TTLL4 glutamyltransferase is a therapeutic target for NPM1-mutated acute myeloid leukemia

NPM1-mutated acute myeloid leukemia (AML) is defined by aberrant cytoplasmic localization of the mutant NPM1c protein, and therapeutic strategies targeting this specific disease remain limited. Here, we identify TTLL4, a mono-glutamate glutamyltransferase, as a selective vulnerability in NPM1c AML. TTLL4 catalyzes post-translational hyper-glutamylation of NPM1c at E126, stabilizes its cytoplasmic localization and promotes a differentiation block in leukemic cells. Multiple genetic TTLL4 inactivation approaches in human NPM1c-mutant cell lines reduce NPM1c glutamylation, trigger myeloid differentiation, and impair proliferation. Transcriptomic analyses show that TTLL4 knockdown pheno-copies NPM1c degradation and aligns with KMT2A and XPO1-targeted gene expression programs. Furthermore, Ttll4 knockout significantly prolonged survival in an NPM1c/NRAS-driven mouse AML model and promoted differentiation. We identify a small molecule, EN7, that selectively inhibits TTLL4 and recapitulates these phenotypes in NPM1c+ cells. These findings identify glutamylation as a new axis of leukemic regulation and highlight TTLL4 as a druggable epigenetic regulator in NPM1c AML.