MARKER-BASED CRISPR SCREENINGS TO CHARACTERIZE KINASES CONTROLLING MACROPHAGE ACTIVATION

Identifying the complete set of components in cellular circuitries is a critical yet remarkably complex task in mammalian cells. Pathways involved in innate immune cell activation in response to various stimuli have been extensively characterized, but it remains unclear whether the full complement of regulators involved in signal transduction and the functional interplay among them have been identified. Kinases form the backbone of most signal transduction cascades, and their pharmacological inhibition is a validated therapeutic strategy in cancer and autoimmune disorders. However, clinically approved kinase inhibitors target only a small fraction of the human kinome, and roughly one-third of kinases remain functionally uncharacterized (so-called dark kinases). To systematically map kinase involvement in macrophage activation, in this project I employed a kinase-targeted CRISPR screen using stimulus-inducible molecules as functional readouts. Two independent screens were performed in bone marrow-derived macrophages (BMDMs) activated with either lipopolysaccharide (LPS) or interleukin 4 (IL-4), inducing distinct transcriptional and functional phenotypes. The screens successfully identified known positive and negative regulators of the LPS and IL-4 pathways. In addition, both subunits of the TGFb receptor emerged as top-ranked negative regulators of RETNLA, the IL-4-inducible marker used in the screen. This unexpected finding prompted a deeper investigation into the role of TGFb signaling in modulating the IL-4-driven transcriptional program. Transcriptomic and proteomic analyses revealed extensive cross-talk between IL-4 and TGFb signaling. While distinct subsets of IL-4- or TGFb-induced genes were reciprocally antagonized, others—including the canonical IL-4 target gene Arginase 1—were synergistically upregulated when both cytokines were present. Further mechanistic analyses, including acetylation profiling, chromatin accessibility assays, and ChIP-seq, pointed to a role for SMAD proteins in mediating these diverging effects. Additionally, metabolic profiling demonstrated a profound rewiring of arginine metabolism upon co-stimulation with IL-4 and TGFb. These findings provide both a modular framework for dissecting kinase-driven signaling networks in immune cells and novel insights into the intricate interplay between IL-4 and TGFb signaling, with potential implications for immunological and metabolic regulation.