PROJECT DESCRIPTION/ABSTRACT Therapeutically resistant triple negative breast cancer (TNBC) is characterized by mesenchymal features that facilitate immune evasion and disease progression, resulting in high rates of death from the disease. New strategies targeting the mesenchymal phenotype to overcome therapeutic resistance are needed. Interactions between the cytoskeleton of cancer cells and the surrounding extracellular matrix play important roles in initiating local invasion and metastasis. The resulting mechanical stimuli activate signaling pathways which promote the transition of non-motile polarized epithelial cells to cells with mesenchymal properties that are able to invade surrounding tissues and evade immune surveillance, a process termed epithelial to mesenchymal transition (EMT). Fibrotic stiffening of the non-cellular stroma resulting from extracellular matrix deposition is a common feature of TNBC and other malignancies that is associated with therapeutic resistance and poor prognosis. How mechanical stress promotes EMT and cancer progression has not been elucidated. Ataxia Telangiectasia Mutated and Rad-3 Related (ATR) is best known as a regulator of the DNA damage response in replicating cells. In our preliminary data, we have found that high ATR protein expression is associated with reduced progression-free survival in TNBC. We discovered that ATR is post-translationally upregulated by a deubiquitinating enzyme, USP21, in response to mechanical stress, and that ATR regulates the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex to promote β-catenin nuclear translocation and mechanical stress-induced EMT. This new role for ATR in mechanical stress and EMT is independent of ATR’s established function in regulating the DNA damage response. In a clinical trial of anti-PD-1 immunotherapy for advanced TNBC, high ATR levels in biopsy specimens was correlated with EMT and reduced responsiveness. ATR inhibition could promote mesenchymal to epithelial transition and anti-tumor immunity in vivo. We hypothesize that ATR regulates the LINC complex following mechanical stress to promote β-catenin mediated EMT, immune evasion, and TNBC progression. We will test this hypothesis through three specific aims. Aim 1 will examine the impact of mechanical stress on ATR deubiquitination and the LINC complex; Aim 2 will evaluate how ATR influences β-catenin pathway activation and EMT; Aim 3 will investigate the role of ATR-SUN2 in stiffness and EMT associated immune evasion.

PROJECT NARRATIVE/LAY SUMMARY We have discovered a new role for a protein called ATR in promoting aggressive triple negative breast cancer (TNBC) behavior and the ability of TNBC to survive by hiding from the immune system. Our goal in this proposal is two-fold: 1) investigate how ATR promotes aggressive TNBC behavior in response to external forces that are commonly applied to cancer cell walls as tumors progress and 2) examine whether targeting ATR and this new ATR pathway can reverse TNBC aggressive features and promote the immune system’s ability to fight TNBC. As there are over 32,000 cases of TNBC diagnosed in the United States each year and nearly all patients with metastatic progression succumb to the disease, our results could have a profound impact on health and society.