Somatic Engineering Of The Mammary Gland For The Development Of Novel Mouse Models Of Triple-Negative Breast Cancer.

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. For these reasons, new technologies are needed to expand the genetic toolbox of cancer biologists and allow a more rapid and systematic in vivo interrogation of gene perturbations. In this regard, the advent of CRISPR/Cas9 technologies for somatic genome editing has already paved the way for a new generation of non-germline animal tumor models. For example, liver-specific gene disruption was achieved by transient delivery of components of the CRISPR/Cas9 system in the tail veins of mice, leading to hepatocellular carcinoma (Xue et al., 2014; Weber et al., 2015). Similar approaches have been used to deliver targeted oncogenic mutations to the lung (Platt et al., 2014; Sanchez-Rivera et al., 2014), brain (Zuckermann et al., 2015), and pancreas (Chiou et al., 2015). We have previously shown that the mammary tissue is also amenable to somatic gene editing using intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in the mammary glands of female mice carrying conditional predisposing alleles (Annunziato et al., 2016). This approach was successfully applied to validate in vivo candidate tumor suppressors implicated in invasive lobular carcinoma (Kas et al., 2017), but it is conceivable that more breast cancer subtypes can be modeled via somatic engineering in mice with distinct predisposing mutations. In this study, we show how somatic engineering via intraductal injection of lentiviruses can be used to develop innovative mouse models of triple-negative breast cancer (TNBC), one of the subtypes with the poorest prognosis in the clinics. Using intraductal injection of Cre-encoding lentiviruses in mice with conditional Brca1 and Trp53 alleles (B1P), we were able to target TNBC-initiating cells from the basal compartment, and induce tumors that retained all the hallmark features of the transgenic Cre-driven tumor counterparts, including latency, histopathology, and DNA copy-number variation (CNV) pattern. Moreover, we could dramatically accelerate tumorigenesis when lentiviral Cre was injected in B1PM mice that harbored, in addition to conditional Brca1 and Trp53 alleles, a conditional knock-in allele overexpressing the Myc oncogene, a candidate driver in BRCA1-associated cancers. Alternatively, somatic Myc expression and loss of BRCA1 and p53 was induced in the mammary glands of B1P mice via intraductal injection of lentiviral vectors encoding Myc and Cre under a viral promoter. In both cases the resulting tumors displayed a reshaped CNV profile that strongly implicated a limited number of recurrent focal amplifications and deletions in the tumorigenic cascade. We reiterated the process by lentiviral overexpression of these candidate driver oncogenes and Cre in the mammary glands of B1PM mice, or by achieving in situ CRISPR/Cas9-mediated somatic gene disruption of one or multiple candidate tumor-suppressor genes in Cas9-transgenic WB1P-Cas9 mice. This versatile somatic platform allows for rapid, flexible, and multiplexable in vivo testing of putative oncogenic hits and deals with the need for increasingly complex mouse models while avoiding the bottlenecks of classical transgenesis. Notably, we show here that we can extend the applicability of non-germline breast cancer modeling in mice to produce genetically and histologically accurate TNBC, which allows in vivo investigation of the underlying biology as well as rapid generation of tailored preclinical models for this aggressive breast cancer subtype. Citation Format: Stefano Annunziato, Julian R. de Ruiter, Chiara S. Brambillasca, Sjors M. Kas, Federica Ferrante, Catrin Lutz, Bjorn Siteur, Bas van Gerwen, Marieke van de Ven, Martine H. van Miltenburg, Linda Henneman, Jos Jonkers. Somatic engineering of the mammary gland for the development of novel mouse models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A01.