FLT3抑制剂quizartinib抑制WT1驱动的KIT-STAT5-PIM信号通路激活并诱导FLT3野生型HL-60细胞凋亡

Background: WT1 (Wilms' tumor 1) is a key transcription factor regulating hematopoietic stem cell self-renewal and differentiation. In acute myeloid leukemia (AML), WT1 overexpression promotes leukemogenesis by blocking differentiation and conferring apoptosis resistance. Although several WT1 targets ( BCL2, c-MYC, IGF2, VEGF ) have been identified, its downstream mechanisms remain unclear. Interestingly, although parental HL-60 cells are FLT3-unmutated and resistant to the FLT3 inhibitor quizartinib, WT1-overexpressing HL-60 cells (HL-60/WT1) exhibited increased sensitivity, suggesting that WT1-induced KIT expression may mediate this effect. Methods: HL-60/WT1 cells were generated via lentiviral transduction, resulting in a 7.5-fold increase in WT1 mRNA expression compared with parental HL-60 cells. To evaluate drug sensitivity, two complementary assays were employed: apoptosis was assessed after quizartinib treatment (700 nM, 48 h) using Annexin V/PI flow cytometry, while dose-dependent cytotoxicity was quantified by XTT assay following 72-hour exposure to graded concentrations of quizartinib. Expression of KIT was measured by RT-PCR, and RNA sequencing was performed to obtain transcriptome-wide profiles and fold-change-based comparisons across conditions, enabling identification of WT1-dependent signaling alterations. Results: Cell growth was comparable between HL-60 and HL-60/WT1 cells (doubling times: 27.3 h and 23.9 h, respectively). HL-60 cells were unresponsive to quizartinib (IC₅₀, not reached), whereas HL-60/WT1 cells exhibited increased sensitivity (IC₅₀ = 665 nM). RT-PCR confirmed KIT expression in HL-60/WT1 but not in parental cells, supporting WT1-driven activation of a KIT -mediated signaling pathway. Quizartinib markedly increased apoptosis in HL-60/WT1 cells (30.0 ± 10.1%) but not in parental HL-60 cells (7.0 ± 1.6%; Mann-Whitney U, p < 0.05). RNA-seq analysis using fold change revealed that WT1 overexpression increased the expression of KIT (1.2-fold), AKT1 (1.4-fold), STAT5A (5.0-fold), and PIM1 (6.2-fold). Notably, quizartinib treatment reduced these transcripts in HL-60/WT1 cells, indicating suppression of a WT1-induced, KIT-driven survival program independent of FLT3. Conclusion: These findings demonstrate that WT1 overexpression establishes a previously unrecognized KIT-STAT5-PIM signaling pathway that promotes AML cell survival. Importantly, this pathway creates a therapeutically exploitable vulnerability: HL-60 cells that are normally resistant to quizartinib become sensitized through WT1-induced KIT activation. Our study provides mechanistic insight into WT1-mediated leukemogenesis and suggests that KIT-targeted strategies may be effective in subsets of WT1-high, FLT3-unmutated AML.