples for comparison. (h) Immunofluorescence γH2AX detection in nuclei of BxPC-3 cells treated with RAD51 siRNA or 2. The percentage of positive cells are reported in the graph of frame (i).
enzyme | % inhibition at 25 μM | references |
---|---|---|
cathepsin B (h) | 1.9 | 26 |
cathepsin D (h) | –6.6 | 27 |
cathepsin E | –25.8 | 27 |
cathepsin S (h) | 12.3 | 28 |
MMP-2 (h) | –2.9 | 29 |
phosphatase CDC25A (h) | –5 | 30 |
phosphatase CDC25B (h) | –0.3 | 30 |
protein serine/threonine phosphatase, PP2A | 5.5 | 31 |
protein tyrosine phosphatase, PTPRC (CD45) | –2.9 | 32, 33 |
Results showing an inhibition or stimulation higher than 50% are considered to represent significant effects of the test compound.
Methods
ELISA Assay
Cell Culture
Crystal Violet Assay
Interaction Index

Western Blot
Rad51 Silencing in BxPC-3 Cells
Transduction of BxPC3 Cells
Western Blot
RT-PCR
Immunofluorescence
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00707.
Supporting Figures S1–S4, virtual screening protocol, and experimental details of all synthetic procedures together with characterization data for all intermediates and final compounds (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgment
We thank the Italian Institute of Technology and Bologna University for financial support. We thank G. Fox for copyediting and proofreading the manuscript. We thank P. G. Pelicci for useful discussions.
References
This article references 33 other publications.
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- 3Lord, C. J. and Ashworth, A. (2016) BRCAness revisited Nat. Rev. Cancer 16, 110– 120 DOI: 10.1038/nrc.2015.21Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xps1Cnsw%253D%253D&md5=c8b7d8b6cb711323711fb9840b416935BRCAness revisitedLord, Christopher J.; Ashworth, AlanNature Reviews Cancer (2016), 16 (2), 110-120CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)Over the past 20 years, there has been considerable progress in our understanding of the biol. functions of the BRCA1 and BRCA2 cancer susceptibility genes. This has led to the development of new therapeutic approaches that target tumors with loss-of-function mutations in either BRCA1 or BRCA2. Tumors that share mol. features of BRCA-mutant tumors - i.e., those with 'BRCAness' - may also respond to similar therapeutic approaches. Several paradigm shifts require a reassessment of the concept of BRCAness, how this property is assayed and its relevance to our understanding of tumor biol. and the treatment of cancer.
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- 5Deeks, E. D. (2015) Olaparib: first global approval Drugs 75, 231– 240 DOI: 10.1007/s40265-015-0345-6Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVejtbg%253D&md5=cd68d932b6532a87c0768590afd81058Olaparib: First Global ApprovalDeeks, Emma D.Drugs (2015), 75 (2), 231-240CODEN: DRUGAY; ISSN:0012-6667. (Springer International Publishing AG)A review. Olaparib (Lynparza) is an oral, small mol., poly (ADP-ribose) polymerase inhibitor being developed by AstraZeneca for the treatment of solid tumors. The primary indication that olaparib is being developed for is BRCA mutation-pos. ovarian cancer. A capsule formulation of the drug has received approval for use in this setting in the EU and USA, and a tablet formulation is in global phase III trials (including in the USA, EU, Australia, Brazil, Canada, China, Israel, Japan, Russia and South Korea). In addn., phase III trials in breast, gastric and pancreatic cancer are underway/planned, and phase I/II investigation is being conducted in other malignancies, including prostate cancer, non-small cell lung cancer, Ewing's sarcoma and advanced cancer. This article summarizes the milestones in the development of olaparib leading to this first approval for ovarian cancer.
- 6Kaelin, W. G. (2009) Synthetic lethality: a framework for the development of wiser cancer therapeutics Genome Med. 1, 99 DOI: 10.1186/gm99Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1Mjos1ajtQ%253D%253D&md5=a99d3271e6197956207403ca957d9bccSynthetic lethality: a framework for the development of wiser cancer therapeuticsKaelin William G JrGenome medicine (2009), 1 (10), 99 ISSN:.The challenge in medical oncology has always been to identify compounds that will kill, or at least tame, cancer cells while leaving normal cells unscathed. Most chemotherapeutic agents in use today were selected primarily for their ability to kill rapidly dividing cancer cells grown in cell culture and in mice, with their selectivity determined empirically during subsequent animal and human testing. Unfortunately, most of the drugs developed in this way have relatively low therapeutic indices (low toxic dose relative to the therapeutic dose). Recent advances in genomics are leading to a more complete picture of the range of mutations, both driver and passenger, present in human cancers. Synthetic lethality provides a conceptual framework for using this information to arrive at drugs that will preferentially kill cancer cells relative to normal cells. It also provides a possible way to tackle 'undruggable' targets. Two genes are synthetically lethal if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes leads to death. If one is a cancer-relevant gene, the task is to discover its synthetic lethal interactors, because targeting these would theoretically kill cancer cells mutant in the cancer-relevant gene while sparing cells with a normal copy of that gene. All cancer drugs in use today, including conventional cytotoxic agents and newer 'targeted' agents, target molecules that are present in both normal cells and cancer cells. Their therapeutic indices almost certainly relate to synthetic lethal interactions, even if those interactions are often poorly understood. Recent technical advances enable unbiased screens for synthetic lethal interactors to be undertaken in human cancer cells. These approaches will hopefully facilitate the discovery of safer, more efficacious anticancer drugs that exploit vulnerabilities that are unique to cancer cells by virtue of the mutations they have accrued during tumor progression.
- 7Fang, B. (2014) Development of synthetic lethality anticancer therapeutics J. Med. Chem. 57, 7859– 7873 DOI: 10.1021/jm500415tGoogle Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpt1Kltr4%253D&md5=a2da13d6bbc402fa474f7da3a9b6084eDevelopment of Synthetic Lethality Anticancer TherapeuticsFang, BingliangJournal of Medicinal Chemistry (2014), 57 (19), 7859-7873CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. The concept of synthetic lethality (the creation of a lethal phenotype from the combined effects of mutations in two or more genes) has recently been exploited in various efforts to develop new genotype-selective anticancer therapeutics. These efforts include screening for novel anticancer agents, identifying novel therapeutic targets, characterizing mechanisms of resistance to targeted therapy, and improving efficacies through the rational design of combination therapy. This review discusses recent developments in synthetic lethality anticancer therapeutics, including poly ADP-ribose polymerase inhibitors for BRCA1- and BRCA2-mutant cancers, checkpoint inhibitors for p53 mutant cancers, and small mol. agents targeting RAS gene mutant cancers. Because cancers are caused by mutations in multiple genes and abnormalities in multiple signaling pathways, synthetic lethality for a specific tumor suppressor gene or oncogene is likely cell context-dependent. Delineation of the mechanisms underlying synthetic lethality and identification of treatment response biomarkers will be crit. for the success of synthetic lethality anticancer therapy.
- 8Dedes, K. J., Wilkerson, P. M., Wetterskog, D., Weigelt, B., Ashworth, A., and Reis-Filho, J. S. (2011) Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations Cell Cycle 10, 1192– 1199 DOI: 10.4161/cc.10.8.15273Google ScholarThere is no corresponding record for this reference.
- 9Wyman, C. and Kanaar, R. (2004) Homologous recombination: Down to the wire Curr. Biol. 14, 629– 631 DOI: 10.1016/j.cub.2004.07.049Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmsVKnu70%253D&md5=5f080ad7434ac685c9a4696e46f6f8b9Homologous recombination: Down to the wireWyman, Claire; Kanaar, RolandCurrent Biology (2004), 14 (15), R629-R631CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)A review. Exchange of strands between homologous DNA mols. is catalyzed by evolutionarily conserved recombinases. These proteins can occur in different quaternary arrangements: rings or helical filaments. Recent results reveal that recombinase function follows from the filamentous form.
- 10Roy, R., Chun, J., and Powell, S. N. (2011) BRCA1 and BRCA2: different roles in a common pathway of genome protection Nat. Rev. Cancer 12, 68– 78 DOI: 10.1038/nrc3181Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38%252FpsFKquw%253D%253D&md5=2118b0146c814a16eb8d91bf6ab48d50BRCA1 and BRCA2: different roles in a common pathway of genome protectionRoy Rohini; Chun Jarin; Powell Simon NNature reviews. Cancer (2011), 12 (1), 68-78 ISSN:.The proteins encoded by the two major breast cancer susceptibility genes, BRCA1 and BRCA2, work in a common pathway of genome protection. However, the two proteins work at different stages in the DNA damage response (DDR) and in DNA repair. BRCA1 is a pleiotropic DDR protein that functions in both checkpoint activation and DNA repair, whereas BRCA2 is a mediator of the core mechanism of homologous recombination. The links between the two proteins are not well understood, but they must exist to explain the marked similarity of human cancer susceptibility that arises with germline mutations in these genes. As discussed here, the proteins work in concert to protect the genome from double-strand DNA damage during DNA replication.
- 11Davies, A. a, Masson, J. Y., McIlwraith, M. J., Stasiak, A. Z., Stasiak, A., Venkitaraman, A. R., and West, S. C. (2001) Role of BRCA2 in control of the RAD51 recombination and DNA repair protein Mol. Cell 7, 273– 282 DOI: 10.1016/S1097-2765(01)00175-7Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXis1Kitrc%253D&md5=9295ae3b5e51e58beeed4f9adb6e20e2Role of BRCA2 in control of the RAD51 recombination and DNA repair proteinDavies, Adelina A.; Masson, Jean-Yves; McIlwraith, Michael J.; Stasiak, Alicja Z.; Stasiak, Andrzej; Venkitaraman, Ashok R.; West, Stephen C.Molecular Cell (2001), 7 (2), 273-282CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-assocd. BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-assocd. BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.
- 12Carreira, A., Hilario, J., Amitani, I., Baskin, R. J., Shivji, M. K. K., Venkitaraman, A. R., and Kowalczykowski, S. C. (2009) The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51 Cell 136, 1032– 1043 DOI: 10.1016/j.cell.2009.02.019Google ScholarThere is no corresponding record for this reference.
- 13Pellegrini, L., Yu, D. S., Lo, T., Anand, S., Lee, M., Blundell, T. L., and Venkitaraman, A. R. (2002) Insights into DNA recombination from the structure of a RAD51-BRCA2 complex Nature 420, 287– 293 DOI: 10.1038/nature01230Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XovVektLY%253D&md5=5dc084f8efb75cf95bc31d4f5b1797e3Insights into DNA recombination from the structure of a RAD51-BRCA2 complexPellegrini, Luca; Yu, David S.; Lo, Thomas; Anand, Shubha; Lee, MiYoung; Blundell, Tom L.; Venkitaraman, Ashok R.Nature (London, United Kingdom) (2002), 420 (6913), 287-293CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The breast cancer susceptibility protein BRCA2 controls the function of RAD51, a recombinase enzyme, in pathways for DNA repair by homologous recombination. We report here the structure of a complex between an evolutionarily conserved sequence in BRCA2 (the BRC repeat) and the RecA-homol. domain of RAD51. The BRC repeat mimics a motif in RAD51 that serves as an interface for oligomerization between individual RAD51 monomers, thus enabling BRCA2 to control the assembly of the RAD51 nucleoprotein filament, which is essential for strand-pairing reactions during DNA recombination. The RAD51 oligomerization motif is highly conserved among RecA-like recombinases, highlighting a common evolutionary origin for the mechanism of nucleoprotein filament formation, mirrored in the BRC repeat. Cancer-assocd. mutations that affect the BRC repeat disrupt its predicted interaction with RAD51, yielding structural insight into mechanisms for cancer susceptibility.
- 14Rajendra, E. and Venkitaraman, A. R. (2010) Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase Nucleic Acids Res. 38, 82– 96 DOI: 10.1093/nar/gkp873Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtF2nuw%253D%253D&md5=8fec23291c3037866be93b27f8a6f685Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinaseRajendra, Eeson; Venkitaraman, Ashok R.Nucleic Acids Research (2010), 38 (1), 82-96CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approx. 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC-RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.
- 15Nomme, J., Renodon-Cornière, A., Asanomi, Y., Sakaguchi, K., Stasiak, A. Z., Stasiak, A., Norden, B., Tran, V., and Takahashi, M. (2010) Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide J. Med. Chem. 53, 5782– 5791 DOI: 10.1021/jm1002974Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXos1ersro%253D&md5=3b6a3ee9962a5dc4966e5dad9653835dDesign of Potent Inhibitors of Human RAD51 Recombinase Based on BRC Motifs of BRCA2 Protein: Modeling and Experimental Validation of a Chimera PeptideNomme, Julian; Renodon-Corniere, Axelle; Asanomi, Yuya; Sakaguchi, Kazuyasu; Stasiak, Alicja Z.; Stasiak, Andrzej; Norden, Bengt; Tran, Vinh; Takahashi, MasayukiJournal of Medicinal Chemistry (2010), 53 (15), 5782-5791CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochem. testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a mol. model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This anal. enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.
- 16Abbott, D. W., Freeman, M. L., and Holt, J. T. (1998) Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells J.Natl.Cancer Inst. 90, 978– 985 DOI: 10.1093/jnci/90.13.978Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXksVSnsbg%253D&md5=b26b75011711ff951d10f6398e7dc31eDouble-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cellsAbbott, Derek W.; Freeman, Michael L.; Holt, Jeffrey T.Journal of the National Cancer Institute (1998), 90 (13), 978-985CODEN: JNCIEQ; ISSN:0027-8874. (Oxford University Press)The protein product of the BRCA2 gene mediates repair of double-strand breaks in DNA. Because a no. of cancer therapies exert cytotoxic effects via the initiation of double-strand breaks, cancers comprised of cells carrying BRCA2 gene mutations may be more amenable to treatment with agents that cause such breaks. We identified a human pancreatic adenocarcinoma cell line lacking one copy of the BRCA2 gene and contg. a mutation (6174delT) in the remaining copy. In vitro and in vivo expts. were conducted with this cell line and with other carcinoma cell lines matched for similar genetic mutations, similar differentiation status, and/or similar carcinoma type to examine double-strand break repair, sensitivity to drugs that induce double-strand breaks, and radiation sensitivity. BRCA2-defective cells were unable to repair the double-strand DNA breaks induced by ionizing radiation. These cells were also markedly sensitive to mitoxantrone, amsacrine, and etoposide (drugs that induce double-strand breaks and to ionizing radiation). Introduction of antisense BRCA2 deoxyribonucleotides into cells possessing normal BRCA2 function led to increased sensitivity to mitoxantrone. Tumors formed by injection of BRCA2-defective cells into nude mice were highly sensitive (>90% tumor size redn.) to both ionizing radiation and mitoxantrone when compared with tumors exhibiting normal BRCA2 function. Histol. anal. of irradiated BRCA2-defective tumors showed a large degree of necrosis compared with that obsd. for control tumors possessing normal BRCA2 function. BRCA2-defective cancer cells are highly sensitive to agents that cause double-strand breaks in DNA.
- 17Jdey, W., Thierry, S., Russo, C., Devun, F., Al Abo, M., Noguiez-Hellin, P., Sun, J.-S., Barillot, E., Zinovyev, A., Kuperstein, I., Pommier, Y., and Dutreix, M. (2017) Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP Inhibitors Clin. Cancer Res. 23, 1001– 1011 DOI: 10.1158/1078-0432.CCR-16-1193Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXisFyktrk%253D&md5=263d4607de066a649953ad8918678b80Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP InhibitorsJdey, Wael; Thierry, Sylvain; Russo, Christophe; Devun, Flavien; Al Abo, Muthana; Noguiez-Hellin, Patricia; Sun, Jian-Sheng; Barillot, Emmanuel; Zinovyev, Andrei; Kuperstein, Inna; Pommier, Yves; Dutreix, MarieClinical Cancer Research (2017), 23 (4), 1001-1011CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Purpose: Cancer treatments using tumor defects in DNA repair pathways have shown promising results but are restricted to small subpopulations of patients. The most advanced drugs in this field are PARP inhibitors (PARPi), which trigger synthetic lethality in tumors with homologous recombination (HR) deficiency. Using AsiDNA, an inhibitor of HR and nonhomologous end joining, together with PARPi should allow bypassing the genetic restriction for PARPi efficacy. Exptl. Design: We characterized the DNA repair inhibition activity of PARPi (olaparib) and AsiDNA by monitoring repair foci formation and DNA damage. We analyzed the cell survival to standalone and combined treatments of 21 tumor cells and three nontumor cells. In 12 breast cancer (BC) cell lines, correlation with sensitivity to each drug and transcriptome were statistically analyzed to identify resistance pathways. Results: Mol. analyses demonstrate that olaparib and AsiDNA resp. prevent recruitment of XRCC1 and RAD51/53BP1 repair enzymes to damage sites. Combination of both drugs increases the accumulation of unrepaired damage resulting in an increase of cell death in all tumor cells. In contrast, nontumor cells do not show an increase of DNA damage nor lethality. Anal. of multilevel omics data from BC cells highlighted different DNA repair and cell-cycle mol. profiles assocd. with resistance to AsiDNA or olaparib, rationalizing combined treatment. Treatment synergy was also confirmed with six other PARPi in development. Conclusions: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Clin Cancer Res; 23(4); 1001-11. ©2016 AACR.
- 18Mistrik, M., Oplustilova, L., Lukas, J., and Bartek, J. (2009) Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis Cell Cycle 8, 2592– 2599 DOI: 10.4161/cc.8.16.9331Google ScholarThere is no corresponding record for this reference.
- 19Vernole, P., Muzi, A., Volpi, A., Dorio, A. S., Terrinoni, A., Shah, G. M., and Graziani, G. (2009) Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cells Mutat. Res., Fundam. Mol. Mech. Mutagen. 664, 39– 47 DOI: 10.1016/j.mrfmmm.2009.02.005Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXltVGjsLo%253D&md5=14729d177ac252141141be6bf7ffb010Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cellsVernole, Patrizia; Muzi, Alessia; Volpi, Antonio; Dorio, Annalisa S.; Terrinoni, Alessandro; Shah, Girish M.; Graziani, GraziaMutation Research, Fundamental and Molecular Mechanisms of Mutagenesis (2009), 664 (1-2), 39-47CODEN: MUREAV; ISSN:0027-5107. (Elsevier B.V.)Mismatch repair (MMR) has been shown to control homologous recombination (HR) by aborting strand exchange between divergent sequences. It has previously been demonstrated that MMR-deficient tumor cells are more resistant to chromosomal damage induced by bleomycin (BLM) during the G2 phase, likely due to the lack of the MMR inhibitory effect on HR. Aim of this study was to investigate whether inhibition of HR by the nucleoside analog BVDU [(E)-5(2-bromovinyl)-2'-deoxyuridine, brivudin], or silencing of genes involved in HR function, might affect sensitivity of MMR-deficient tumor cells to DNA damage induced by BLM in G2. The results indicated that BVDU increased chromatid damage and DNA double strand breaks induced by BLM only in MMR-deficient MT-1, HL-60R, HCT116 cells, which are more resistant to BLM with respect to MMR-proficient TK-6, HL-60S and HCT116/3-6 lines. Silencing of RAD51, a key component of HR, increased sensitivity of MMR-deficient HCT-15 cells to BLM clastogenicity; in this case combined treatment with BVDU had no addnl. effect. Similarly, treatment with BVDU did not affect BLM clastogenicity in CAPAN-1 cells, characterized by a defective HR due to BRCA2 mutations. Conversely, BVDU increased chromatid breaks induced by BLM in HCT-15 cells transiently silenced for DNA-PK catalytic subunit, which plays a key role in non-homologous end joining. The BVDU-mediated increase of chromatid breaks in MMR-deficient cells did not depend on its previously reported inhibitory effect on poly(ADP-ribose) polymerase (PARP). In fact, it was obsd. also in cells stably silenced for PARP-1, which is responsible for most of cellular PARP activity. These data support the suggestion that the higher sensitivity of MMR-proficient vs. MMR-deficient cells to BLM-induced chromatid breaks in the G2 phase is a consequence of the inhibition of HR by MMR. In MMR-deficient cells, BVDU attenuates the repair of BLM-induced DSBs and this is likely to occur via inhibition of HR.
- 20Maxwell, K. N. and Domchek, S. M. (2012) Cancer treatment according to BRCA1 and BRCA2 mutations Nat. Rev. Clin. Oncol. 9, 520– 528 DOI: 10.1038/nrclinonc.2012.123Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1Kjs7zM&md5=c6da652ff478b6175e184f98cc7c13c2Cancer treatment according to BRCA1 and BRCA2 mutationsMaxwell, Kara N.; Domchek, Susan M.Nature Reviews Clinical Oncology (2012), 9 (9), 520-528CODEN: NRCOAA; ISSN:1759-4774. (Nature Publishing Group)A review. Identification of germline mutations assocd. with significant cancer susceptibility has the potential to change all aspects of an individual's care, from screening to cancer treatment. For example, women with germline mutations in BRCA1 and BRCA2 have markedly elevated risks of breast and ovarian cancer and the identification of these germline mutations has led to specific screening and prevention strategies. More recently, advances in the understanding of the biol. function of BRCA1 and BRCA2 have led to clin. trials testing targeted therapies in this population, particularly poly(ADP-ribose) polymerase (PARP) inhibitors. Unfortunately, the development of PARP inhibitors has not been as rapid as anticipated and has been more challenging than expected. Somatic mutations identified in many cancer types have allowed the development of therapeutics that target these mutated genes, and many of these agents obtained rapid regulatory approval and are currently in widespread clin. practice. Diagnostic testing has a central role in targeted cancer therapeutics for both somatic and germline mutations. Although the era of mol. medicine and targeted therapies has led to significant changes in the practice of oncol., new challenges continue to arise.
- 21Banerjee, S., Kaye, S. B., and Ashworth, A. (2010) Making the best of PARP inhibitors in ovarian cancer Nat. Rev. Clin. Oncol. 7, 508– 519 DOI: 10.1038/nrclinonc.2010.116Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVyjsr%252FM&md5=c88eb3b20aeffa2e1875aee7cfb18594Making the best of PARP inhibitors in ovarian cancerBanerjee, Susana; Kaye, Stan B.; Ashworth, AlanNature Reviews Clinical Oncology (2010), 7 (9), 508-519CODEN: NRCOAA; ISSN:1759-4774. (Nature Publishing Group)A review. Inhibitors of poly(ADP-ribose)polymerase (PARP) have shown promise as therapeutic agents for the treatment of ovarian cancers with mutations in BRCA1 or BRCA2. By exploiting the synthetic lethal interaction that exists between PARP inhibition and BRCA mutations, these agents specifically kill cancer cells by targeting their DNA repair system. The authors of this Review describe the importance of BRCA mutations for the efficacy of PARP inhibitors. They also discuss the preclin. and clin. trial results of PARP inhibitors, the challenges related to the use of these agents, and future directions. Drugs that inhibit the enzyme poly(ADP-ribose)polymerase (PARP) are showing considerable promise for the treatment of cancers that have mutations in the BRCA1 or BRCA2 tumor suppressors. This therapeutic approach exploits a synthetic lethal strategy to target the specific DNA repair pathway in these tumors. High-grade ovarian cancers have a generally poor prognosis, and accumulating evidence suggests that mutations in BRCA1 or BRCA2, or silencing of BRCA1 by promoter methylation, may be common in this disease. Here, we consider how the potential benefit of PARP inhibitors might be maximized in ovarian cancer. We suggest that it will be crucial to explore novel therapeutic trial strategies and drug combinations, and incorporate robust biomarkers predictive of response if these drugs are to reach their full potential.
- 22Montoni, A., Robu, M., Pouliot, É., and Shah, G. M. (2013) Resistance to PARP-inhibitors in cancer therapy Front. Pharmacol. 4 (FEB) 1– 7 DOI: 10.3389/fphar.2013.00018Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht12iu7fN&md5=bda6a7ac6dfb80c4d6be6fe130202c7dResistance to PARP-inhibitors in cancer therapyMontoni, Alicia; Robu, Mihaela; Pouliot, Emilie; Shah, Girish M.Frontiers in Pharmacology (2013), 4 (), 1-7CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)A review. The pharmacol. inhibitors of poly(ADP-ribose) polymerase (PARP) family of proteins have shown promising results in preclin. studies and clin. trials as a monotherapy or in combination therapy for some cancers. Thus, usage of PARP-inhibitors (PARPi) in cancer therapy is bound to increase with time, but resistance of cancer cells to PARPi is also beginning to be obsd. Here we review different known and potential mechanisms by which: (i) PARPi kill cancer cells; and (ii) cancer cells develop resistance to PARPi. Understanding the lethality caused by PARPi and the countermeasures deployed by cancers cells to survive PARPi will help us rationalize the use of this new 1class of drugs in cancer therapy.
- 23Liu, Y., Burness, M. L., Martin-Trevino, R., Guy, J., Bai, S., Harouaka, R., Brooks, M. D., Shang, L., Fox, A., Luther, T. K., Davis, A., Baker, T. L., Colacino, J., Clouthier, S. G., Shao, Z., Wicha, M. S., and Liu, S. (2017) RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer Clin. Cancer Res. 23, 514– 522 DOI: 10.1158/1078-0432.CCR-15-1348Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVeitLs%253D&md5=56dce7989dcf9edb314d3927adf180dfRAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast CancerLiu, Yajing; Burness, Monika L.; Martin-Trevino, Rachel; Guy, Joey; Bai, Shoumin; Harouaka, Ramdane; Brooks, Michael D.; Shang, Li; Fox, Alex; Luther, Tahra K.; Davis, April; Baker, Trenton L.; Colacino, Justin; Clouthier, Shawn G.; Shao, Zhi-ming; Wicha, Max S.; Liu, SulingClinical Cancer Research (2017), 23 (2), 514-522CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Introduction: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)-deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-neg. breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display "cancer stem cell" properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. Exptl. Design: We detd. the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. Results: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1-wild-type cells were relatively resistant to PARP inhibition alone, but redn. of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. Conclusions: Our data suggest that in both BRCA1-mutant and BRCA1-wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi.
- 24Klein, H. L. (2008) The consequences of Rad51 overexpression for normal and tumor cells DNA Repair 7, 686– 693 DOI: 10.1016/j.dnarep.2007.12.008Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXls1Sgsbw%253D&md5=824aee339ab1c58745af64778d24378aThe consequences of Rad51 overexpression for normal and tumor cellsKlein, Hannah L.DNA Repair (2008), 7 (5), 686-693CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)A review. The Rad51 recombinase is an essential factor for homologous recombination and the repair of DNA double strand breaks, binding transiently to both single stranded and double stranded DNA during the recombination reaction. The use of a homologous recombination mechanism to repair DNA damage is controlled at several levels, including the binding of Rad51 to single stranded DNA to form the Rad51 nucleofilament, which is controlled through the action of DNA helicases that can counteract nucleofilament formation. Overexpression of Rad51 in different organisms and cell types has a wide assortment of consequences, ranging from increased homologous recombination and increased resistance to DNA damaging agents to disruption of the cell cycle and apoptotic cell death. Rad51 expression is increased in p53-neg. cells, and since p53 is often mutated in tumor cells, there is a tendency for Rad51 to be overexpressed in tumor cells, leading to increased resistance to DNA damage and drugs used in chemotherapies. As cells with increased Rad51 levels are more resistant to DNA damage, there is a selection for tumor cells to have higher Rad51 levels. While increased Rad51 can provide drug resistance, it also leads to increased genomic instability and may contribute to carcinogenesis.
- 25Dos Santos Ferreira, A. C., Fernandes, R. A., Kwee, J. K., and Klumb, C. E. (2012) Histone deacetylase inhibitor potentiates chemotherapy-induced apoptosis through Bim upregulation in Burkitt’s lymphoma cells J. Cancer Res. Clin. Oncol. 138, 317– 325 DOI: 10.1007/s00432-011-1093-yGoogle ScholarThere is no corresponding record for this reference.
- 26Barrett, A. J. and Kirschke, H. (1981) Cathepsin B, cathepsin H, and cathepsin L Methods Enzymol. 80, 535– 561 DOI: 10.1016/S0076-6879(81)80043-2Google ScholarThere is no corresponding record for this reference.
- 27Yasuda, Y., Kageyama, T., Akamine, A., Shibata, M., Kominami, E., Uchiyama, Y., and Yamamoto, K. (1999) Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D J. Biochem. 125, 1137– 1143 DOI: 10.1093/oxfordjournals.jbchem.a022396Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXlsVygsr4%253D&md5=23d0e8c891cf0ed5c38fa1191d18ec9aCharacterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin DYasuda, Yoshiyuki; Kageyama, Takashi; Akamine, Akifumi; Shibata, Masahiro; Kominami, Eiki; Uchiyama, Yasuo; Yamamoto, KenjiJournal of Biochemistry (1999), 125 (6), 1137-1143CODEN: JOBIAO; ISSN:0021-924X. (Japanese Biochemical Society)Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiol. and pathol. degrdn. of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the sepn. of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)γ-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 μM-1·s-1 for cathepsin E and 15.6 μM-1·s-1 for cathepsin D). A very acidic pH optimum of 4.0 was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(Dnp)γ-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 μM-1·s-1 for cathepsin E and 16.3 μM-1·s-1 for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive detn. of the precise levels of cathepsins E and D in crude cell exts. of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.
- 28Brömme, D. and Mcgrath, M. E. (1996) High level expression and crystallization of recombinant human cathepsin S Protein Sci. 5, 789– 791 DOI: 10.1002/pro.5560050426Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XitFSgsL8%253D&md5=f5940b367e9810071c5e100c74179586High level expression and crystallization of recombinant human cathepsin SBromme, Dieter; McGrath, Mary E.Protein Science (1996), 5 (4), 789-91CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)The authors have expressed active human cathepsin S to 60 mg/L in Sf9 cells using a baculovirus system. Prodn. of milligram quantities has facilitated crystallog. studies to det. the structure of this enzyme, which has unique properties among lysosomal cysteine proteinases. Recombinant, irreversibly inhibited cathepsin S was crystd. from ammonium phosphate at 17°. The crystals diffract to at least 2.3 Å, and belong to the orthorhombic crystal system with a primitive lattice. Approx. cell dimensions are: a = 37.7 Å, b = 73.9 Å, and c = 106.7 Å. There is most likely one mol. per asym. unit.
- 29Nagase, H., Fields, C. G., and Fields, G. B. (1994) Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3) J. Biol. Chem. 269, 20952– 20957Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmsV2hsrY%253D&md5=6b997ba5965f2335020a79fa6b681deeDesign and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3)Nagase, Hideaki; Fields, Cynthia G.; Fields, Gregg B.Journal of Biological Chemistry (1994), 269 (33), 20952-7CODEN: JBCHA3; ISSN:0021-9258.Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. The authors examd. known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km ∼11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation expts. were additive, the authors found discrepancies between predicted and exptl. kcat/Km values, one on the order of 2000-5000. Thus, the design of addnl. discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.
- 30Tierno, M. B., Johnston, P. a, Foster, C., Skoko, J. J., Shinde, S. N., Shun, T. Y., and Lazo, J. S. (2007) Development and optimization of high-throughput in vitro protein phosphatase screening assays Nat. Protoc. 2, 1134– 1144 DOI: 10.1038/nprot.2007.155Google ScholarThere is no corresponding record for this reference.
- 31Davis, M. I., Ronesi, J., and Lovinger, D. M. (2003) A predominant role for inhibition of the adenylate cyclase/protein kinase A pathway in ERK activation by cannabinoid receptor 1 in N1E-115 neuroblastoma cells J. Biol. Chem. 278, 48973– 48980 DOI: 10.1074/jbc.M305697200Google ScholarThere is no corresponding record for this reference.
- 32Montalibet, J., Skorey, K. I., and Kennedy, B. P. (2005) Protein tyrosine phosphatase: Enzymatic assays Methods 35, 2– 8 DOI: 10.1016/j.ymeth.2004.07.002Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVGgu77I&md5=95ed7b0f7de47a71e0aa5194597c6576Protein tyrosine phosphatase: enzymatic assaysMontalibet, Jacqueline; Skorey, Kathryn I.; Kennedy, Brian P.Methods (San Diego, CA, United States) (2005), 35 (1), 2-8CODEN: MTHDE9; ISSN:1046-2023. (Elsevier)Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger mols. such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the detns. of the rate of dephosphorylation and calcns. of kinetic consts. such as kcat and Km. These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.
- 33Welte, S., Baringhaus, K. H., Schmider, W., Müller, G., Petry, S., and Tennagels, N. (2005) 6,8-Difluoro-4-methylumbiliferyl phosphate: A fluorogenic substrate for protein tyrosine phosphatases Anal. Biochem. 338, 32– 38 DOI: 10.1016/j.ab.2004.11.047Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlenurk%253D&md5=8fbf39bd52aacefebe63ba75d1d9d5da6,8-Difluoro-4-methylumbiliferyl phosphate: a fluorogenic substrate for protein tyrosine phosphatasesWelte, Stefan; Baringhaus, Karl-Heinz; Schmider, Wolfgang; Mueller, Guenter; Petry, Stefan; Tennagels, NorbertAnalytical Biochemistry (2005), 338 (1), 32-38CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor consts. for DIFMUP cleavage were comparable with those of the widely used but less discriminative and practicable substrates, para-nitrophenylphosphate and phosphotyrosine-contg. peptides, resp. Furthermore, the continuous and highly sensitive assay allows fast and accurate investigations of the type, kinetic behavior, and binding mode of small-mol. inhibitors. We discuss the validation of this assay system for various PTPs and its use in inhibitor screening for PTP1B.
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Abstract
Figure 1
Figure 1. (a) Rad51–BRCA2 BRC repeat complex. Rad51 is represented as a surface, BRCA as a cartoon. The two hot spots of the interaction between the proteins (Phe1524 and Phe1546) are highlighted in sticks. (b) SiteMap hydrophobic (green), hydrogen-bond donor (blue), and hydrogen-bond acceptor (red) maps for 1N0W in the proximity of Phe1524. (c) Schematic representation of the virtual screening protocol. The numbers on the left column represent the number of compounds in each step; the filters used to narrow down the compounds are reported on the right column.
Figure 2
Figure 2. Experiments on cell cultures. (a) Effect of 7 days of Olaparib treatment on the viability of BxPC-3 and Capan-1 cells. (b, c) Dose–response curves assessed in BxPC-3 (b) and Capan-1 (c) cells exposed to 2. The compound doses used for combination experiments have been sketched with dark symbols. (d, e) Combination experiments of Olaparib with 2 in BxPC-3 (d) and Capan-1 (e). In the BxPC-3 cell line, the results obtained with Olaparib were compared with those obtained with Olaparib in combination with 2 using two-way ANOVA; for both 20 and 30 μM 2, p values < 0.001 were obtained at 3–6 days of treatment. (f) Combination experiment of Olaparib with 2, in BxPC-3 cells after stable Rad51 silencing, compared with scrambled controls. The effect was evaluated after a 96 h treatment. Results obtained in siRad51 cultures were statistically analyzed by ANOVA followed by Bonferroni post test. (g) Densitometric reading of immunoblots evaluating γH2AX expression (normalized on Actin levels) in cells treated with Cisplatin (12 μM) and 2 at the same doses used in the Olaparib combination experiments. For a detailed explanation, see the text. Vertical lines indicate the samples for comparison. (h) Immunofluorescence γH2AX detection in nuclei of BxPC-3 cells treated with RAD51 siRNA or 2. The percentage of positive cells are reported in the graph of frame (i).
References
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- 7Fang, B. (2014) Development of synthetic lethality anticancer therapeutics J. Med. Chem. 57, 7859– 7873 DOI: 10.1021/jm500415t7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpt1Kltr4%253D&md5=a2da13d6bbc402fa474f7da3a9b6084eDevelopment of Synthetic Lethality Anticancer TherapeuticsFang, BingliangJournal of Medicinal Chemistry (2014), 57 (19), 7859-7873CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. The concept of synthetic lethality (the creation of a lethal phenotype from the combined effects of mutations in two or more genes) has recently been exploited in various efforts to develop new genotype-selective anticancer therapeutics. These efforts include screening for novel anticancer agents, identifying novel therapeutic targets, characterizing mechanisms of resistance to targeted therapy, and improving efficacies through the rational design of combination therapy. This review discusses recent developments in synthetic lethality anticancer therapeutics, including poly ADP-ribose polymerase inhibitors for BRCA1- and BRCA2-mutant cancers, checkpoint inhibitors for p53 mutant cancers, and small mol. agents targeting RAS gene mutant cancers. Because cancers are caused by mutations in multiple genes and abnormalities in multiple signaling pathways, synthetic lethality for a specific tumor suppressor gene or oncogene is likely cell context-dependent. Delineation of the mechanisms underlying synthetic lethality and identification of treatment response biomarkers will be crit. for the success of synthetic lethality anticancer therapy.
- 8Dedes, K. J., Wilkerson, P. M., Wetterskog, D., Weigelt, B., Ashworth, A., and Reis-Filho, J. S. (2011) Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations Cell Cycle 10, 1192– 1199 DOI: 10.4161/cc.10.8.15273There is no corresponding record for this reference.
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- 10Roy, R., Chun, J., and Powell, S. N. (2011) BRCA1 and BRCA2: different roles in a common pathway of genome protection Nat. Rev. Cancer 12, 68– 78 DOI: 10.1038/nrc318110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38%252FpsFKquw%253D%253D&md5=2118b0146c814a16eb8d91bf6ab48d50BRCA1 and BRCA2: different roles in a common pathway of genome protectionRoy Rohini; Chun Jarin; Powell Simon NNature reviews. Cancer (2011), 12 (1), 68-78 ISSN:.The proteins encoded by the two major breast cancer susceptibility genes, BRCA1 and BRCA2, work in a common pathway of genome protection. However, the two proteins work at different stages in the DNA damage response (DDR) and in DNA repair. BRCA1 is a pleiotropic DDR protein that functions in both checkpoint activation and DNA repair, whereas BRCA2 is a mediator of the core mechanism of homologous recombination. The links between the two proteins are not well understood, but they must exist to explain the marked similarity of human cancer susceptibility that arises with germline mutations in these genes. As discussed here, the proteins work in concert to protect the genome from double-strand DNA damage during DNA replication.
- 11Davies, A. a, Masson, J. Y., McIlwraith, M. J., Stasiak, A. Z., Stasiak, A., Venkitaraman, A. R., and West, S. C. (2001) Role of BRCA2 in control of the RAD51 recombination and DNA repair protein Mol. Cell 7, 273– 282 DOI: 10.1016/S1097-2765(01)00175-711https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXis1Kitrc%253D&md5=9295ae3b5e51e58beeed4f9adb6e20e2Role of BRCA2 in control of the RAD51 recombination and DNA repair proteinDavies, Adelina A.; Masson, Jean-Yves; McIlwraith, Michael J.; Stasiak, Alicja Z.; Stasiak, Andrzej; Venkitaraman, Ashok R.; West, Stephen C.Molecular Cell (2001), 7 (2), 273-282CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-assocd. BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-assocd. BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.
- 12Carreira, A., Hilario, J., Amitani, I., Baskin, R. J., Shivji, M. K. K., Venkitaraman, A. R., and Kowalczykowski, S. C. (2009) The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51 Cell 136, 1032– 1043 DOI: 10.1016/j.cell.2009.02.019There is no corresponding record for this reference.
- 13Pellegrini, L., Yu, D. S., Lo, T., Anand, S., Lee, M., Blundell, T. L., and Venkitaraman, A. R. (2002) Insights into DNA recombination from the structure of a RAD51-BRCA2 complex Nature 420, 287– 293 DOI: 10.1038/nature0123013https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XovVektLY%253D&md5=5dc084f8efb75cf95bc31d4f5b1797e3Insights into DNA recombination from the structure of a RAD51-BRCA2 complexPellegrini, Luca; Yu, David S.; Lo, Thomas; Anand, Shubha; Lee, MiYoung; Blundell, Tom L.; Venkitaraman, Ashok R.Nature (London, United Kingdom) (2002), 420 (6913), 287-293CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The breast cancer susceptibility protein BRCA2 controls the function of RAD51, a recombinase enzyme, in pathways for DNA repair by homologous recombination. We report here the structure of a complex between an evolutionarily conserved sequence in BRCA2 (the BRC repeat) and the RecA-homol. domain of RAD51. The BRC repeat mimics a motif in RAD51 that serves as an interface for oligomerization between individual RAD51 monomers, thus enabling BRCA2 to control the assembly of the RAD51 nucleoprotein filament, which is essential for strand-pairing reactions during DNA recombination. The RAD51 oligomerization motif is highly conserved among RecA-like recombinases, highlighting a common evolutionary origin for the mechanism of nucleoprotein filament formation, mirrored in the BRC repeat. Cancer-assocd. mutations that affect the BRC repeat disrupt its predicted interaction with RAD51, yielding structural insight into mechanisms for cancer susceptibility.
- 14Rajendra, E. and Venkitaraman, A. R. (2010) Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase Nucleic Acids Res. 38, 82– 96 DOI: 10.1093/nar/gkp87314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtF2nuw%253D%253D&md5=8fec23291c3037866be93b27f8a6f685Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinaseRajendra, Eeson; Venkitaraman, Ashok R.Nucleic Acids Research (2010), 38 (1), 82-96CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approx. 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC-RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.
- 15Nomme, J., Renodon-Cornière, A., Asanomi, Y., Sakaguchi, K., Stasiak, A. Z., Stasiak, A., Norden, B., Tran, V., and Takahashi, M. (2010) Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide J. Med. Chem. 53, 5782– 5791 DOI: 10.1021/jm100297415https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXos1ersro%253D&md5=3b6a3ee9962a5dc4966e5dad9653835dDesign of Potent Inhibitors of Human RAD51 Recombinase Based on BRC Motifs of BRCA2 Protein: Modeling and Experimental Validation of a Chimera PeptideNomme, Julian; Renodon-Corniere, Axelle; Asanomi, Yuya; Sakaguchi, Kazuyasu; Stasiak, Alicja Z.; Stasiak, Andrzej; Norden, Bengt; Tran, Vinh; Takahashi, MasayukiJournal of Medicinal Chemistry (2010), 53 (15), 5782-5791CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochem. testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a mol. model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This anal. enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.
- 16Abbott, D. W., Freeman, M. L., and Holt, J. T. (1998) Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells J.Natl.Cancer Inst. 90, 978– 985 DOI: 10.1093/jnci/90.13.97816https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXksVSnsbg%253D&md5=b26b75011711ff951d10f6398e7dc31eDouble-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cellsAbbott, Derek W.; Freeman, Michael L.; Holt, Jeffrey T.Journal of the National Cancer Institute (1998), 90 (13), 978-985CODEN: JNCIEQ; ISSN:0027-8874. (Oxford University Press)The protein product of the BRCA2 gene mediates repair of double-strand breaks in DNA. Because a no. of cancer therapies exert cytotoxic effects via the initiation of double-strand breaks, cancers comprised of cells carrying BRCA2 gene mutations may be more amenable to treatment with agents that cause such breaks. We identified a human pancreatic adenocarcinoma cell line lacking one copy of the BRCA2 gene and contg. a mutation (6174delT) in the remaining copy. In vitro and in vivo expts. were conducted with this cell line and with other carcinoma cell lines matched for similar genetic mutations, similar differentiation status, and/or similar carcinoma type to examine double-strand break repair, sensitivity to drugs that induce double-strand breaks, and radiation sensitivity. BRCA2-defective cells were unable to repair the double-strand DNA breaks induced by ionizing radiation. These cells were also markedly sensitive to mitoxantrone, amsacrine, and etoposide (drugs that induce double-strand breaks and to ionizing radiation). Introduction of antisense BRCA2 deoxyribonucleotides into cells possessing normal BRCA2 function led to increased sensitivity to mitoxantrone. Tumors formed by injection of BRCA2-defective cells into nude mice were highly sensitive (>90% tumor size redn.) to both ionizing radiation and mitoxantrone when compared with tumors exhibiting normal BRCA2 function. Histol. anal. of irradiated BRCA2-defective tumors showed a large degree of necrosis compared with that obsd. for control tumors possessing normal BRCA2 function. BRCA2-defective cancer cells are highly sensitive to agents that cause double-strand breaks in DNA.
- 17Jdey, W., Thierry, S., Russo, C., Devun, F., Al Abo, M., Noguiez-Hellin, P., Sun, J.-S., Barillot, E., Zinovyev, A., Kuperstein, I., Pommier, Y., and Dutreix, M. (2017) Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP Inhibitors Clin. Cancer Res. 23, 1001– 1011 DOI: 10.1158/1078-0432.CCR-16-119317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXisFyktrk%253D&md5=263d4607de066a649953ad8918678b80Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP InhibitorsJdey, Wael; Thierry, Sylvain; Russo, Christophe; Devun, Flavien; Al Abo, Muthana; Noguiez-Hellin, Patricia; Sun, Jian-Sheng; Barillot, Emmanuel; Zinovyev, Andrei; Kuperstein, Inna; Pommier, Yves; Dutreix, MarieClinical Cancer Research (2017), 23 (4), 1001-1011CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Purpose: Cancer treatments using tumor defects in DNA repair pathways have shown promising results but are restricted to small subpopulations of patients. The most advanced drugs in this field are PARP inhibitors (PARPi), which trigger synthetic lethality in tumors with homologous recombination (HR) deficiency. Using AsiDNA, an inhibitor of HR and nonhomologous end joining, together with PARPi should allow bypassing the genetic restriction for PARPi efficacy. Exptl. Design: We characterized the DNA repair inhibition activity of PARPi (olaparib) and AsiDNA by monitoring repair foci formation and DNA damage. We analyzed the cell survival to standalone and combined treatments of 21 tumor cells and three nontumor cells. In 12 breast cancer (BC) cell lines, correlation with sensitivity to each drug and transcriptome were statistically analyzed to identify resistance pathways. Results: Mol. analyses demonstrate that olaparib and AsiDNA resp. prevent recruitment of XRCC1 and RAD51/53BP1 repair enzymes to damage sites. Combination of both drugs increases the accumulation of unrepaired damage resulting in an increase of cell death in all tumor cells. In contrast, nontumor cells do not show an increase of DNA damage nor lethality. Anal. of multilevel omics data from BC cells highlighted different DNA repair and cell-cycle mol. profiles assocd. with resistance to AsiDNA or olaparib, rationalizing combined treatment. Treatment synergy was also confirmed with six other PARPi in development. Conclusions: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Clin Cancer Res; 23(4); 1001-11. ©2016 AACR.
- 18Mistrik, M., Oplustilova, L., Lukas, J., and Bartek, J. (2009) Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis Cell Cycle 8, 2592– 2599 DOI: 10.4161/cc.8.16.9331There is no corresponding record for this reference.
- 19Vernole, P., Muzi, A., Volpi, A., Dorio, A. S., Terrinoni, A., Shah, G. M., and Graziani, G. (2009) Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cells Mutat. Res., Fundam. Mol. Mech. Mutagen. 664, 39– 47 DOI: 10.1016/j.mrfmmm.2009.02.00519https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXltVGjsLo%253D&md5=14729d177ac252141141be6bf7ffb010Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cellsVernole, Patrizia; Muzi, Alessia; Volpi, Antonio; Dorio, Annalisa S.; Terrinoni, Alessandro; Shah, Girish M.; Graziani, GraziaMutation Research, Fundamental and Molecular Mechanisms of Mutagenesis (2009), 664 (1-2), 39-47CODEN: MUREAV; ISSN:0027-5107. (Elsevier B.V.)Mismatch repair (MMR) has been shown to control homologous recombination (HR) by aborting strand exchange between divergent sequences. It has previously been demonstrated that MMR-deficient tumor cells are more resistant to chromosomal damage induced by bleomycin (BLM) during the G2 phase, likely due to the lack of the MMR inhibitory effect on HR. Aim of this study was to investigate whether inhibition of HR by the nucleoside analog BVDU [(E)-5(2-bromovinyl)-2'-deoxyuridine, brivudin], or silencing of genes involved in HR function, might affect sensitivity of MMR-deficient tumor cells to DNA damage induced by BLM in G2. The results indicated that BVDU increased chromatid damage and DNA double strand breaks induced by BLM only in MMR-deficient MT-1, HL-60R, HCT116 cells, which are more resistant to BLM with respect to MMR-proficient TK-6, HL-60S and HCT116/3-6 lines. Silencing of RAD51, a key component of HR, increased sensitivity of MMR-deficient HCT-15 cells to BLM clastogenicity; in this case combined treatment with BVDU had no addnl. effect. Similarly, treatment with BVDU did not affect BLM clastogenicity in CAPAN-1 cells, characterized by a defective HR due to BRCA2 mutations. Conversely, BVDU increased chromatid breaks induced by BLM in HCT-15 cells transiently silenced for DNA-PK catalytic subunit, which plays a key role in non-homologous end joining. The BVDU-mediated increase of chromatid breaks in MMR-deficient cells did not depend on its previously reported inhibitory effect on poly(ADP-ribose) polymerase (PARP). In fact, it was obsd. also in cells stably silenced for PARP-1, which is responsible for most of cellular PARP activity. These data support the suggestion that the higher sensitivity of MMR-proficient vs. MMR-deficient cells to BLM-induced chromatid breaks in the G2 phase is a consequence of the inhibition of HR by MMR. In MMR-deficient cells, BVDU attenuates the repair of BLM-induced DSBs and this is likely to occur via inhibition of HR.
- 20Maxwell, K. N. and Domchek, S. M. (2012) Cancer treatment according to BRCA1 and BRCA2 mutations Nat. Rev. Clin. Oncol. 9, 520– 528 DOI: 10.1038/nrclinonc.2012.12320https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1Kjs7zM&md5=c6da652ff478b6175e184f98cc7c13c2Cancer treatment according to BRCA1 and BRCA2 mutationsMaxwell, Kara N.; Domchek, Susan M.Nature Reviews Clinical Oncology (2012), 9 (9), 520-528CODEN: NRCOAA; ISSN:1759-4774. (Nature Publishing Group)A review. Identification of germline mutations assocd. with significant cancer susceptibility has the potential to change all aspects of an individual's care, from screening to cancer treatment. For example, women with germline mutations in BRCA1 and BRCA2 have markedly elevated risks of breast and ovarian cancer and the identification of these germline mutations has led to specific screening and prevention strategies. More recently, advances in the understanding of the biol. function of BRCA1 and BRCA2 have led to clin. trials testing targeted therapies in this population, particularly poly(ADP-ribose) polymerase (PARP) inhibitors. Unfortunately, the development of PARP inhibitors has not been as rapid as anticipated and has been more challenging than expected. Somatic mutations identified in many cancer types have allowed the development of therapeutics that target these mutated genes, and many of these agents obtained rapid regulatory approval and are currently in widespread clin. practice. Diagnostic testing has a central role in targeted cancer therapeutics for both somatic and germline mutations. Although the era of mol. medicine and targeted therapies has led to significant changes in the practice of oncol., new challenges continue to arise.
- 21Banerjee, S., Kaye, S. B., and Ashworth, A. (2010) Making the best of PARP inhibitors in ovarian cancer Nat. Rev. Clin. Oncol. 7, 508– 519 DOI: 10.1038/nrclinonc.2010.11621https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVyjsr%252FM&md5=c88eb3b20aeffa2e1875aee7cfb18594Making the best of PARP inhibitors in ovarian cancerBanerjee, Susana; Kaye, Stan B.; Ashworth, AlanNature Reviews Clinical Oncology (2010), 7 (9), 508-519CODEN: NRCOAA; ISSN:1759-4774. (Nature Publishing Group)A review. Inhibitors of poly(ADP-ribose)polymerase (PARP) have shown promise as therapeutic agents for the treatment of ovarian cancers with mutations in BRCA1 or BRCA2. By exploiting the synthetic lethal interaction that exists between PARP inhibition and BRCA mutations, these agents specifically kill cancer cells by targeting their DNA repair system. The authors of this Review describe the importance of BRCA mutations for the efficacy of PARP inhibitors. They also discuss the preclin. and clin. trial results of PARP inhibitors, the challenges related to the use of these agents, and future directions. Drugs that inhibit the enzyme poly(ADP-ribose)polymerase (PARP) are showing considerable promise for the treatment of cancers that have mutations in the BRCA1 or BRCA2 tumor suppressors. This therapeutic approach exploits a synthetic lethal strategy to target the specific DNA repair pathway in these tumors. High-grade ovarian cancers have a generally poor prognosis, and accumulating evidence suggests that mutations in BRCA1 or BRCA2, or silencing of BRCA1 by promoter methylation, may be common in this disease. Here, we consider how the potential benefit of PARP inhibitors might be maximized in ovarian cancer. We suggest that it will be crucial to explore novel therapeutic trial strategies and drug combinations, and incorporate robust biomarkers predictive of response if these drugs are to reach their full potential.
- 22Montoni, A., Robu, M., Pouliot, É., and Shah, G. M. (2013) Resistance to PARP-inhibitors in cancer therapy Front. Pharmacol. 4 (FEB) 1– 7 DOI: 10.3389/fphar.2013.0001822https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht12iu7fN&md5=bda6a7ac6dfb80c4d6be6fe130202c7dResistance to PARP-inhibitors in cancer therapyMontoni, Alicia; Robu, Mihaela; Pouliot, Emilie; Shah, Girish M.Frontiers in Pharmacology (2013), 4 (), 1-7CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)A review. The pharmacol. inhibitors of poly(ADP-ribose) polymerase (PARP) family of proteins have shown promising results in preclin. studies and clin. trials as a monotherapy or in combination therapy for some cancers. Thus, usage of PARP-inhibitors (PARPi) in cancer therapy is bound to increase with time, but resistance of cancer cells to PARPi is also beginning to be obsd. Here we review different known and potential mechanisms by which: (i) PARPi kill cancer cells; and (ii) cancer cells develop resistance to PARPi. Understanding the lethality caused by PARPi and the countermeasures deployed by cancers cells to survive PARPi will help us rationalize the use of this new 1class of drugs in cancer therapy.
- 23Liu, Y., Burness, M. L., Martin-Trevino, R., Guy, J., Bai, S., Harouaka, R., Brooks, M. D., Shang, L., Fox, A., Luther, T. K., Davis, A., Baker, T. L., Colacino, J., Clouthier, S. G., Shao, Z., Wicha, M. S., and Liu, S. (2017) RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer Clin. Cancer Res. 23, 514– 522 DOI: 10.1158/1078-0432.CCR-15-134823https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVeitLs%253D&md5=56dce7989dcf9edb314d3927adf180dfRAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast CancerLiu, Yajing; Burness, Monika L.; Martin-Trevino, Rachel; Guy, Joey; Bai, Shoumin; Harouaka, Ramdane; Brooks, Michael D.; Shang, Li; Fox, Alex; Luther, Tahra K.; Davis, April; Baker, Trenton L.; Colacino, Justin; Clouthier, Shawn G.; Shao, Zhi-ming; Wicha, Max S.; Liu, SulingClinical Cancer Research (2017), 23 (2), 514-522CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Introduction: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)-deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-neg. breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display "cancer stem cell" properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. Exptl. Design: We detd. the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. Results: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1-wild-type cells were relatively resistant to PARP inhibition alone, but redn. of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. Conclusions: Our data suggest that in both BRCA1-mutant and BRCA1-wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi.
- 24Klein, H. L. (2008) The consequences of Rad51 overexpression for normal and tumor cells DNA Repair 7, 686– 693 DOI: 10.1016/j.dnarep.2007.12.00824https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXls1Sgsbw%253D&md5=824aee339ab1c58745af64778d24378aThe consequences of Rad51 overexpression for normal and tumor cellsKlein, Hannah L.DNA Repair (2008), 7 (5), 686-693CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)A review. The Rad51 recombinase is an essential factor for homologous recombination and the repair of DNA double strand breaks, binding transiently to both single stranded and double stranded DNA during the recombination reaction. The use of a homologous recombination mechanism to repair DNA damage is controlled at several levels, including the binding of Rad51 to single stranded DNA to form the Rad51 nucleofilament, which is controlled through the action of DNA helicases that can counteract nucleofilament formation. Overexpression of Rad51 in different organisms and cell types has a wide assortment of consequences, ranging from increased homologous recombination and increased resistance to DNA damaging agents to disruption of the cell cycle and apoptotic cell death. Rad51 expression is increased in p53-neg. cells, and since p53 is often mutated in tumor cells, there is a tendency for Rad51 to be overexpressed in tumor cells, leading to increased resistance to DNA damage and drugs used in chemotherapies. As cells with increased Rad51 levels are more resistant to DNA damage, there is a selection for tumor cells to have higher Rad51 levels. While increased Rad51 can provide drug resistance, it also leads to increased genomic instability and may contribute to carcinogenesis.
- 25Dos Santos Ferreira, A. C., Fernandes, R. A., Kwee, J. K., and Klumb, C. E. (2012) Histone deacetylase inhibitor potentiates chemotherapy-induced apoptosis through Bim upregulation in Burkitt’s lymphoma cells J. Cancer Res. Clin. Oncol. 138, 317– 325 DOI: 10.1007/s00432-011-1093-yThere is no corresponding record for this reference.
- 26Barrett, A. J. and Kirschke, H. (1981) Cathepsin B, cathepsin H, and cathepsin L Methods Enzymol. 80, 535– 561 DOI: 10.1016/S0076-6879(81)80043-2There is no corresponding record for this reference.
- 27Yasuda, Y., Kageyama, T., Akamine, A., Shibata, M., Kominami, E., Uchiyama, Y., and Yamamoto, K. (1999) Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D J. Biochem. 125, 1137– 1143 DOI: 10.1093/oxfordjournals.jbchem.a02239627https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXlsVygsr4%253D&md5=23d0e8c891cf0ed5c38fa1191d18ec9aCharacterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin DYasuda, Yoshiyuki; Kageyama, Takashi; Akamine, Akifumi; Shibata, Masahiro; Kominami, Eiki; Uchiyama, Yasuo; Yamamoto, KenjiJournal of Biochemistry (1999), 125 (6), 1137-1143CODEN: JOBIAO; ISSN:0021-924X. (Japanese Biochemical Society)Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiol. and pathol. degrdn. of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the sepn. of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)γ-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 μM-1·s-1 for cathepsin E and 15.6 μM-1·s-1 for cathepsin D). A very acidic pH optimum of 4.0 was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(Dnp)γ-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 μM-1·s-1 for cathepsin E and 16.3 μM-1·s-1 for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive detn. of the precise levels of cathepsins E and D in crude cell exts. of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.
- 28Brömme, D. and Mcgrath, M. E. (1996) High level expression and crystallization of recombinant human cathepsin S Protein Sci. 5, 789– 791 DOI: 10.1002/pro.556005042628https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XitFSgsL8%253D&md5=f5940b367e9810071c5e100c74179586High level expression and crystallization of recombinant human cathepsin SBromme, Dieter; McGrath, Mary E.Protein Science (1996), 5 (4), 789-91CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)The authors have expressed active human cathepsin S to 60 mg/L in Sf9 cells using a baculovirus system. Prodn. of milligram quantities has facilitated crystallog. studies to det. the structure of this enzyme, which has unique properties among lysosomal cysteine proteinases. Recombinant, irreversibly inhibited cathepsin S was crystd. from ammonium phosphate at 17°. The crystals diffract to at least 2.3 Å, and belong to the orthorhombic crystal system with a primitive lattice. Approx. cell dimensions are: a = 37.7 Å, b = 73.9 Å, and c = 106.7 Å. There is most likely one mol. per asym. unit.
- 29Nagase, H., Fields, C. G., and Fields, G. B. (1994) Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3) J. Biol. Chem. 269, 20952– 2095729https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmsV2hsrY%253D&md5=6b997ba5965f2335020a79fa6b681deeDesign and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3)Nagase, Hideaki; Fields, Cynthia G.; Fields, Gregg B.Journal of Biological Chemistry (1994), 269 (33), 20952-7CODEN: JBCHA3; ISSN:0021-9258.Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. The authors examd. known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km ∼11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation expts. were additive, the authors found discrepancies between predicted and exptl. kcat/Km values, one on the order of 2000-5000. Thus, the design of addnl. discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.
- 30Tierno, M. B., Johnston, P. a, Foster, C., Skoko, J. J., Shinde, S. N., Shun, T. Y., and Lazo, J. S. (2007) Development and optimization of high-throughput in vitro protein phosphatase screening assays Nat. Protoc. 2, 1134– 1144 DOI: 10.1038/nprot.2007.155There is no corresponding record for this reference.
- 31Davis, M. I., Ronesi, J., and Lovinger, D. M. (2003) A predominant role for inhibition of the adenylate cyclase/protein kinase A pathway in ERK activation by cannabinoid receptor 1 in N1E-115 neuroblastoma cells J. Biol. Chem. 278, 48973– 48980 DOI: 10.1074/jbc.M305697200There is no corresponding record for this reference.
- 32Montalibet, J., Skorey, K. I., and Kennedy, B. P. (2005) Protein tyrosine phosphatase: Enzymatic assays Methods 35, 2– 8 DOI: 10.1016/j.ymeth.2004.07.00232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVGgu77I&md5=95ed7b0f7de47a71e0aa5194597c6576Protein tyrosine phosphatase: enzymatic assaysMontalibet, Jacqueline; Skorey, Kathryn I.; Kennedy, Brian P.Methods (San Diego, CA, United States) (2005), 35 (1), 2-8CODEN: MTHDE9; ISSN:1046-2023. (Elsevier)Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger mols. such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the detns. of the rate of dephosphorylation and calcns. of kinetic consts. such as kcat and Km. These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.
- 33Welte, S., Baringhaus, K. H., Schmider, W., Müller, G., Petry, S., and Tennagels, N. (2005) 6,8-Difluoro-4-methylumbiliferyl phosphate: A fluorogenic substrate for protein tyrosine phosphatases Anal. Biochem. 338, 32– 38 DOI: 10.1016/j.ab.2004.11.04733https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlenurk%253D&md5=8fbf39bd52aacefebe63ba75d1d9d5da6,8-Difluoro-4-methylumbiliferyl phosphate: a fluorogenic substrate for protein tyrosine phosphatasesWelte, Stefan; Baringhaus, Karl-Heinz; Schmider, Wolfgang; Mueller, Guenter; Petry, Stefan; Tennagels, NorbertAnalytical Biochemistry (2005), 338 (1), 32-38CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor consts. for DIFMUP cleavage were comparable with those of the widely used but less discriminative and practicable substrates, para-nitrophenylphosphate and phosphotyrosine-contg. peptides, resp. Furthermore, the continuous and highly sensitive assay allows fast and accurate investigations of the type, kinetic behavior, and binding mode of small-mol. inhibitors. We discuss the validation of this assay system for various PTPs and its use in inhibitor screening for PTP1B.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00707.
Supporting Figures S1–S4, virtual screening protocol, and experimental details of all synthetic procedures together with characterization data for all intermediates and final compounds (PDF)
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