Wednesday, May 1, 2024
5/1/2024
Project Summary Natural killer (NK) cells are innate lymphoid cells that are crucial for host defense from viral infections and tumor cells. The ability of NK cells to recognize and kill malignant cells provides a strong premise to advance them as a cellular therapy in the clinical setting. While NK cell-based therapies are being explored in clinical trials, issues with persistence and in vivo functionality have been identified as limitations to their clinical application. Our lab pioneered cytokine-induced memory-like (ML) NK cells that are generated after brief activation with IL-12, IL-15, and IL-18. This overcomes many of the shortcomings of conventional NK cells as a cellular therapy. ML NK cells exhibited enhanced functionality, cytotoxicity, and persistence in a phase I clinical trial for acute myeloid leukemia (AML), with many patients achieving complete remissions from this novel intervention. Despite the translational advancement, there is little known about the underlying mechanisms of ML reprogramming in NK cells. Clarifying the molecular programs that drive the ML phenotype will have broad implications for optimizing NK cells as a cellular-based therapy for hematological malignancies. The proposed project aims to better understand the mechanisms of ML NK cell differentiation with a focus on the role of EOMES. EOMES is a T-box transcription factor (TF) that is essential for NK cell development from hematopoietic stem cells and has been implicated in generating memory in CD8+ T cells. Data from our lab demonstrated that EOMES deletion prior to activation with cytokines abrogated ML NK cell differentiation, suggesting that EOMES plays a key role in ML reprogramming. We hypothesize that activation with IL-12/15/18 triggers a molecular reprogramming of conventional NK cells to ML NK cells mediated by EOMES to induce a unique transcriptional profile and poised epigenetic state, which facilitates the enhanced functionality of ML NK cells upon restimulation by cytokines or a tumor cell. Aim 1 of this proposal focuses on determining the memory differentiation phase that requires EOMES. Specifically, we will assess the requirement of EOMES during initiation (prior to IL-12/15/18 activation) and differentiation (after IL-12/15/18 activation) using CRISPR/Cas9 deletion. Aim 2 focuses on defining the transcriptional and epigenetic landscape induced by EOMES in the different phases of ML reprogramming through CRISPR/Cas9 knockdown, single-cell RNAseq, and single-nuclei ATACseq, as well as defining the genes regulated by EOMES in ML NK cells using Cut&Run. Together, these results will reveal mechanisms of ML NK cell differentiation driven by EOMES, further advancing the potential of ML NK cell therapies and enhancing our understanding of ML NK cell biology.
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