Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer (BC) with few targeted therapies. To identify novel genetic modifiers of TNBC, we created a murine model incorporating high levels of genetic and phenotypic diversity. C3(1)-T-antigen ("C3Tag") mice, which develop spontaneous basal-like TNBC tumors, were systematically crossed with a large set of sequenced BXD recombinant inbred strains to produce isogenic hybrids segregating for C3Tag. The severity of TNBC traits including tumor latency, multiplicity, and survival was highly variable and heritable. We mapped modifiers of TNBC and identified loci on chromosomes 16 and 10 associated with tumor multiplicity and latency, respectively. Candidate genes were prioritized including a lysosomal enzyme involved in cell proliferation, Gns; tumor suppressor Rassf3; and Rab-modifying Tbc1d30. In tumors from BC patients, higher GNS, RASSF3, and TBC1D30 expression associated with poor overall survival. In sum, we developed a clinically relevant, BXD-BC model which provides robust genetic heterogeneity enabling the identification of conserved modifiers and mediators of BC.

Breast cancer (BC) is the most common cancer and the second cause of death in US women. Our lack of understanding of how genetic variants affect molecular mechanisms that mediate BC aggression poses a substantial obstacle to advancements in cancer diagnosis and therapy. To examine genetic variants on BC traits, a novel murine model was created with robust phenotypic and genomic variation. The FVB C3(1)-T-antigen (“C3Tag”) mouse develops spontaneous tumors in the mammary glands of female mice with a mean latency of 4-5 months of age. This genetically engineered mouse model (GEMM) is well established to resemble human basal-like TNBC. TNBC is an aggressive subtype with few clinical approaches and poor patient outcomes. Thus, to model human heterogeneity in BC outcomes, we systematically crossed the C3Tag GEMM into the BXD recombinant inbred family – the largest and best characterized genetic reference population. The new model is termed “BXD-BC” and F1 hybrids of the cross have isogenic genomes that are reproducible. BXD-BCs are a potent tool to determine the impact of genetic modifiers on BC tumor traits. We hypothesized that examination of BXD-BC GEMMs will enable the identification of susceptibility loci, candidate genes, and molecular networks that underlie variation of multiple BC phenotypes. Using N=29 BXD-BC strains, we demonstrated significant heritable variations in the severity of TNBC characteristics such as tumor latency, multiplicity, and survival. Interestingly, 2 BXD-BC strains never developed tumors out to 1 year of age. Thus, BXD-BC strains demonstrate variance in cancer susceptibility and progression compared to the parent C3Tag GEMM, indicating the presence of genetic modifiers. Through an unbiased systematic quantification of breast cancer severity across BXD-BC hybrids, we identified several significant quantitative trait loci (QTL) and candidate genes for specific tumor traits. In combination with public human GWAS datasets, we defined syntenic regions, candidate genes, and underlying networks through cross-species systems genetics analyses to demonstrate the translational validity of conserved, biologically relevant, and targetable candidates. Our findings suggest conserved candidates predicting TNBC patient survival. In sum, the BXD-BC resource is an innovative, reliable, and robust preclinical model that reflects robust genetic heterogeneity. Using cutting edge systems genetics, we have identified genetic modifiers of BC phenotypic variation that could be targeted to advance therapeutic limitations or as biomarkers of risk or response to therapy.### Competing Interest StatementThe authors have declared no competing interest.

Bariatric surgery is a sustainable weight loss approach, including vertical sleeve gastrectomy (VSG). Obesity exacerbates tumor growth, while diet-induced weight loss impairs progression. It remains unknown how bariatric surgery-induced weight loss impacts cancer progression or alters response to therapy. Using a pre-clinical model of obesity followed by VSG or diet-induced weight loss, breast cancer progression and immune checkpoint blockade therapy were investigated. Weight loss by VSG or weight-matched dietary intervention before tumor engraftment protected against obesity-exacerbated tumor progression. However, VSG was not as effective as diet in reducing tumor burden despite achieving similar weight and adiposity loss. Leptin did not associate with changes in tumor burden; however, circulating IL-6 was elevated in VSG mice. Uniquely, VSG tumors displayed elevated inflammation and immune checkpoint ligand PD-L1+ myeloid and non-immune cells. VSG tumors also had reduced T lymphocytes and markers of cytolysis, suggesting an ineffective anti-tumor microenvironment which prompted investigation of immune checkpoint blockade. While obese mice were resistant to immune checkpoint blockade, anti-PD-L1 potently impaired tumor progression after VSG through improved anti-tumor immunity. Thus, in formerly obese mice, surgical weight loss followed by immunotherapy reduced breast cancer burden. Finally, we compared transcriptomic changes in adipose tissue after bariatric surgery from patients and mouse models. A conserved bariatric surgery-associated weight loss signature (BSAS) was identified which significantly associated with decreased tumor volume. Findings demonstrate conserved impacts of obesity and bariatric surgery-induced weight loss pathways associated with breast cancer progression.

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor outcomes. This is a grave clinical challenge for the ~30,000 patients diagnosed with this disease every year. Discovering genetic modifiers of differential TNBC vulnerability and disease progression is critical to improving predictive and personalized treatments. We hypothesized that using a well-established recombinant inbred strain, novel genetic modifiers of TNBC risk and aggression will be identified. The C3(1)-T antigen (C3Tag) genetically engineered mouse model (GEMM) recapitulates many facets of human basal-like TNBC to demonstrate promoting effects of exposures on tumor phenotypes. However, GEMM are highly constrained by their inbred genotype and do not allow a robust interrogation of the manner in which individual genetic variation might impact tumor initiation, progression, and response to therapy. Therefore, we developed a novel murine model of TNBC in the background of the largest and best characterized genetic reference population. Systems genetics is used to identify gene candidates. Cross-species comparison of our findings with publicly available human GWAS and genomic databases is an effective approach to validate conserved biologically relevant and targetable pathways. To our knowledge, this is the first study to explore modifier genes for TNBC phenotypes using a systems genetics approach in a GEMM for TNBC. Our results will contribute to significant advances in understanding risk and improving outcomes for breast cancer. Citation Format: Laura M. Sipe, Emily B. Korba, Lu Lu, Robert W. Williams, David G. Ashbrook, Liza Makowski. Novel pre-clinical model to identify genetic modifiers of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2919.

Background: Obesity leads to a higher risk of cancer invasion, metastases, recurrence, mortality, and impaired therapeutic response through various mechanisms. One of these mechanisms is microenvironment dysfunction, where changes in immune cells, microbes, metabolites, and growth factors contribute to tumor aggressiveness. We study obesity-induced changes to the tumor microenvironment in Triple Negative Breast Cancer (TNBC), an aggressive subtype associated with obesity. The goal to compare tumor progression in lean vs. obese vs. weight loss manipulations to identify causal and targetable pathways associated with reprogramming the tumor microenvironment. Clinically, bariatric surgery induced weight loss reduced the risk of BC, with the greatest benefit detected in pre-menopausal patients with ER- tumors, like TNBC. We hypothesize surgically-induced weight loss will diminish obesity-associated tumor progression. Methods: To study weight gain and loss that best mimics human adiposity, we utilized obesogenic C57BL/6 females with a syngeneic orthotopic transplant of TNBC cells. Female mice placed on a high fat diet (HFD) at weaning became obese compared to mice on a low fat diet (LFD). After 16 weeks on an HFD diet, mice underwent the bariatric surgery Vertical Sleeve Gastrectomy (VSG). The VSG resulted in reduced body weight, adiposity, and correction of metabolic profiles compared to obese mice. Two weeks post-surgery, TNBC cells were OT into the mammary fat. Results: As expected, tumor growth was increased in obese mice compared to lean. Importantly, surgical weight loss rescued obese tumor progression. We identified key changes in tumor infiltrating of immune cells that could be responsible for the beneficial effects of bariatric surgery on tumor progression. We then exploited these changes with immunotherapy, which was uniquely effective in mice that underwent bariatric surgery. Conclusions: In conclusion obesity promotes a pro-tumor microenvironment, that can be corrected through surgically induced weight loss. Citation Format: Laura Sipe, Mehdi Chaib, Emily B Korba, Mary C Lovely, Jared C Clements, Neena A John, Ajeeth K Pingili, Joseph F Pierre, Liza Makowski. Surgically induced weight loss corrects obesity associated tumor progression and improves responsiveness to immunotherapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-29.