Krawczyk Group – Turning DNA repair against cancer
The mechanisms involved in repair of DNA double-strand breaks (DSBs) guard genome integrity in healthy cells but they can also protect cancer cells against the therapies that target their DNA. The long-term research goal of our group is exploring the cellular responses to DNA damage to develop and improve anticancer strategies relying on manipulation of DNA repair in normal and cancer cells.
Contact: P.M. Krawczyk (email@example.com)
1. Uncovering the molecular choreography of DNA repair responses
DSB repair and cancer. Many successful anticancer agents, including ionizing radiation and multiple chemotherapeutics, exert cytotoxic effects by inducing double-strand breaks (DSBs) in DNA of tumor cells. However, mammals evolved robust DSB repair (DSBR) mechanisms which reduce the efficacy of these agents. Therefore, inhibition of DSBR and exploitation of DSBR defects are among attractive anticancer strategies. These strategies hinge on deep understanding of DSBR mechanisms in cancer as well as in healthy cells, because optimal therapies should target the former and spare the latter.
Problem. Most known DSBR proteins localize to DSB sites, where they establish complex structures, interacting with each other and with the adjacent chromatin. These dynamic processes, termed here ‘DSBR choreography’, can be visualized in live cells (Figure 2), providing crucial information about the underlying molecular mechanisms . Over the last decade, independent groups inspected individual DSBR proteins at damaged DNA or at neighboring chromatin. However, the vast majority of these studies focused on unrelated cancer cell lines and used diverse artefact-prone, clinically irrelevant methods of DSB induction. As a consequence, the available data are fragmented, incomplete and cannot be related to each other. Importantly – and perhaps surprisingly – basic parameters of DSBR (e.g. the temporal sequence of repair events, real-time interactions between individual DSBR proteins and spatial organization of repair activities in the context of chromatin) in normal human cells remain practically unexplored. Because of this profound deficit in our understanding of repair processes in normal cells, it is currently not possible to determine how they are altered by oncogenic transformation.
Aim. We are investigating, for the first time, DSBR choreography in normal human cells, and how it changes in cancer cells. In particular, we focus on comparing: (I) the kinetics (mobility) of most known DSBR proteins in undamaged cell nuclei and at damaged DNA/chromatin; (II) the temporal sequence of accumulation of these proteins at well-characterized DNA lesions induced by clinically-relevant X-ray microirradiation; (III) super-resolution localization of proteins during DSBR (Figure 3); (IV) the interactions between individual proteins in irradiated living cells (DSBR interactome). These experiments will address multiple open questions and expose some properties of DSBR processes that are crucial for understanding cellular responses to DSB-inducing therapies. How do DSBR proteins interact with intact chromatin (which is essential for sensing DNA damage) and with structures they establish at DSB sites (which is important for repair)? What is the temporal sequence of repair events in live cells? How is the DSBR machinery organized at damaged DNA/chromatin and how does this organization change during repair? How do DSBR proteins interact with each other in the absence of DNA damage and at DSB sites, and how do these interactions change as repair unfolds? And most importantly, whether and how these aspects of DSBR change in transformed cells?
2. Improving hyperthermia-based clinical cancer treatments.
Hyperthermia and cancer. Hyperthermia (HT) – temporary elevation of tumor temperature to 41-43 °C – alters many aspects of cellular metabolism, but its effects on DNA repair are of special interest in the context of cancer research and treatment. HT inhibits repair of DNA double-strand breaks (DSBs), making it a powerful radio- and chemosensitizer with proven clinical track record in combination therapy for various types of cancer, including breast, bladder, head, neck, melanoma, soft tissue sarcoma and cervix.
Problem. The efficacy of HT treatments is negatively affected by a number of factors, such as insufficient thermal dose, temporary nature of HT effects (resulting in a short therapeutic window) and thermotolerance (induction of a temporary resistance to subsequent HT treatments). It is thus evident that outcomes of HT therapies would benefit from strategies to: (I) Increase HT efficacy at lower temperatures and shorter treatments; (II) extend the duration of the therapeutic window and (III) eliminate or reduce thermotolerance.
Results. Recently, we found that a short incubation of cervix cancer cells in the presence of a HSP90 inhibitor ganetespib enhances the effects of concomitant HT treatment in vitro, nearly without affecting non-heated cells. In particular, ganetespib (i) potentiated cytotoxic as well as radiosensitizing and chemosensitizing effects of HT; (ii) enhanced HT-mediated induction of DNA damage; (ii) reduced thermotolerance and (iii) prolonged and enhanced the effects of HT on DSB repair. Our preliminary results thus establish HSP90 inhibition as a straightforward and efficient approach to improve HT treatment efficacy with no or limited systemic toxicity.
Aims. We are currently investigating the effects of HSP90 inhibition on the efficacy of anticancer treatments involving HT in animal models of cancer, in vivo. The long-term goal of our study is boosting the effectiveness of HT treatments in the clinic.
Relevance. We are confident that positive results of our current in vivo studies will find their way into clinical practice. Together with clinicians of AMC and EMC, we are already considering various groups of patients that would most benefit from our improved treatments. Since ganetespib has a favorable safety profile and is already used clinically, we are convinced that the first trials could be initiated in the near future.
Dr. P.M. Krawczyk – group leader (firstname.lastname@example.org)
Dr. J. Stap – senior scientist (email@example.com)
Dr. A. Jonker – senior scientist (firstname.lastname@example.org)
Dr. J.A. Kochan – postdoc (email@example.com)
Mr. E. Scutigliani – PhD student (firstname.lastname@example.org)
Ms. Julia Raaiman – student
Ms. Julia van der Lippe – student
Mr. Matthias van der Belt – student
Ms. Farangis Sharifi – student
Dr. Jacob Aten
Dr. Jan Verhoeven
Ing. Carel van Oven
- Kochan JA, Desclos ECB, Bosch R, Meister L, Vriend LEM, van Attikum H, Krawczyk PM. Meta-analysis of DNA double-strand break response kinetics. Nucleic Acids Res. 2017 Dec 15;45(22):12625-12637. dx.doi.org/10.1093/nar/gkx1128 www.ncbi.nlm.nih.gov/pubmed/29182755
- Vriend LEM, van den Tempel N, Oei AL, L’Acosta M, Pieterson FJ, Franken NAP, Kanaar R, Krawczyk PM. Boosting the effects of hyperthermia-based anticancer treatments by HSP90 inhibition. Oncotarget. 2017 Oct 27;8(57):97490-97503. dx.doi.org/10.18632/oncotarget.22142 www.ncbi.nlm.nih.gov/pubmed/29228626
- van Dijk IA, Ferrando ML, van der Wijk AE, Hoebe RA, Nazmi K, de Jonge WJ, Krawczyk PM, Bolscher JGM, Veerman ECI, Stap J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function. FASEB J. 2017 Sep;31(9):3922-3933. dx.doi.org/10.1096/fj.201700180R www.ncbi.nlm.nih.gov/pubmed/28522595
- Oei AL, van Leeuwen CM, Ahire VR, Rodermond HM, Ten Cate R, Westermann AM, Stalpers LJA, Crezee J, Kok HP, Krawczyk PM, Kanaar R, Franken NAP. Enhancing synthetic lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia. Oncotarget. 2017 Apr 25;8(17):28116-28124. dx.doi.org/10.18632/oncotarget.15922 www.ncbi.nlm.nih.gov/pubmed/28427225
- van Dijk IA, Beker AF, Jellema W, Nazmi K, Wu G, Wismeijer D, Krawczyk PM, Bolscher JG, Veerman EC, Stap J. Histatin 1 Enhances Cell Adhesion to Titanium in an Implant Integration Model. J Dent Res. 2017 Apr;96(4):430-436. dx.doi.org/10.1177/0022034516681761 www.ncbi.nlm.nih.gov/pubmed/27941125
- Oei AL, Vriend LE, van Leeuwen CM, Rodermond HM, Ten Cate R, Westermann AM, Stalpers LJ, Crezee J, Kanaar R, Kok HP, Krawczyk PM, Franken NA. Sensitizing thermochemotherapy with a PARP1-inhibitor. Oncotarget. 2017 Mar 7;8(10):16303-16312. dx.doi.org/10.18632/oncotarget.11422 www.ncbi.nlm.nih.gov/pubmed/27557507
- Oei AL, Vriend LE, Krawczyk PM, Horsman MR, Franken NA, Crezee J. Targeting therapy-resistant cancer stem cells by hyperthermia. Int J Hyperthermia. 2017 Feb 2:1-12. dx.doi.org/10.1080/02656736.2017.1279757 www.ncbi.nlm.nih.gov/pubmed/28100096
- Vriend LE, Krawczyk PM. Nick-initiated homologous recombination: Protecting the genome, one strand at a time. DNA Repair (Amst). 2017 Feb;50:1-13. dx.doi.org/10.1016/j.dnarep.2016.12.005 www.ncbi.nlm.nih.gov/pubmed/28087249
- Vriend LE, Prakash R, Chen CC, Vanoli F, Cavallo F, Zhang Y, Jasin M, Krawczyk PM. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks. Nucleic Acids Res. 2016 Jun 20;44(11):5204-17. dx.doi.org/10.1093/nar/gkw179 www.ncbi.nlm.nih.gov/pubmed/27001513
- van Montfort T, Thomas AA, Krawczyk PM, Berkhout B, Sanders RW, Paxton WA. Reactivation of Neutralized HIV-1 by Dendritic Cells Is Dependent on the Epitope Bound by the Antibody. J Immunol. 2015 Oct 15;195(8):3759-68. dx.doi.org/10.4049/jimmunol.1402344. Epub 2015 Sep 9. https://www.ncbi.nlm.nih.gov/pubmed/26355155.
- Oei AL, Vriend LE, Crezee J, Franken NA, Krawczyk PM. Effects of hyperthermia on DNA repair pathways: one treatment to inhibit them all. Radiat Oncol. 2015 Aug 7;10:165. dx.doi.org/10.1186/s13014-015-0462-0 www.ncbi.nlm.nih.gov/pubmed/26245485
- Zhang Y, Vanoli F, LaRocque JR, Krawczyk PM, Jasin M. Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system. Methods. 2014 Sep;69(2):171-178. dx.doi.org/10.1016/j.ymeth.2014.05.003 www.ncbi.nlm.nih.gov/pubmed/24929070
- Vriend LE, Jasin M, Krawczyk PM. Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases. Methods Enzymol. 2014;546:175-91. dx.doi.org/10.1016/B978-0-12-801185-0.00009-X www.ncbi.nlm.nih.gov/pubmed/25398341
- Bergs JW, Krawczyk PM, Borovski T, ten Cate R, Rodermond HM, Stap J, Medema JP, Haveman J, Essers J, van Bree C, Stalpers LJ, Kanaar R, Aten JA, Franken NA. Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining. DNA Repair (Amst). 2013 Jan 1;12(1):38-45. dx.doi.org/10.1016/j.dnarep.2012.10.008 www.ncbi.nlm.nih.gov/pubmed/ 23237939
- Krawczyk PM, Borovski T, Stap J, Cijsouw T, ten Cate R, Medema JP, Kanaar R, Franken NA, Aten JA. Chromatin mobility is increased at sites of DNA double-strand breaks. J Cell Sci. 2012 May 1;125(Pt 9):2127-33. dx.doi.org/10.1242/jcs.089847 www.ncbi.nlm.nih.gov/pubmed/22328517
- Eppink B, Krawczyk PM, Stap J, Kanaar R. Hyperthermia-induced DNA repair deficiency suggests novel therapeutic anti-cancer strategies. Int J Hyperthermia. 2012;28(6):509-17. dx.doi.org/10.3109/02656736.2012.695427 www.ncbi.nlm.nih.gov/pubmed/22834701
- Krawczyk PM, Eppink B, Essers J, Stap J, Rodermond H, Odijk H, Zelensky A, van Bree C, Stalpers LJ, Buist MR, Soullie T, Rens J, Verhagen HJ, O’Connor MJ, Franken NA, Ten Hagen TL, Kanaar R, Aten JA. Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition. Proc Natl Acad Sci U S A. 2011 Jun 14;108(24):9851-6. dx.doi.org/10.1073/pnas.1101053108 www.ncbi.nlm.nih.gov/pubmed/21555554
- Franken NA, ten Cate R, Krawczyk PM, Stap J, Haveman J, Aten J, Barendsen GW. Comparison of RBE values of high-LET Î±-particles for the induction of DNA-DSBs, chromosome aberrations and cell reproductive death. Radiat Oncol. 2011 Jun 8;6:64. dx.doi.org/10.1186/1748-717X-6-64 www.ncbi.nlm.nih.gov/pubmed/21651780
- Mir SE, De Witt Hamer PC, Krawczyk PM, Balaj L, Claes A, Niers JM, Van Tilborg AA, Zwinderman AH, Geerts D, Kaspers GJ, Peter Vandertop W, Cloos J, Tannous BA, Wesseling P, Aten JA, Noske DP, Van Noorden CJ, Wurdinger T. In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma. Cancer Cell. 2010 Sep 14;18(3):244-57. dx.doi.org/10.1016/j.ccr.2010.08.011 www.ncbi.nlm.nih.gov/pubmed/20832752
- van Oven C, Krawczyk PM, Stap J, Melo AM, Piazzetta MH, Gobbi AL, van Veen HA, Verhoeven J, Aten JA. An ultrasoft X-ray multi-microbeam irradiation system for studies of DNA damage responses by fixed- and live-cell fluorescence microscopy. Eur Biophys J. 2009 Jul;38(6):721-8. dx.doi.org/10.1007/s00249-009-0472-7 www.ncbi.nlm.nih.gov/pubmed/19495740
- Stap J, Krawczyk PM, Van Oven CH, Barendsen GW, Essers J, Kanaar R, Aten JA. Induction of linear tracks of DNA double-strand breaks by alpha-particle irradiation of cells. Nat Methods. 2008 Mar;5(3):261-6. dx.doi.org/10.1038/nmeth.f.206 www.ncbi.nlm.nih.gov/pubmed/18309310
- Krol HA, Krawczyk PM, Bosch KS, Aten JA, Hol EM, Reits EA. Polyglutamine expansion accelerates the dynamics of ataxin-1 and does not result in aggregate formation. PLoS One. 2008 Jan 30;3(1):e1503. dx.doi.org/10.1371/journal.pone.0001503 www.ncbi.nlm.nih.gov/pubmed/18231590
- Krawczyk PM, Stap J, Hoebe RA, van Oven CH, Kanaar R, Aten JA. Analysis of the mobility of DNA double-strand break-containing chromosome domains in living mammalian cells. Methods Mol Biol. 2008;463:309-20. dx.doi.org/10.1007/978-1-59745-406-3_19 www.ncbi.nlm.nih.gov/pubmed/18951175
- Williams ES, Stap J, Essers J, Ponnaiya B, Luijsterburg MS, Krawczyk PM, Ullrich RL, Aten JA, Bailey SM. DNA double-strand breaks are not sufficient to initiate recruitment of TRF2. Nat Genet. 2007 Jun;39(6):696-8; dx.doi.org/10.1038/ng0607-696 www.ncbi.nlm.nih.gov/pubmed/17534357
- Krawczyk PM, Stap J, van Oven C, Hoebe R, Aten JA. Clustering of double strand break-containing chromosome domains is not inhibited by inactivation of major repair proteins. Radiat Prot Dosimetry. 2006;122(1-4):150-3. doi.org/10.1093/rpd/ncl479 www.ncbi.nlm.nih.gov/pubmed/17229782
- Aten JA, Stap J, Krawczyk PM, van Oven CH, Hoebe RA, Essers J, Kanaar R. Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains. Science. 2004 Jan 2;303(5654):92-5. doi.org/10.1126/science.1088845 www.ncbi.nlm.nih.gov/pubmed/14704429