Project Summary/Abstract Most of breast cancer related mortality is related to metastatic disease. While NK cells play an essential role in the control of metastatic breast cancer we do not fully understand key mechanistic and temporal events of NK cell immunosurveillance. To address this need, I created novel 3D co-culture assays of NK cells and invasive breast cancer organoids and cell clusters. Using these models I demonstrated NK cells limit breast cancer metastasis through induction of apoptosis in invasive breast cancer cells. We discovered micrometastatic cancer cells reprogram NK cells to promote metastatic outgrowth. Using bulk RNA-seq, we identified KLRG1 as a key expressed gene by teNK cells. Blocking KLRG1 restores anti-metastatic activity. teNK cells also express high levels of DNA methyltransferases (DNMTs). Inhibition of DNMTs in teNK cells achieves anti- metastatic results. Combination KLRG1 blockade and DNMT inhibition further reduces tumor growth. Building on these exciting data, we hypothesize: 1) reprogrammed teNK cell KLRG1 expression is regulated by DNMTs and targeting both in combination will reverse NK cell reprogramming; 2) reprogrammed human NK cells increase human breast cancer metastasis and blocking prioritized NK cell inhibitory receptors restores the cytotoxic activity of metastases-reprogrammed human NK cells. Aim 1 will determine how blocking DNMTs and KLRG1 synergistically reverses NK cell reprogramming. We have observed blocking KLRG1 and DNMTs together in teNK cells synergistically reduces colony formation. We will first test these ex vivo findings in in vivo models of metastasis. Next, we will determine the link between NK cell reprogramming and DNMTs using novel functional ex vivo and in vivo models of metastasis using CRISPR-Cas9 molecular tools. Aim 2 will determine the function of metastases-reprogrammed NK cells in human breast cancer. First, we showed that human NK cells can be reprogrammed in culture to actively promote metastatic outgrowth. We will leverage preliminary transcriptomic analysis of reprogrammed NK cells to identify markers of metastases- reprogrammed NK cells. We will apply these tools to FFPE sections of patients with metastatic breast cancer to define the abundance of reprogrammed NK cells. Together with patient samples, single-cell RNA-seq, and ex vivo and in vivo models that capture the progression from anti-metastatic NK cells to metastases-promoting NK cells, we will further characterize the function and molecular profile of reprogrammed NK cells in human breast cancer. Finally, using a preliminary list of targets, we will test effective individual or combination treatments that can restore human NK cell cytotoxicity against metastatic breast cancer cells. Impact: Completing these aims will deepen our fundamental understanding of NK cell biology and have the potential to bring new NK cell directed immunotherapies to our patients.

Project Narrative I discovered that invasive breast cancer cells can reprogram natural killer (NK) cells into being metastasis- promoters. I propose to determine the mechanisms driving metastases-reprogrammed NK cells and to determine its significance in human breast cancer. NK cell directed therapies could provide alternative immunotherapies for metastatic breast cancer patients and I will complete key preclinical experiments needed to translate these insights to patient benefit.