Abstract B43: NF1 deficiency induces aggressive mammary carcinomas in a CRISPR rat model and correlates with poor outcome in sporadic human breast cancer
Authors: Dischinger Patrick , Tovar Elizabeth , Essenburg Curt , Gardner Eve , Callaghan Megan , Bowman Megan , Madaj Zachary , Turner Ashley , Challa Anil , Kempston Tristan , Eagleson Bryn , Bronson Roderick , Kesterson Robert , Steensma Matthew , Graveel Carrie 
Published: August 2018

Abstract
Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome caused by germline mutations in the NF1 gene that affects1 in 3,000 births. The NF1 gene encodes for neurofibromin, a GTPase that negatively regulates RAS. Women with NF1 have a 10-fold increased risk of developing breast cancer under the age of 40 and an overall lifetime risk of 18%. NF1-related breast cancers are associated with higher grade, poor prognostic factors, and poor survival (5-year survival is 68%). Comprehensive genomic analyses have also revealed that NF1 is commonly mutated in sporadic breast cancers and is a suspected driver gene. Much of our understanding of the mechanisms underlying the functional loss of NF1 comes from genetically engineered mouse models that do not completely recapitulate the phenotypes of human NF1. To examine the effect of functional NF1 loss on tumorigenesis, we induced a deletion in the NF1 GAP-related domain (GRD) in Sprague-Dawley rats using a CRISPR-Cas9 editing strategy. Because CRISPR modification results in variability at the guide site, two classes of Nf1-deficient rats were generated: larger “in-frame” indels (i.e., 63 bp) and smaller “premature stop” indels (i.e., -11). Both types of Nf1 indels induced highly penetrant and aggressive mammary adenocarcinomas in multiple rat founder lines. Mammary tumors were observed in 100% (11/11) of G0 females in mammary glands 1-5 at the average age of 51 days. Notably, two male founders also developed mammary adenocarcinomas at 14-16 months of age. This aggressive mammary phenotype was highly penetrant and multifocal mammary tumors were observed in the subsequent F1 and F2 generations from each of the Nf1 lines. Histopathologic analysis of the mammary tumors revealed that both Nf1 in-frame deletions and premature stop indels induced a wide variety of histopathologic mammary tumor types (i.e., acinar, solid, ductular, and cystic). To understand how the distinct Nf1 indels affect disease burden and survival, we examined the overall survival of females with in-frame indels (n=35) compared to premature stop indels (n=24) in three lines (Nf1IF-57/+, Nf1IF-57/PS-8, Nf1IF-54/PS 11). Kaplan-Meier analysis revealed that animals with a premature stop codon in exon 20 died due to tumor burden significantly faster than animals with an in-frame deletion (p &lt; 0.0002). These results suggest that the Nf1 in-frame deletions may maintain some functional activity (compared to premature stop indels) that delays tumorigenesis. To our knowledge, this is the first model of NF1-related breast cancer and one of the few animal models of male breast cancer. Moreover, the Nf1 CRISPR rat is a novel model in which to explore the role of NF1 and deregulated Ras signaling in breast cancer initiation and progression.
To examine the frequency of NF1 alterations in sporadic human breast cancer, we analyzed the METABRIC breast cancer dataset. NF1 mutations were identified in 3.8% of patients, whereas 24.5% of cancers harbored shallow deletions in NF1. Survival analysis (accounting for ER status and age at diagnosis) revealed that patients with NF1 shallow deletions are 1.65X (p &lt; 1.6e-05) more likely to die within the first 10 years after diagnosis. These results reveal that NF1 deficiency may drive a more aggressive breast cancer phenotype in approximately 25% of all sporadic breast cancers.
Citation Format: Patrick Dischinger, Elizabeth Tovar, Curt Essenburg, Eve Gardner, Megan Callaghan, Megan Bowman, Zachary Madaj, Ashley Turner, Anil Challa, Tristan Kempston, Bryn Eagleson, Roderick Bronson, Robert Kesterson, Matthew Steensma, Carrie Graveel. NF1 deficiency induces aggressive mammary carcinomas in a CRISPR rat model and correlates with poor outcome in sporadic human breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B43.