Replication stress response defects predict and enhance immune checkpoint therapy response in triple negative breast cancer

Project Summary The lack of specific targets for the treatment of triple-negative breast cancer (TNBC) is a major challenge, as many TNBCs do not respond to cytotoxic chemotherapies. Immune checkpoint blockade (ICB) has yielded promising results in both advanced and early-stage TNBC and is expected to substantially improve the overall

prognosis of patients with this disease. However, since TNBC is not inherently immunogenic, it is important to identify patients who would benefit most from immunotherapy and to identify agents that can prime the tumor microenvironment to enhance the therapeutic effects. TNBC is known to exhibit high levels of replication stress,

which occurs when the DNA replication machinery encounters obstacles that impede the replication process. In normal cells, replication stress activates the replication stress response (RSR) to maintain genome integrity. Defective RSR allows cells with high replication stress to survive and proliferate. Recently, we have identified a

gene signature that represents defects in RSR (RSRD). We found this RSRD signature to be highly enriched in TNBC cells. Furthermore, RSRD-high TNBC cells accumulate cytoplasmic DNA and induce STING-dependent cytokine production, which is required for the effectiveness of ICB. Intriguingly, the RSRD signature score

correlates perfectly with the response of TNBC to ICB in syngeneic mouse models, and it accurately predicts ICB response across 5 low–mutation-burden tumor lineages. All these intriguing findings support the hypotheses that RSRD may act as a key determinant of ICB outcomes in low–mutation-burden cancers, including TNBC,

and that RSRD-enhancing drugs may sensitize ICB-resistant TNBC to immunotherapy. These hypotheses will be tested via 3 specific aims. (1) To determine how the immune microenvironment is modified in RSR-defective TNBC. We will use a highly multiplexed imaging mass cytometry panel to determine how RSRD remodels the

immune microenvironment of TNBC and induces susceptibility to ICB. In addition, we will manipulate the RSR status in TNBC cells to assess the relationship between RSR defects and immunotherapy response. (2) To identify causative drivers of RSRD-high–mediated ICB responsiveness in TNBC. Our preliminary studies suggest

that RSR defects may drive immunotherapy response through accumulation of immunostimulatory cytosolic single-stranded DNA (ssDNA). We will, therefore, seek to manipulate the cytosolic ssDNA level in TNBC models to determine whether cytosolic ssDNA is indeed a causative driver of ICB responsiveness in TNBC. In addition,

to understand why our RSRD gene signature predicts response to ICB in TNBC, we will apply an in vivo CRISPR screen to determine what transcriptional changes contained within our RSRD gene signature cause this response. (3) To develop novel combination therapy to convert RSRD-low TNBC to RSRD-high to improve their

response to ICB. Using cutting-edge systems and bioinformatics approaches, we have identified many potential RSRD-inducing agents. We will assess the 6 most promising candidates and identify the best candidate compound that can effectively sensitize RSRD-low TNBC to ICB.