Abstract：
Internal tandem duplication mutations of FLT3 (FLT3/ITD) confer poor prognosis in AML. FLT3 tyrosine kinase inhibitors (TKIs) alone have limited and transient clinical efficacy thus calling for new targets for more effective combination therapy. In a loss-of-function RNAi screen, we identified NOTCH4 as one such potential target whose inhibition proved cytotoxic to AML cells, and also sensitized them to FLT3 inhibition. Further investigation found increased NOTCH4 expression in FLT3/ITD AML cell lines and primary patient samples. Inhibition of NOTCH4 by shRNA knockdown, CRISPR-Cas9-based knockout or γ-secretase inhibitors synergized with FLT3 TKIs to kill FLT3/ITD AML cells in vitro. NOTCH4 inhibition sensitized TKI-resistant FLT3/ITD cells to FLT3 TKI inhibition. The combination reduced phospho-ERK and phospho-AKT, indicating inhibition of MAPK and PI3K/AKT signaling pathways. It also led to changes in expression of genes involved in regulating cell cycling, DNA repair and transcription. A patient-derived xenograft model showed that the combination reduced both the level of leukemic involvement of primary human FLT3/ITD AML cells and their ability to engraft secondary recipients. In summary, these results demonstrate that NOTCH4 inhibition synergizes with FLT3 TKIs to eliminate FLT3/ITD AML cells, providing a new therapeutic target for AML with FLT3/ITD mutations.
摘要：
FLT3的内部串联重复突变（FLT3/ITD）导致AML预后不良。单独的FLT3酪氨酸激酶抑制剂(TKIs)具有有限且短暂的临床疗效,因此需要更有效的联合治疗的新靶标。在功能丧失的RNAi筛选中，我们确定NOTCH4是一个潜在的目标，其抑制被证明对AML细胞有细胞毒性，并使它们对FLT3抑制敏感。进一步的研究发现，在FLT3/ITDAML细胞系和原发性患者样本中NOTCH4表达增加。通过shRNA敲低抑制NOTCH4，基于CRISPR-Cas9的敲除或γ-分泌酶抑制剂与FLT3TKI协同体外杀死FLT3/ITDAML细胞。NOTCH4抑制使TKI抗性FLT3/ITD细胞对FLT3TKI抑制敏感。该组合减少了磷酸-ERK和磷酸-AKT，显示MAPK和PI3K/AKT信号通路的抑制。它还导致调节细胞周期的基因表达发生变化，DNA修复和转录。患者来源的异种移植模型显示，该组合降低了原发性人类FLT3/ITDAML细胞的白血病参与水平及其移植次级受体的能力。总之,这些结果表明，NOTCH4抑制与FLT3TKIs协同消除FLT3/ITDAML细胞，为具有FLT3/ITD突变的AML提供新的治疗靶标。
