Stefano Annunziato

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.

Cancer Gene Discovery: Exploiting Insertional Mutagenesis

Insertional mutagenesis has been used as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. Retroviruses have been used for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using retrovirus-based approaches. Most recently, lentiviral vectors have appeared on the scene for use in cancer gene screens. Lentiviral vectors are replication-defective integrating vectors that have the advantage of being able to infect nondividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations, and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future. Visual Overview: http://mcr.aacrjournals.org/content/11/10/1141/F1.large.jpg. Mol Cancer Res; 11(10); 1141–58. ©2013 AACR. Visual Overview

Insertional mutagenesis identifies drivers of a novel oncogenic pathway in invasive lobular breast carcinoma

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8–14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.

Lentiviral Vector-based Insertional Mutagenesis Identifies Genes Involved in the Resistance to Targeted Anticancer Therapies

The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2(+) breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors.

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.

Genetic Dissection of Cancer Development, Therapy Response, and Resistance in Mouse Models of Breast Cancer.

The cancer genomics revolution has rapidly expanded the inventory of somatic mutations characterizing human malignancies, highlighting a previously underappreciated extent of molecular variability between and within patients. Also in breast cancer, the most commonly diagnosed malignancy in women, this heterogeneity complicates the understanding of the stepwise sequence of pathogenic events and the design of effective and long-lasting target therapies. To disentangle this complexity and pinpoint which molecular perturbations are crucial to hijack the cellular machinery and lead to tumorigenesis and drug resistance, functional studies are needed in model systems that faithfully and comprehensively recapitulate all the salient aspects of their cognate human counterparts. Mouse models of breast cancer have been instrumental for the study of tumor initiation and drug response but also involve cost and time limitations that represent serious bottlenecks in translational research. To keep pace with the overwhelming amount of hypotheses that warrant in vivo testing, continuous refinement of current breast cancer models and implementation of new technologies is crucial. In this review, we summarize the current state of the art in modeling human breast cancer in mice, and we put forward our vision for future developments.

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.

Abstract 5115: Establishing tumoroid and mouse models for functional validation of progression markers in DCIS

Ductal Carcinoma in Situ (DCIS) is a starting lesion in the milk duct of the breast, which accounts for 25% of all ‘breast cancers9 detected since the introduction of breast screening. DCIS is usually treated by surgery combined with radiotherapy, which can have a large impact on the life of patients. However, there is little to no evidence that treatment of low and intermediate grade DCIS reduces mortality, while women diagnosed with DCIS do perceive their risk of dying the same as patients with invasive disease. To reduce the negative perception and the overtreatment of DCIS, but assure proper treatment for high risk DCIS, it is critical to identify which factors can predict whether DCIS stays indolent or becomes invasive. We have successfully shown that it is possible to propagate tumor cells and organoids in vivo using intraductal injections. Adult female NSG and nude mice were injected intraductally into the mammary gland with suspensions of breast cancer cell and organoid lines as well as DCIS cell lines. Moreover, we were able to instigate a protocol for deriving fresh patient DCIS material to create in vitro primary tumoroid cultures, which we transplanted as suspended cells into NSG and nude mice. Tumor formation was examined via palpation and bioluminescence imaging. The resulting tumors will be compared to the original patient material to show that we are able to create a suitable in vivo model, retaining the morphologic and genomic features of the patient. In addition to these transplantation models we sought to take advantage of existing non-germline models for establishing a genetic DCIS model. To establish these, genes were selected from literature which are suspected to initiate DCIS formation. These genes were then incorporated in lentiviral vectors and injected in the mammary gland of immunocompetent mice. The mice will be sacrificed at set time points to follow DCIS initiation and progression to Invasive Ductal Carcinoma (IDC). For characterization, all these models will be subjected to in‐depth histopathologic, transcriptomic, mutational, proteomic, immunologic and methylomic characterization, such as copy number sequencing, RNA sequencing and reverse-phase protein assays. Together these models will aid in finding factors initiating DCIS and finding the key characteristics driving DCIS to IDC switch, and ultimately making it possible to distinguish between high risk and low risk DCIS. On top of this, the in vitro and in vivo human DCIS models will place a unique opportunity for high-throughput drug screening to obtain sensitivity profiles. These will be combined with -omics data to identify (epi)genetic determinants of drug sensitivity. Citation Format: Stefan J. Hutten, Catrin Lutz, Stefano Annunziato, Ellen Tanger, Jelle Wesseling, Jos Jonkers. Establishing tumoroid and mouse models for functional validation of progression markers in DCIS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5115.