Ute Boon

Loss of 53BP1 Causes PARP Inhibitor Resistance in Brca1-Mutated Mouse Mammary Tumors

UNLABELLED Inhibition of PARP is a promising therapeutic strategy for homologous recombination-deficient tumors, such as BRCA1-associated cancers. We previously reported that BRCA1-deficient mouse mammary tumors may acquire resistance to the clinical PARP inhibitor (PARPi) olaparib through activation of the P-glycoprotein drug efflux transporter. Here, we show that tumor-specific genetic inactivation of P-glycoprotein increases the long-term response of BRCA1-deficient mouse mammary tumors to olaparib, but these tumors eventually developed PARPi resistance. In a fraction of cases, this resistance is caused by partial restoration of homologous recombination due to somatic loss of 53BP1. Importantly, PARPi resistance was minimized by long-term treatment with the novel PARP inhibitor AZD2461, which is a poor P-glycoprotein substrate. Together, our data suggest that restoration of homologous recombination is an important mechanism for PARPi resistance in BRCA1-deficient mammary tumors and that the risk of relapse of BRCA1-deficient tumors can be effectively minimized by using optimized PARP inhibitors. SIGNIFICANCE In this study, we show that loss of 53BP1 causes resistance to PARP inhibition in mouse mammary tumors that are deficient in BRCA1. We hypothesize that low expression or absence of 53BP1 also reduces the response of patients with BRCA1-deficient tumors to PARP inhibitors.

BRCA1 RING function is essential for tumor suppression but dispensable for therapy resistance.

Hereditary breast cancers are frequently caused by germline BRCA1 mutations. The BRCA1(C61G) mutation in the BRCA1 RING domain is a common pathogenic missense variant, which reduces BRCA1/BARD1 heterodimerization and abrogates its ubiquitin ligase activity. To investigate the role of BRCA1 RING function in tumor suppression and therapy response, we introduced the Brca1(C61G) mutation in a conditional mouse model for BRCA1-associated breast cancer. In contrast to BRCA1-deficient mammary carcinomas, tumors carrying the Brca1(C61G) mutation responded poorly to platinum drugs and PARP inhibition and rapidly developed resistance while retaining the Brca1(C61G) mutation. These findings point to hypomorphic activity of the BRCA1-C61G protein that, although unable to prevent tumor development, affects response to therapy.

Mechanisms of Therapy Resistance in Patient-Derived Xenograft Models of BRCA1-Deficient Breast Cancer.

BACKGROUND Although BRCA1-deficient tumors are extremely sensitive to DNA-damaging drugs and poly(ADP-ribose) polymerase (PARP) inhibitors, recurrences do occur and, consequently, resistance to therapy remains a serious clinical problem. To study the underlying mechanisms, we induced therapy resistance in patient-derived xenograft (PDX) models of BRCA1-mutated and BRCA1-methylated triple-negative breast cancer. METHODS A cohort of 75 mice carrying BRCA1-deficient breast PDX tumors was treated with cisplatin, melphalan, nimustine, or olaparib, and treatment sensitivity was determined. In tumors that acquired therapy resistance, BRCA1 expression was investigated using quantitative real-time polymerase chain reaction and immunoblotting. Next-generation sequencing, methylation-specific multiplex ligation-dependent probe amplification (MLPA) and Target Locus Amplification (TLA)-based sequencing were used to determine mechanisms of BRCA1 re-expression in therapy-resistant tumors. RESULTS BRCA1 protein was not detected in therapy-sensitive tumors but was found in 31 out of 42 resistant cases. Apart from previously described mechanisms involving BRCA1-intragenic deletions and loss of BRCA1 promoter hypermethylation, a novel resistance mechanism was identified in four out of seven BRCA1-methylated PDX tumors that re-expressed BRCA1 but retained BRCA1 promoter hypermethylation. In these tumors, we found de novo gene fusions that placed BRCA1 under the transcriptional control of a heterologous promoter, resulting in re-expression of BRCA1 and acquisition of therapy resistance. CONCLUSIONS In addition to previously described clinically relevant resistance mechanisms in BRCA1-deficient tumors, we describe a novel resistance mechanism in BRCA1-methylated PDX tumors involving de novo rearrangements at the BRCA1 locus, demonstrating that BRCA1-methylated breast cancers may acquire therapy resistance via both epigenetic and genetic mechanisms.

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.

Mps1 inhibitors synergise with low doses of taxanes in promoting tumour cell death by enhancement of errors in cell division

BackgroundChromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice.MethodsTo investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1−/−;TP53−/− mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination.ResultsThe enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment.ConclusionsOur study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.

Combination therapy of the EZH2 inhibitor GSK126 and the PARP inhibitor Olaparib shows in vivo synergy in a patient-derived xenograft model of BRCA1-deficient breast cancer

Background: Current treatment options for BRCA1-deficient breast cancer are limited highlighting the need for novel targeted therapies. Our group previously demonstrated that EZH2 is overexpressed in BRCA1-deficient breast tumors and a promising target for this subtype. EZH2 is the catalytic subunit of polycomb repressive complex 2 and involved in gene silencing through methylation of histone H3K27. Recently, GSK126, a highly selective inhibitor of EZH2 methyltransferase activity, was discovered. Additionally, PARP inhibitors are effective in breast tumors with defects in homologous recombination due to BRCA1 mutations. Methods: To study a possible synergy between the EZH2 inhibitor GSK126 and PARP inhibitor Olaparib we made use of a patient-derived xenograft (PDX) model from triple negative breast cancer with BRCA1 mutation. Cell lines were derived from a genetically modified mouse model for hereditary breast cancer (K14cre;Brca1F/F;p53F/F). Results: Combined inhibition of EZH2 and PARP in BRCA1-deficient cell lines resulted in delayed cell growth compared to single treatments. In the BRCA1-deficient PDX model the EZH2 inhibitor GSK126 alone attenuated the tumor growth modestly. Treatment with Olaparib shows tumor stasis. However, dual EZH2 and PARP inhibition with GSK126 and Olaparib reduced the tumor volume substantially. Western blot analysis of tumor lysates demonstrate target inhibition of GSK126 by significantly reduced H3K27me3-levels. Conclusion: Here, we report that the combination of EZH2 inhibition with a PARP inhibitor provides in vivo synergy in a PDX-mouse model for BRCA1-deficient breast tumors. Our findings suggest that this candidate combination might be an effective treatment of BRCA1-related tumors to be tested in further trials.

Abstract PR02: Genomic rearrangements and promoter demethylation drive therapy resistance in patient-derived xenograft models of BRCA1-deficient breast cancer

Background: BRCA1 mutated breast tumor cells are defective in DNA repair by homologous recombination and therefore especially sensitive to treatment with DNA double-strand break (DSB) inducing agents, such as alkylators and PARP inhibitors. However, such tumors can eventually develop therapy resistance. Understanding the underlying mechanisms may help in designing strategies to avoid or overcome acquired therapy resistance. Methods: We have developed patient derived xenograft (PDX) models of BRCA1-deficient triple-negative breast cancer (TNBC) by implantating fresh human breast tumor pieces and subsequent serial passaging. These models show epigenetic loss of BRCA1 due to promoter hypermethylation or genetic inactivation of BRCA1 due to a frameshift mutation (c.2210delC) resulting in a premature stop codon (p.Thr737LeufsX15). We have used these 3 BRCA1-deficient TNBC models to study response and acquisition of resistance to alkylating therapy (cisplatin, melphalan, nimustine) and the clinical PARP inhibitor olaparib. Results: Treated tumors responded well to the alkylators cisplatin, melphalan and nimustine or the PARP inhibitor olaparib, in some cases resulting in complete remission with no palpable tumor left. However, relapses did occur in most cases and repeated treatment of recurrent tumors eventually led to aquired resistance. Since restoration of BRCA1 function has been suggested as a mechanism of therapy resistance (Swisher et al., Cancer Res 2008; 68: 2581), we determined BRCA1 expression in therapy-sensitive and -resistant tumors by Western blot analysis. While no full length BRCA1 protein could be detected in the therapy-sensitive tumors, expression of full length BRCA1 protein was found in the majority of alkylator resistant and olaparib resistant tumors. BRCA1 re-expression in the therapy-resistant BRCA1-c.2210delC tumors was caused in most cases by genetic restoration of the reading frame due to additional deletions near the c.2210delC mutation. In therapy-resistant TNBC xenografts that showed epigenetic loss of BRCA1 before treatment, resistance was also often associated with expression of BRCA1. In many of these tumors, loss of BRCA1 promoter hypermethylation was detected. Some of the therapy resistant tumors that showed BRCA1 expression, did not show loss of BRCA1 promoter hypermethylation. In these tumors, complex rearrangements at the BRCA1 locus were found. Conclusion: Although BRCA1-deficient TNBC xenografts are initially very sensitive to alkylating agents and olaparib, resistance to treatment develops in almost all treated tumors. This acquired resistance is frequently associated with re-expression of BRCA1 due to secondary mutations in BRCA1 mutated tumors. In breast tumors that showed epigenetic loss of BRCA1, acquired resistance is associated with loss of promoter methylation or complex rearrangements at the BRCA1 locus. This abstract is also presented as Poster B010. Citation Format: Petra ter Brugge, Eline van der Burg, Petra Kristel, Ute Boon, Ian Majewski, Catia Moutinho, Manel Esteller, Frans Hogervorst, Heidrun Gevensleben, Nick Turner, Wigard Kloosterman, Esther Lips, Jelle Wesseling. Genomic rearrangements and promoter demethylation drive therapy resistance in patient-derived xenograft models of BRCA1-deficient breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR02.

Abstract PR3: Patient derived BRCA1-deficient triple-negative breast cancer xenografts develop resistance to DNA damaging agents via genetic and epigenetic mechanisms

Background: BRCA1 mutated breast tumor cells are defective in DNA repair by homologous recombination and therefore especially sensitive to treatment with DNA double-strand break (DSB) inducing agents, such as alkylators and PARP inhibitors. However, such tumors can eventually develop therapy resistance. Understanding the underlying mechanisms may help in designing strategies to avoid or overcome acquired therapy resistance. Methods: We have developed patient derived BRCA1-deficient triple-negative breast cancer (TNBC) xenograft models by implantation of fresh human breast tumor pieces and subsequent serial passaging. These models show epigenetic loss of BRCA1 expression due to promoter hypermethylation or genetic inactivation of BRCA1 due to a frameshift mutation (c.2210delC) resulting in a premature stop codon (p.Thr737LeufsX15). We have used these 3 BRCA1-deficient TNBC models to study response and acquisition of resistance to alkylating therapy (cisplatin, melphalan, nimustine) and the clinical PARP inhibitor olaparib. Results: Treated tumors responded well to the alkylators cisplatin, melphalan and nimustine or the PARP inhibitor olaparib, in some cases resulting in complete remission with no palpable tumor left. However, relapses did occur in most cases and repeated treatment of recurrent tumors eventually led to acquired resistance. Since restoration of BRCA1 function has been suggested as a mechanism of therapy resistance (Swisher et al., Cancer Res 2008; 68: 2581), we determined BRCA1 expression in therapy-sensitive and -resistant tumors by Western blot analysis. While no full length BRCA1 protein could be detected in the therapy-sensitive tumors, expression of full length BRCA1 protein was found in the majority of alkylator resistant and olaparib resistant tumors. BRCA1 re-expression in the therapy-resistant BRCA1-c.2210delC tumors was caused in most cases by genetic restoration of the reading frame due to additional deletions near the c.2210delC mutation. In therapy-resistant TNBC xenografts that showed epigenetic loss of BRCA1 before treatment, resistance was also often associated with expression of BRCA1. In many of these tumors, loss of BRCA1 promoter hypermethylation was detected. Some of the therapy resistant tumors that showed BRCA1 expression did not show loss of BRCA1 promoter hypermethylation. In these tumors, complex rearrangements at the BRCA1 locus were found. These rearrangements placed the BRCA1 gene behind an alternative promoter, which led to expression of BRCA1 in these tumors. Conclusion: Although BRCA1-deficient TNBC xenografts are initially very sensitive to alkylating agents and olaparib, resistance to treatment develops in almost all treated tumors. This acquired resistance is frequently associated with re-expression of BRCA1 due to secondary mutations in BRCA1 mutated tumors. In breast tumors that showed epigenetic loss of BRCA1, acquired resistance is associated with loss of promoter methylation or complex rearrangements at the BRCA1 locus. This proffered talk is also presented as Poster A40.