Cancer chess: molecular insights into PARP inhibitor resistance

The clinical potential of applying synthetic lethality to cancer treatment is famously demonstrated by the BRCA1/PARP1 paradigm: a tumor specific defect in BRCA1 – a component of the DNA double-strand break (DSB) repair pathway homologous recombination (HR) – results in a remarkable sensitivity to PARP1 inhibition (PARPi). Despite spectacular initial responses in patients, resistance to PARPi treatment may develop and must be overcome to maximally exploit this interaction in the clinic. Genetically engineered (mouse) model systems have shown that PARPi resistance may arise through inactivation of the 53BP1 pathway. The 53BP1 pathway normally protects DSB ends from resection and the removal of this “brake” restores HR in the absence of BRCA1. However, how the 53BP1 pathway protects DSB ends from resection has remained elusive. In this thesis, advances in 3D tumor organoid culture protocols and CRISPR/Cas9 (screening) technology were applied to identify and validate new components of the 53BP1 pathway that render BRCA1 deficient cells resistant to PARPi upon their loss. Furthermore, a new acquired vulnerability that can be therapeutically exploited to deplete such PARPi resistant cells is described. Together, this thesis provides mechanistic insight in DSB repair and illustrates how such fundamental knowledge may stand at the basis to combat resistance.The research described in this thesis was performed at the Division of Molecular Pathology of the Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital (NKI-AVL) and Oncode Institute, Amsterdam, The Netherlands, and was financially supported by the Dutch Cancer Society (KWF 2011-5220 and 2014-6532 to S. Rottenberg and J. Jonkers), the Netherlands Organization for Scientific Research (VICI 91814643, NGI 93512009, Cancer Genomics Netherlands, and a National Roadmap Grant for Large-Scale Research Facilities to J. Jonkers), the Swiss National Science Foundation (310030_179360 to S. Rottenberg), the Swiss Cancer League (KLS-4282-08-2017 to S. Rottenberg); and the European Union (ERC CoG-681572 to S. Rottenberg and ERC Synergy Grant 319661 to J. Jonkers).Drug Delivery Technolog

The CST Complex Mediates End Protection at Double-Strand Breaks and Promotes PARP Inhibitor Sensitivity in BRCA1-Deficient Cells

Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection. Using CRISPR/SpCas9-based loss-of-function screens, Barazas et al. show that loss of the CTC1-STN1-TEN1 (CST) complex promotes PARP inhibitor resistance in BRCA1-deficient cells. Mechanistically, the CST complex maintains double-strand break end stability in addition to its role in protecting telomeric ends

The CST complex mediates end protection at double-strand breaks and promotes PARP inhibitor sensitivity in BRCA1-deficient cells

Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection

Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality (vol 33, pg 1078, 2018)

Genome Instability and Cance

Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality (vol 33, pg 1078, 2018)

Genome Instability and Cance

Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality

Genome Instability and Cance

Selective loss of PARG restores PARylation and counteracts PARP inhibitor-mediated synthetic lethality

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically

REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway(1). In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers(2,3). Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration(4). Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases(5). In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent endresection of DSBs in BRCA1 deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance(6). Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells