ZEB2 is a Master Switch Controlling the Tumor-Associated Macrophage Program [KO_screen]

Tumor-associated macrophages (TAMs) are major players in regulating the immunosuppressive tumor environment, and their abundance is highly correlated with poor clinical outcomes. Here, we constructed a TAM regulatory network by integrating scRNA-seq data across human solid tumors with a dedicated CRISPR knockout screen. Using a deep generative model capable of learning a local representation for each candidate regulator, we constructed a gene perturbation network that linked individual target genes with prototypical functional modules in TAMs. We identified non-redundant pathways regulating distinct TAM functions, showing modular circuitry. For instance, the complement module is repressed by Stat6, Zeb2, Gpnmb, and Spp1, while the Tgfbr1-Smad2/4 pathway induces this program in TAMs. Importantly, we identified Zeb2 as the master regulator of pro-tumor functions in TAMs, orchestrating the suppression of type I interferon response and antigen presentation alongside the activation of immune suppression programs. Genetic ablation of Zeb2 reprogrammed TAMs identity on chromatin, RNA, and protein levels. In human tumors with high macrophage content, ZEB2 expression was associated with poor prognosis in solid cancers, including lung and bladder. Functional macrophage coculturing assays defined Zeb2 as a critical regulator of TAM immunosuppression activity by inhibiting T cell proliferation and activation. Selective in vivo targeting of Zeb2 in macrophages using a CpGsiRNAZeb2 DNA hybrid reprogrammed TAMs and mobilized systematic anti-tumoral T cell responses, achieving complete tumor clearance as a monotherapy. Overall, our study generated a detailed genetic roadmap of TAM gene circuits and identified ZEB2 as a master switch of TAMs with potential therapeutic implications for macrophage-based immunotherapies.