Abstract A21: Dissecting MYC immunoregulatory activity in BRCA1-associated breast cancer
Authors: Brambillasca Chiara Svetlana , Henneman Linda , Ruiter Julian de , Wellenstein Max , Drenth Anne Paulien , Visser Karin de , Jonkers Jos 
Published: May 2018

Abstract
Breast cancer is the most common cancer among women in the Western world. Approximately 8% of all breast cancer cases may be caused by inheritance of mutations in breast cancer susceptibility genes. Of these, BRCA1 is the most important one, contributing to approximately half of all familial breast cancer cases. BRCA1-related breast tumors show a triple-negative (TBNC) basal-like phenotype, which correlates with aggressive characteristics and poor prognosis. MYC has been identified as a potential driver gene in BRCA1-associated breast cancer. In line with this, we found MYC to be frequently amplified in both mouse and human BRCA1-deficient breast cancers. Thus, evaluating the contribution of MYC to BRCA1-associated breast cancer might unravel new mechanistic insights and potentially open the way to new therapeutic approaches.
To investigate a causal role of MYC in BRCA1-associated breast cancer, we generated WAPCre;Brca1F/F;Trp53F/F (WB1P) and WAPCre;Brca1F/F;Trp53F/F;invCAG-Myc (WB1P-Myc) mouse models, which were monitored for tumor development. Whereas WB1P female mice developed mammary tumors after 300 days, WB1P-Myc mice showed a dramatic acceleration in tumor development with a median survival of 100 days. Tumors of both models were histopathologically classified as poorly differentiated solid carcinomas resembling TNBC. To characterize the differences between the WB1P and WB1P-Myc tumors, we performed whole-transcriptome analysis using RNAseq. Unsupervised hierarchical cluster analysis showed that WB1P and WB1P-Myc tumors cluster separately. Interestingly, Gene Set Enrichment Analysis (GSEA) showed that the most significant deregulated pathway is the cytokine-cytokine interaction pathway. Compared to WB1P tumors, WB1P-Myc tumors showed downregulation of different molecules involved in immune cell chemoattraction and antigen presentation. Characterization of the immune infiltrate of WB1P and WB1P-Myc tumors showed that MYC overexpression leads to depletion of different tumor-infiltrating immune cells, including leukocytes, T cells, myeloid cells, and NK cells.
To further investigate the role of MYC on the tumor microenvironment, we generated different Myc inducible platforms, including a “double-layered” system for Cre-conditional and doxycycline-regulatable overexpression of the MYC gene in WB1P mice. We also obtained tumor organoids from the WB1P and WB1P-Myc tumors which can be manipulated in vitro and orthotopically transplanted in vivo. We transduced the WB1P organoids with a lentiviral MycERT2 construct, enabling us to switch on and off MYC in organoids and study if its overexpression is the cause of the differences observed in the WB1P-Myc tumors. In addition, we are studying if MYC upregulation induces secretion of immune-suppressive factors and/or upregulation of molecules that can create an immunosuppressive microenvironment. Moreover, we are studying the secretome of WB1P and WB1P-Myc organoids in vitro.
Of note, we discovered that WB1P-Myc tumors strongly downregulate the CIITA-CD74-MHCII axis, which is important for T-cell mediated antitumor activity. Taking advantage of the aforementioned Myc-inducible platforms, we are currently investigating if this axis is the underlying cause of the immune-depleted microenvironment we observed in WB1P-Myc tumors. In addition, we are somatically inducing CIITA expression in the mammary epithelium of WB1P-Myc mice to investigate if this leads to delayed mammary tumor formation and/or increased influx of immune cells in the resulting tumors.
Citation Format: Chiara Svetlana Brambillasca, Linda Henneman, Julian de Ruiter, Max Wellenstein, Anne Paulien Drenth, Karin de Visser, Jos Jonkers. Dissecting MYC immunoregulatory activity in BRCA1-associated 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 A21.