Abstract P3-08-01: Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system
Authors: Wallis Deeann , Fay Christian , Bradley Kelley , Westin Erik , Bradley William , Liu Hui , Lambert Laura , Crossman David , Foote Jeremy , Kesterson Robert 
Published: March 2023

Abstract
Loss of NF1 plays a major role as an oncogenic driver in many cancer types and can be found in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. In addition, certain NF1 genotypes may increase cancer risks, while others do not. NF1 is largely perceived as a classic Ras-opathy syndrome due to inactivating mutations in neurofibromin affecting RAS-MAPK signaling. However, recently it has been shown that NF1 binds estrogen receptor alpha (ER) and acts as a transcriptional corepressor. This helps explain some BC findings specifically in ER+ BC. In this model, specific changes to NF1 that abrogate ER signaling lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics. A preclinical mammalian model of NF1 loss and BC would be helpful in both evaluating the role of NF1/ER transcriptional signaling in BC, evaluating the role of immune cells in BC, and testing therapeutics. Novel Nf1 rat models have a very robust ER+ BC phenotype, therefore more closely recapitulating clinical tumors compared to other preclinical models. Our models include a pathogenic patient missense allele c.3827G&gt;A, p.R1276Q (knockin or KI), associated in humans with familial spinal NF1 and malignancy, as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 females develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset while het KI rats tend to develop fewer tumors with later onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as by 1 year 76% of KI females survive yet only 58% of KO females survive. However, by 2 years, both alleles have 54% survival. The divergence in phenotype between patient and null alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. While het male rats also develop mammary tumors at low rates, they experience longer survival times (76% of KO males survive 1 year and 98% of KI males survive 1 year) and males also develop tumors in other locations. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response and compare/contrast them with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options for treatment.
Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, Laura Lambert, David Crossman, Jeremy Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-01.