Marieke van de Ven

Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.

BRCA1185delAG tumors may acquire therapy resistance through expression of RING-less BRCA1

Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.

EZH2 promotes degradation of stalled replication forks by recruiting MUS81 through histone H3 trimethylation

The emergence of resistance to poly-ADP-ribose polymerase inhibitors (PARPi) poses a threat to the treatment of BRCA1 and BRCA2 (BRCA1/2)-deficient tumours. Stabilization of stalled DNA replication forks is a recently identified PARPi-resistance mechanism that promotes genomic stability in BRCA1/2-deficient cancers. Dissecting the molecular pathways controlling genomic stability at stalled forks is critical. Here we show that EZH2 localizes at stalled forks where it methylates Lys27 on histone 3 (H3K27me3), mediating recruitment of the MUS81 nuclease. Low EZH2 levels reduce H3K27 methylation, prevent MUS81 recruitment at stalled forks and cause fork stabilization. As a consequence, loss of function of the EZH2/MUS81 axis promotes PARPi resistance in BRCA2-deficient cells. Accordingly, low EZH2 or MUS81 expression levels predict chemoresistance and poor outcome in patients with BRCA2-mutated tumours. Moreover, inhibition of Ezh2 in a murine Brca2−/− breast tumour model is associated with acquired PARPi resistance. Our findings identify EZH2 as a critical regulator of genomic stability at stalled forks that couples histone modifications to nuclease recruitment. Our data identify EZH2 expression as a biomarker of BRCA2-deficient tumour response toxa0chemotherapy.

Fancf-deficient mice are prone to develop ovarian tumours.

Fanconi anaemia (FA) is a rare recessive disorder marked by developmental abnormalities, bone marrow failure, and a high risk for the development of leukaemia and solid tumours. The inactivation of FA genes, in particular FANCF, has also been documented in sporadic tumours in non‐FA patients. To study whether there is a causal relationship between FA pathway defects and tumour development, we have generated a mouse model with a targeted disruption of the FA core complex gene Fancf. Fancf‐deficient mouse embryonic fibroblasts displayed a phenotype typical for FA cells: they showed an aberrant response to DNA cross‐linking agents as manifested by G2 arrest, chromosomal aberrations, reduced survival, and an inability to monoubiquitinate FANCD2. Fancf homozygous mice were viable, born following a normal Mendelian distribution, and showed no growth retardation or developmental abnormalities. The gonads of Fancf mutant mice functioned abnormally, showing compromised follicle development and spermatogenesis as has been observed in other FA mouse models and in FA patients. In a cohort of Fancf‐deficient mice, we observed decreased overall survival and increased tumour incidence. Notably, in seven female mice, six ovarian tumours developed: five granulosa cell tumours and one luteoma. One mouse had developed tumours in both ovaries. High‐resolution array comparative genomic hybridization (aCGH) on these tumours suggests that the increased incidence of ovarian tumours correlates with the infertility in Fancf‐deficient mice and the genomic instability characteristic of FA pathway deficiency. Copyright

A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma.

Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.

BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.

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

Summary 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

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.

Prophylactic window therapy with the clinical poly(ADP-ribose) polymerase inhibitor olaparib delays BRCA1-deficient mammary tumour formation in mice: Prophylactic treatments against BRCA1-deficient breast cancer

Women with heterozygous germline mutations in the BRCA1 tumour suppressor gene are strongly predisposed to developing early‐onset breast cancer through loss of the remaining wild‐type BRCA1 allele and inactivation of TP53. Although tumour prevention strategies in BRCA1‐mutation carriers are still limited to prophylactic surgery, several therapeutic strategies have been developed to target the DNA repair defects (also known as ‘BRCAness’) of BRCA1‐deficient tumours. In particular, DNA‐damaging agents such as platinum drugs and poly(ADP‐ribose) polymerase (PARP) inhibitors show strong activity against BRCA1‐mutated tumours. However, it is unclear whether drugs that target BRCAness can also be used to prevent tumour formation in BRCA1‐mutation carriers, especially as loss of wild‐type BRCA1 may not be the first event in BRCA1‐associated tumourigenesis. We performed prophylactic treatments in a genetically engineered mouse model in which de novo development of BRCA1‐deficient mammary tumours is induced by stochastic loss of BRCA1 and p53. We found that prophylactic window therapy with nimustine, cisplatin or olaparib reduced the amount and size of mammary gland lesions, and significantly increased the median tumour latency. Similar results were obtained with intermittent prophylactic treatment with olaparib. Importantly, prophylactic window therapy with nimustine and cisplatin resulted in an increased fraction of BRCA1‐proficient mammary tumours, suggesting selective survival and malignant transformation of BRCA1‐proficient lesions upon prophylactic treatment with DNA‐damaging agents. Prophylactic therapy with olaparib significantly prolonged mammary tumour‐free survival without any significant increase in the fraction of BRCA1‐proficient tumours, warranting the evaluation of this PARP inhibitor in prophylactic trials in BRCA1‐mutation carriers. Copyright

Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors

The homologous recombination (HR) defect in BRCA1-associated cancers can be therapeutically exploited by the treatment with DNA-damaging agents and poly (ADP-ribose) polymerase (PARP) inhibitors. We and others previously reported that BRCA1-deficient tumors are initially hypersensitive to the inhibition of topoisomerase I/II and PARP, but acquire drug resistance through restoration of HR activity by the loss of end-resection antagonists of the 53BP1/RIF1/REV7/Shieldin/CST pathway. Here, we identified radiotherapy as an acquired vulnerability of 53BP1;BRCA1-deficient cells in vitro and in vivo. In contrast to the radioresistance caused by HR restoration through BRCA1 reconstitution, HR restoration by 53BP1 pathway inactivation further increases radiosensitivity. This highlights the relevance of this pathway for the repair of radiotherapy-induced damage. Moreover, our data show that BRCA1-mutated tumors that acquired drug resistance due to BRCA1-independent HR restoration can be targeted by radiotherapy. STATEMENT OF SIGNIFICANCE In this study, we uncovered radiosensitivity as a novel therapeutically exploitable vulnerability of BRCA1-deficient mouse mammary cells that have acquired drug resistance due to the loss of the 53BP1 pathway.