Ariena Kersbergen

High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs

Whereas target-specific drugs are available for treating ERBB2-overexpressing and hormone receptor-positive breast cancers, no tailored therapy exists for hormone receptor- and ERBB2-negative (“triple-negative”) mammary carcinomas. Triple-negative tumors account for 15% of all breast cancers and frequently harbor defects in DNA double-strand break repair through homologous recombination (HR), such as BRCA1 dysfunction. The DNA-repair defects characteristic of BRCA1-deficient cells confer sensitivity to poly(ADP-ribose) polymerase 1 (PARP1) inhibition, which could be relevant to treatment of triple-negative tumors. To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor AZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.

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.

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. 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. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. 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 end resection 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. 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.

6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

Moderate Increase in Mdr1a/1b Expression Causes In vivo Resistance to Doxorubicin in a Mouse Model for Hereditary Breast Cancer

We have found previously that acquired doxorubicin resistance in a genetically engineered mouse model for BRCA1-related breast cancer was associated with increased expression of the mouse multidrug resistance (Mdr1) genes, which encode the drug efflux transporter ATP-binding cassette B1/P-glycoprotein (P-gp). Here, we show that even moderate increases of Mdr1 expression (as low as 5-fold) are sufficient to cause doxorubicin resistance. These moderately elevated tumor P-gp levels are below those found in some normal tissues, such as the gut. The resistant phenotype could be completely reversed by the third-generation P-gp inhibitor tariquidar, which provides a useful strategy to circumvent this type of acquired doxorubicin resistance. The presence of MDR1A in drug-resistant tumors with a moderate increase in Mdr1a transcripts could be shown with a newly generated chicken antibody against a mouse P-gp peptide. Our data show the usefulness of realistic preclinical models to characterize levels of Mdr1 gene expression that are sufficient to cause resistance.

Sensitivity and Acquired Resistance of BRCA1;p53-Deficient Mouse Mammary Tumors to the Topoisomerase I Inhibitor Topotecan

There is no tailored therapy yet for human basal-like mammary carcinomas. However, BRCA1 dysfunction is frequently present in these malignancies, compromising homology-directed DNA repair. This defect may serve as the tumors Achilles heel and make the tumor hypersensitive to DNA breaks. We have evaluated this putative synthetic lethality in a genetically engineered mouse model for BRCA1-associated breast cancer, using the topoisomerase I (Top1) poison topotecan as monotherapy and in combination with poly(ADP-ribose) polymerase inhibition by olaparib. All 20 tumors tested were topotecan sensitive, but response heterogeneity was substantial. Although topotecan increased mouse survival, all tumors eventually acquired resistance. As mechanisms of in vivo resistance, we identified overexpression of Abcg2/Bcrp and markedly reduced protein levels of the drug target Top1 (without altered mRNA levels). Tumor-specific genetic ablation of Abcg2 significantly increased overall survival of topotecan-treated animals (P < 0.001), confirming the in vivo relevance of ABCG2 for topotecan resistance in a novel approach. Despite the lack of ABCG2, a putative tumor-initiating cell marker, none of the 11 Abcg2(-/-);Brca1(-/-);p53(-/-) tumors were eradicated, not even by the combination topotecan-olaparib. We find that olaparib substantially increases topotecan toxicity in this model, and we suggest that this might also happen in humans.

Impact of Intertumoral Heterogeneity on Predicting Chemotherapy Response of BRCA1-Deficient Mammary Tumors

The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy.

BRCA2-Deficient Sarcomatoid Mammary Tumors Exhibit Multidrug Resistance

Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this.

Noninvasive functional imaging of P-glycoprotein-mediated doxorubicin resistance in a mouse model of hereditary breast cancer to predict response, and assign P-gp inhibitor sensitivity

PurposeUsing a “spontaneous” mammary mouse tumor model we set out to develop diagnostic approaches for non-invasive P-glycoprotein (P-gp) staging and response prediction.Methods99mTc-MIBI efflux rates were measured using a gamma camera in three Brca1−/−; p53−/− mouse mammary tumors that have different Mdr1a/b expression levels. The efflux rates were quantified in the 10–30-min period after injection. In addition to the P-gp-mediated efflux measurements in untreated tumors, efflux measurements were performed in the presence of the P-gp inhibitor tariquidar. Volumetric doxorubicin response patterns for the different tumors were determined and correlated with the efflux rates.ResultsCombined pre- and post-inhibitor treatment imaging of P-gp-mediated efflux correlated with Mdr1a/b expression: basal (0.0026, p = 0.16), 3-fold Mdr1a/b (0.0074, p = 0.02), and 17-fold Mdr1a and 46-fold Mdr1b (0.012, p = 0.002). Based on the doxorubicin response of these tumors, we generated a computer-aided diagnosis model that predicts the likelihood of drug resistance.ConclusionsQuantified 99mTc-MIBI efflux has potential to: (1) noninvasively assign Mdr1 expression levels, (2) predict the therapeutic impact of a P-gp inhibitor, and (3) noninvasively assess the probability of drug resistance.

Lack of ABCG2 shortens latency of BRCA1-deficient mammary tumors and this is not affected by genistein or resveratrol

In addition to their role in drug resistance, the ATP-binding cassette (ABC) transporters ABCG2 and ABCB1 have been suggested to protect cells from a broad range of substances that may foster tumorigenesis. Phytoestrogens or their metabolites are substrates of these transporters and the influence of these compounds on breast cancer development is controversial. Estrogen-like properties might accelerate tumorigenesis on the one hand, whereas their proposed health-protective properties might antagonize tumorigenesis on the other. To address this issue, we used a newer generation mouse model of BRCA1-mutated breast cancer and examined tumor latency in K14cre;Brca1F/F; p53F/F, Abcb1a/b−/−;K14cre;Brca1F/F; p53F/F, or Abcg2−/−;K14cre;Brca1F/F; p53F/F animals, fed with genistein- or resveratrol-supplemented diets. Ovariectomized K14cre;Brca1F/F; p53F/F animals were included to evaluate whether any estrogen-mimicking effects can restore mammary tumor development in the absence of endogenous estrogens. Compared with the ABC transporter proficient model, ABCG2-deficient animals showed a reduced median tumor latency of 17.5 days (P < 0.001), whereas no significant difference was observed for ABCB1-deficient animals. Neither genistein nor resveratrol altered this latency reduction in Abcg2−/−;K14cre;Brca1F/F; p53F/F animals. Ovariectomy resulted in nearly complete loss of mammary tumor development, which was not restored by genistein or resveratrol. Our results show that ABCG2 contributes to the protection of genetically instable epithelial cells against carcinogenesis. Diets containing high levels of genistein or resveratrol had no effect on mammary tumorigenesis, whether mice were lacking ABCG2 or not. Because genistein and resveratrol only delayed skin tumor development of ovariectomized animals, we conclude that these phytoestrogens are no effective modulators of mammary tumor development in our mouse model. Cancer Prev Res; 5(8); 1053–60. ©2012 AACR.