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Supplementary Figures S1 - S5

PDF file - 1.2MB, This file contains additional data on tumor responses to olaparib treatment (Fig. S1 and S2), pH2AX stainings on treated and untreated tumors (Fig. S3), analysis of mutations in 53BP1 (Fig. S4 and S7B), characterization of cell lines (Fig. S5), RAD51 and 53BP1 IRIFs in tumors and cell lines (Fig. S6 and S7), 53BP1 in topotecan-resistant tumors (Fig. S8) and tumor responses to AZD2461 treatment (Fig. S9 and S10).

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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. Cancer Discov; 3(1); 68–81. ©2012 AACR.See related commentary by Fojo and Bates, p. 20This article is highlighted in the In This Issue feature, p. 1

PDF file - 28K, Information on all antibodies and primers that were used in this study

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC.Significance:The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis.This article is highlighted in the In This Issue feature, p. 2945

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084–94. ©2016 AACR.

Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concentration-dependent potentiation of MMS (PF50 ratios). Suppl Table 2. Permeability of AZD2461 and olaparib were assessed. Suppl Table 3. Activity of olaparib and AZD2461 alone or in combination with the P-gp inhibitor verapamil in the colorectal cancer cell line HCT-15, which expresses high levels of P-gp. Suppl Fig. 1. Immunofluorescence for poly(ADP-ribose) in human A549 cells. Suppl Fig. 2. Analysis of both in vivo pharmacokinetics (PK) and pharmacodynamics (PD) in tumors taken from SW620 (human colon carcinoma) xenograft-bearing mice. Suppl Fig. 3. Colony formation (clonogenic) assays in the intrinsically high P-gp-expressing MRE11-deficient HCT-15 human colorectal cell line. Suppl. Fig. 4. Combination efficacy studies. Suppl Fig. 5. Relative body weight changes in mice following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide. Suppl. Fig. 6. A serial dilution of RNA concentration was used to establish a standard curve for determining the reaction efficiency of PARP1, PARP2, PARP3, and housekeeper gene (PPIA: cyclophilin A) TaqMan RT-PCR assays in both rat and mouse species; Determination of optimal reference gene for rat-mouse data normalization. Suppl Fig. 7.A. A serial dilution of reference RNA (Qiagen) concentration was used to establish a standard curve for determining the reaction efficiency of PARP3 and housekeeper genes (PPIA: cyclophilin and YWHAZ: tyrosine 3-monooxygenase/tryptohpan 5-monooxygenase activation protein, zeta polypeptide) SybrGreen RT-PCR assays in both rat and human species; Determination of optimal reference gene for rat-human data normalization. Suppl Fig. 8. Relative body weight changes in rat following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide; Relative body weight changes in rat following treatment with 20 mg/kg AZD2461 and olaparib in combination with temozolomide.

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Supplementary Figure 3 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Supplementary Tables 1-2 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Supplementary Figure 5 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

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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. Cancer Res; 70(15); 6268–76. ©2010 AACR.

Supplementary Tables 1-4 from Sensitivity and Acquired Resistance of BRCA1;p53-Deficient Mouse Mammary Tumors to the Topoisomerase I Inhibitor Topotecan

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. Cancer Res; 72(9); 2350–61. ©2012 AACR.

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Supplementary Figure 1 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor 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. Cancer Res; 75(4); 732–41. ©2014 AACR.

Supplementary Figure 7 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

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

Supplementary Figure 2 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

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

Supplementary Figure 4 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. Significance: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084–94. ©2016 AACR.

Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy against hematologic malignancies. Antitumor activity of CAR T cells, however, needs to be improved to increase therapeutic efficacy in both hematologic and solid cancers. Limitations to overcome are 'on-target, off-tumor' toxicity, antigen escape, short CAR T cell persistence, little expansion, trafficking to the tumor and inhibition of T cell activity by an inhibitory tumor microenvironment. Here we will discuss how optimizing the design of CAR T cells through genetic engineering addresses these limitations and improves the antitumor efficacy of CAR T cell therapy in pre-clinical models.

INTRODUCTION: Anatomic imaging alone is often inadequate for tuning systemic treatment for individual tumor response. Optically based techniques could potentially contribute to fast and objective response monitoring in personalized cancer therapy. In the present study, we evaluated the feasibility of dual-modality diffuse reflectance spectroscopy–autofluorescence spectroscopy (DRS-AFS) to monitor the effects of systemic treatment in a mouse model for hereditary breast cancer. METHODS: Brca1−/−; p53−/− mammary tumors were grown in 36 mice, half of which were treated with a single dose of cisplatin. Changes in the tumor physiology and morphology were measured for a period of 1 week using dual-modality DRS-AFS. Liver and muscle tissues were also measured to distinguish tumor-specific alterations from systemic changes. Model-based analyses were used to derive different optical parameters like the scattering and absorption coefficients, as well as sources of intrinsic fluorescence. Histopathologic analysis was performed for cross-validation with trends in optically based parameters. RESULTS: Treated tumors showed a significant decrease in Mie-scattering slope and Mie-to-total scattering fraction and an increase in both fat volume fraction and tissue oxygenation after 2 days of follow-up. Additionally, significant tumor-specific changes in the fluorescence spectra were seen. These longitudinal trends were consistent with changes observed in the histopathologic analysis, such as vital tumor content and formation of fibrosis. CONCLUSIONS: This study demonstrates that dual-modality DRS-AFS provides quantitative functional information that corresponds well with the degree of pathologic response. DRS-AFS, in conjunction with other imaging modalities, could be used to optimize systemic cancer treatment on the basis of early individual tumor response.

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

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. Cancer Discov; 3(1); 68–81. ©2012 AACR. See related commentary by Fojo and Bates, p. 20 This article is highlighted in the In This Issue feature, p. 1

Breast cancer arising in female BRCA1 mutation carriers is characterized by an aggressive phenotype and early age of onset. We performed tandem mass spectrometry-based proteomics of secretomes and exosome-like extracellular vesicles from BRCA1-deficient and BRCA1-proficient murine breast tumor models to identify extracellular protein biomarkers, which can be used as an adjunct to current diagnostic modalities in patients with BRCA1-deficient breast cancer. We identified 2,107 proteins, of which 215 were highly enriched in the BRCA1-deficient secretome. We demonstrated that BRCA1-deficient secretome proteins could cluster most human BRCA1- and BRCA2-related breast carcinomas at the transcriptome level. Topoisomerase I (TOP1) and P-cadherin (CDH3) expression was investigated by immunohistochemistry on tissue microarrays of a large panel of 253 human breast carcinomas with and without BRCA1/2 mutations. We showed that expression of TOP1 and CDH3 was significantly increased in human BRCA1-related breast carcinomas relative to sporadic cases (p = 0.002 and p < 0.001, respectively). Multiple logistic regression showed that TOP1 (adjusted odds ratio [OR] 3.75; 95% confidence interval [95% CI], 1.85 - 7.71, p < 0.001) as well as CDH3 positivity (adjusted OR 2.45; 95% CI, 1.08 - 5.49, p = 0.032) were associated with BRCA1/2-related breast carcinomas after adjustment for triple-negative phenotype and age. In conclusion, proteome profiling of secretome using murine breast tumor models is a powerful strategy to identify non-invasive candidate biomarkers of BRCA1-deficient breast cancer. We demonstrate that TOP1 and CDH3 are closely associated to BRCA1-deficient breast cancer. These data merit further investigation for early detection of tumors arising in BRCA1 mutation carriers.

Breast cancer is the most common malignancy among women in developed countries, affecting more than a million women per year worldwide. Over the last decades, our increasing understanding of breast cancer biology has led to the development of endocrine agents against hormone receptor-positive tumors and targeted therapeutics against HER2-expressing tumors. However, no targeted therapy is available for patients with triple-negative breast cancer, lacking expression of hormone receptors and HER2. Overlap between BRCA1-mutated breast cancers and triple-negative tumors suggests that an important part of the triple-negative tumors may respond to therapeutics targeting BRCA1-deficient cells. Here, we review the features shared between triple-negative, basal-like and BRCA1-related breast cancers. We also discuss the development of novel therapeutic strategies to target BRCA1-mutated tumors and triple-negative tumors with BRCA1-like features. Finally, we highlight the utility of mouse models for BRCA1-mutated breast cancer to optimize (combination) therapy and to understand drug resistance.

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.

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. Cancer Res; 75(4); 732–41. ©2014 AACR.

Breast cancer 1, early onset (BRCA1) hereditary breast cancer, a type of cancer with defects in the homology-directed DNA repair pathway, would benefit from the identification of proteins for diagnosis, which might also be of potential use as screening, prognostic, or predictive markers. Sporadic breast cancers with defects in the BRCA1 pathway might also be diagnosed. We employed proteomics based on one-dimensional gel electrophoresis in combination with nano-LC-MS/MS and spectral counting to compare the protein profiles of mammary tumor tissues of genetic mouse models either deficient or proficient in BRCA1. We identified a total of 3,545 proteins, of which 801 were significantly differentially regulated between the BRCA1-deficient and -proficient breast tumors. Pathway and protein complex analysis identified DNA repair and related functions as the major processes associated with the up-regulated proteins in the BRCA1-deficient tumors. In addition, by selecting highly connected nodes, we identified a BRCA1 deficiency signature of 45 proteins that enriches for homology-directed DNA repair deficiency in human gene expression breast cancer data sets. This signature also exhibits prognostic power across multiple data sets, with optimal performance in a data set enriched in tumors deficient in homology-directed DNA repair. In conclusion, by comparing mouse proteomes from BRCA1-proficient and -deficient mammary tumors, we were able to identify several markers associated with BRCA1 deficiency and a prognostic signature for human breast cancer deficient in homology-directed DNA repair.

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. Cancer Res; 72(9); 2350–61. ©2012 AACR.

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. Cancer Res; 70(15); 6268–76. ©2010 AACR.

Tyrosine kinase inhibitors (TKI) targeting angiogenesis via inhibition of the vascular endothelial growth factor pathway have changed the medical management of metastatic renal cell carcinoma. Although treatment with TKIs has shown clinical benefit, these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. To address this question, we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response to sunitinib and eventually progressed with therapy. Tumor pieces were grafted s.c. in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density, and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor-derived cell line was established and assessed in vitro for potential direct antitumor effects of sunitinib. To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg, sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histologic examination of the original skin metastasis revealed evidence of epithelial to mesenchymal transition, whereas the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. In conclusion, the histologic examination in this xenograft study suggests that reversible epithelial to mesenchymal transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. Mol Cancer Ther; 9(6); 1525–35. ©2010 AACR.

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 tumor's 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. Cancer Res; 70(4); 1700–10

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.

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.

We have previously shown that mammary tumors spontaneously arising in a genetically engineered mouse model for human BRCA1-associated breast cancer (K14Cre;Brca1F/F;p53F/F) are highly sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib due to their deficiency in homology-directed DNA repair (Rottenberg et al. PNAS 2008;105:17079-84). Despite the initial sensitivity, tumors are not eradicated and eventually develop drug resistance. In this project we aim to identify mechanisms that explain this resistance. In particular we are interested in alterations that result in restoration of DNA repair by homologous recombination (HR). For this purpose we performed a loss-of-function shRNA screen using Brca1; p53-deficient cell lines derived from a mouse mammary tumor. The CGH profile of these cell lines is highly similar to that of the original tumor. Moreover, these cells have successfully been transplanted orthotopically into syngeneic mice and they recapitulate the morphology and olaparib response of the donor tumor. In our screen we included 2000 hairpins from the Sigma Mission library which target 409 mouse genes involved in the DNA damage response. As a result we found enrichments for hairpins targeting Rev7 in olaparib-surviving colonies. This finding was validated with 3 individual hairpins which caused substantially decreased Rev7 mRNA levels. Moreover, Rev7-deficient Brca1−/−;p53−/− tumor cells are less sensitive to doxorubicin, carboplatin and cisplatin. Loss of Rev7 rescued mES cells from Brca1 deficiency and also correlated with the formation of Rad51 foci in both Brca1-deficient mES cells and Brca1−/−;p53−/− tumor cells after irradiation. We therefore think that homology-directed DNA repair is restored in Rev7-depleted Brca1−/−;p53−/− cells. We are currently confirming this finding using HR reporter assays and we are investigating whether the loss of Rev7 causes olaparib resistance in mouse tumors. Our data suggest that absence of REV7 function, like lack of 53BP1, contributes to PARP inhibitor resistance in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-392. doi:1538-7445.AM2012-LB-392

Background: Breast cancer is the most common malignancy in women in the western world. The outcome of breast cancer would be strongly improved if patients could be diagnosed and treated early. This especially holds for patients with hereditary breast cancer. Aim: Our aim is to identify proteins that are associated with BRCA1 deficient hereditary breast cancer. These proteins could have potential use as screening, prognostic or predictive biomarkers. Approach: In-depth proteome profiling of tumor tissues of mouse breast cancer models was employed to identify BRCA1-associated proteins. To this end, tumor tissue lysates of five BRCA1 deficient and five proficient mouse models (Liu et al., 2007) were fractionated using SDS-PAGE followed by tryptic in-gel digestion, nanoLC-MS/MS and database searching. Normalized spectral counting was used for protein quantification and beta binomial statistics to discover significantly regulated proteins. Ingenuity Pathway Analysis and COFECO protein complex analysis was used to support data interpretation. The prognostic power was assessed by the use of publicly available gene expression human breast cancer data sets. Results: We identified a total of 3614 proteins, of which 804 were differential between the genomic instable BRCA1-deficient and the genomic stable BRCA1-proficient breast tumors. Pathway analysis and protein complex identified DNA-repair and associated function like chromatin modeling and RNA processing as the major functions associated with the upregulated proteins of the BRCA1 deficient tumors. We created a core DNA-repair protein signature using pathway and protein Complex analysis. When the signature proteins were mapped to human breast cancer gene-expression data sets, they were shown to have prognostic power. Conclusion: Proteomics of genetic mouse models for familial breast cancer is a powerful strategy to discover novel candidate BRCA1 deficiency proteins with human relevance. The major upregulation of DNA-repair complexes may indicate a compensatory mechanism for loss of BRCA1 DNA-repair. Further validation studies are required to investigate prognostic candidates in tissue microarrays. Integration with data on tumor and cancer cell-secreted proteins will pinpoint which of the BRCA1 deficiency associated proteins may have potential for screening of BRCA1 tumors and whether they have predictive value for sensitivity to PARP1 inhibition and Cisplatin treatment in BRCA1 as well as non-BRCA1-deficient tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5109. doi:10.1158/1538-7445.AM2011-5109

Although ABC transporters are brilliant in causing drug resistance in cultured cells, their contribution to drug resistance in patients has remained controversial. We have tackled this problem in a mammary tumor model in mice. These Brca1 / , p53 / tumors arise ‘spontaneously’ and closely resemble human breast cancer in BRCA1 carriers (1). These tumors respond to a range of standard chemotherapeutic agents (1), but eventually end up being resistant (1e3) to most drugs, but not to cisplatin (1,4). Resistance to doxorubicin in this tumor model is nearly always due to P-glycoprotein upregulation at the transcriptional level (1,3). A 5-fold increase in the low basal level of P-glycoprotein RNA in these tumors is sufficient for complete resistance. These levels of P-glycoprotein are not detectable with standard immunocytochemistry (3), showing that our current tools are inadequate to detect relevant amounts of P-gp in clinical samples. Crossing in null alleles for Mdr1a/b made the tumors hypersensitive to doxorubicin and docetaxel, showing the important contribution of P-gp to resistance in this tumor model. P-gp also causes resistance to the PARP inhibitor olaparib (2). In about half of the mice treated with topotecan, resistance is associated with upregulation of Abcg2 (Bcrp) (5). Tumors in Abcg2 / mice take longer to become resistant, showing the importance of Abcg2 as a defense system (5). We have not observed upregulation of any of the other ABC-transporters associated with topotecan resistance in cultured cells, such as Abcc2 and 4. Notwithstanding the complete remissions often obtained with some drugs, we are rarely able to eradicate the tumor (1e5). Our evidence indicates that this is not due to the hypothetical unique properties of tumor stem cells. We are also studying potential markers that determine initial drug response, but detect none at the RNA level before tumor treatment. References: (1) Rottenberg S et al. Proc Natl Acad Sci U S A 2007;104:12117e22. (2) Rottenberg S et al. Proc Natl Acad Sci U S A 2008;105:17079e84. (3) Pajic M et al. Cancer Res 2009;69:6396e404. (4) Borst P, Rottenberg S, Jonkers J. Cell Cycle 2008;7:1353e9. (5) Zander S et al. Cancer Res 2010;70: 1700e10. GOBAL DNA METHYLATION IN FETAL HUMAN GERM CELLS AND GERM CELL TUMORS: CORRELATION WITH DIFFERENTIATION AND CISPLATIN RESISTANCE

The contribution of ABC-transporters to resistance in patients has remained controversial. We have tackled this problem in a mammary mouse tumor model. These Brca1−/−, p53−/− tumors arise “spontaneously” and closely resemble human breast cancer in BRCA1+/− carriers (Rottenberg, PNAS 2007;104:12117–22; 2008;105:17079–84). The tumors respond to chemotherapeutic agents used in human breast cancer, but eventually end up being resistant to most drugs. Resistance to doxorubicin in this tumor model is nearly always due to P-glycoprotein upregulation at the transcriptional level. A 5-fold increase in the low basal level of P-glycoprotein RNA in these tumors is sufficient for complete resistance. These levels of P-glycoprotein are not detectable with standard immunocytochemistry using the C219 Mab (Pajic, Cancer Res 2009;69:6396–404). Crossing in null alleles for Mdr1a/b made the tumors hypersensitive to doxorubicin, docetaxel, and to the PARP inhibitor olaparib. In about half of the mice treated with topotecan, resistance is associated with upregulation of Abcg2 (Bcrp). Tumors in Abcg2−/− mice take longer to become resistant (Zander, Cancer Res 2009 in press). Notwithstanding the complete remissions often obtained with some drugs, we are rarely able to eradicate the tumor. This is not due to the hypothetical unique properties of tumor stem cells. Supported by Dutch Cancer Foundation and EU Framework Program. Citation Information: Clin Cancer Res 2010;16(7 Suppl):PL6-1

Purpose: Tyrosine kinase inhibitors (TKIs) targeting angiogenesis via inhibition of the vascular endothelial growth factor (VEGF) pathway have changed the medical management of metastatic renal cell carcinoma. While the treatment with TKIs has demonstrated clinical benefit these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. Experimental Design: To address this question we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response on sunitinib and eventually progressed on therapy. Tumor pieces were grafted subcutaneously in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor derived cell line was established and assessed in vitro for a potential direct antitumor effects of sunitinib. Results: To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histological examination of the original skin metastasis revealed evidence of epithelial-to-mesenchymal-transition while the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. Conclusions: The histological examination in this xenograft study suggests that reversible epithelial-to mesenchymal-transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 388.

14633 Background: Bevacizumab has shown efficacy in combination with chemotherapy. We previously found that Bevacizumab is taken up by platelets and neutralizes platelet VEGF. Platelets transport V...