FLT3-ITD scaffolds PKCι-STAT1 to drive noncanonical S727 phosphorylation and CD276-driven CD8+ T-cell exhaustion in AML

FLT3-ITD mutation is associated with poor prognosis in acute myeloid leukemia (AML), yet its kinase-independent mechanisms remain unclear. To investigate kinase-independent immunosuppressive mechanisms in FLT3-ITD AML, we integrated single-cell RNA sequencing from two public datasets and multiparameter flow cytometry data from 104 primary patient samples, identifying profound CD8+ T cell exhaustion as a hallmark of the FLT3-ITD immune microenvironment. Mechanistically, FLT3-ITD acts as a mutation-specific scaffold that assembles a ternary complex with PKCι and STAT1, as demonstrated by co-immunoprecipitation and intracellular colocalization. This complex enables PKCι-mediated phosphorylation of STAT1 specifically at serine 727 (S727), driving CD276 transcription independent of the canonical tyrosine 701 (Y701) site. Chromatin immunoprecipitation, electrophoretic mobility shift, promoter-reporter assays, and phosphosite-mutant constructs confirmed that S727 phosphorylation is necessary and sufficient for CD276 transactivation. Multiplex immunohistochemistry of patient bone marrow validated co-elevation of pS727-STAT1 and CD276 in FLT3-ITD blasts, accompanied by CD8+ T cell depletion. Functionally, CD276 upregulation induced profound CD8+ T cell exhaustion, characterized by reduced cytotoxicity, impaired proliferation, diminished IFN-γ production and elevated inhibitory checkpoints expression. Targeting CD276 restored CD8+ T cell function by 1.2-1.7-fold (cytotoxicity), 1.4-1.7-fold (proliferation), 1.5-1.8-fold (IFN-γ secretion) and 25.4%-67.6% (checkpoints expression) in ex vivo co-culture. In patient-derived xenograft models, co-treatment with FLT3i (quizartinib) and CD276-targeting agents led to 72.9%-80.4% tumor burden reduction and enhanced CD8+ T cell function, outperforming quizartinib monotherapy. These findings define a scaffolded PKCι-pS727-STAT1 signaling axis that promotes immune evasion in FLT3-ITD AML, supporting combined FLT3 and CD276 targeting as a promising translational strategy in this aggressive leukemia subtype.