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

Abstract

Abstract The internal tandem duplications in FMS-like tyrosine kinase 3 (FLT3-ITD) are 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 2 data sets and multiparameter flow cytometry data from 104 primary patient samples and identified 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 protein kinase C iota (PKCι) and STAT1, as demonstrated by coimmunoprecipitation and colocalization. This complex enables PKCι-mediated phosphorylation of STAT1, at serine 727 (S727), thereby driving CD276 transcription independent of the canonical tyrosine 701 (Y701) site. Chromatin immunoprecipitation, electrophoretic mobility shift assays, promoter-reporter assays, and phosphosite-mutant constructs confirmed that S727 phosphorylation is necessary and sufficient for CD276 transactivation. Multiplex immunohistochemistry of bone marrow validated coelevation 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 interferon-γ (IFN-γ) production, and elevated inhibitory checkpoint expression. Targeting CD276 restored CD8+ T-cell function by 1.2- to 1.7-fold (cytotoxicity), 1.4- to 1.7-fold (proliferation), 1.5- to 1.8-fold (IFN-γ secretion), and 25.4% to 67.6% (checkpoint expression) in ex vivo coculture. In patient–derived xenograft models, cotreatment with an FLT3 inhibitor (quizartinib) and CD276-targeting agents led to 72.9% to 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.