Activation of Inflammatory Responses Upon Replication Stress in Basal-Like Breast Cancer - Project Summary Breast cancer accounts for the second most cancer-related deaths in U.S. women despite the availability of improved treatment options and increased screening. A particularly aggressive subtype that represents a disproportionate number of these mortality cases is basal-like breast cancer (BLBC). The high mortality of BLBC stems from a lack of effective, sustainable chemotherapeutic treatment options with tumors often developing resistance to treatment despite encouraging treatment responses. This higher relapse rate among BLBC patients calls for the development of more effective therapeutic regimens targeting distinct molecular alterations of basal-like tumors. Strikingly, we find that BLBC, but not luminal breast cancer cell lines, exhibit a severe sensitivity to partial suppression of leading strand replicative polymerase, DNA polymerase epsilon (POLE). POLE suppression in BLBC cells leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. Our preliminary data, which form the premise of my application, reveals that suppression of POLE in a BLBC cell line, but not other breast cancer cell lines, results in an increase in inflammatory cytokine transcripts known to be targeted by the NF-𝜅B transcription factor, a phenotype that is dependent upon RELA expression. This cytokine production is sufficient to impact the tumor immune microenvironment, as I observed significant infiltration of F4/80 positive macrophages in BLBC xenografts upon POLE suppression. Therefore, my central hypothesis is that NF-𝜅B activation upon POLE inhibition activates a cytokine secretion program selectively in BLBC cells. In aim 1, I will analyze activation of pattern recognition receptors (PRRs) and NF-𝜅B via immunoblot, immunofluorescence for NF- 𝜅B subcellular localization, and an NF-𝜅B luciferase reporter. I will delineate which PRRs (cGAS- STING, MAVS-RIG-I) and NF-𝜅B components (IKK-g, p65, RELB) are triggered by replication stress, and use CRISPR/Cas9 to generate cell lines lacking key signaling components to uncover those responsible for NF-𝜅B activation. In aim 2, I will determine the impact of POLE suppression on intra-tumoral cytokine expression and tumor cell composition. If immune infiltration is altered, I will determine the degree of cooperation with immune checkpoint inhibitors. This work is impactful because it will discriminate whether replication stress induced by inhibiting leading strand replication triggers an immunostimulatory or immunosuppressive environment that could be targeted to enhance future anti-cancer therapies.
