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Abstract：
BACKGROUND: Approximately 25-30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML.
METHODS: We performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, β-catenin, PP2A, and GSK3β. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results.
RESULTS: We demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates β-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit β-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3β (GSK3β) pathway.
CONCLUSIONS: These findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.
METHODS: We performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, β-catenin, PP2A, and GSK3β. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results.
RESULTS: We demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates β-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit β-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3β (GSK3β) pathway.
CONCLUSIONS: These findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.
摘要：
背景：大约25-30%的急性髓性白血病（AML）患者存在FMS样受体酪氨酸激酶-3（FLT3）突变，这些突变导致疾病进展和不良预后。长期暴露于FLT3酪氨酸激酶抑制剂(TKIs)通常由于不同的代偿生存信号而导致有限的临床反应。因此,迫切需要阐明FLT3TKI耐药的潜在机制.异常调节的鞘脂代谢经常导致癌症进展和不良的治疗反应。然而,在FLT3突变的AML中,其与TKI敏感性的关系尚不清楚.因此，我们旨在评估AML中FLT3TKI耐药的机制.
方法：我们进行了脂质组学分析，RNA-seq,qRT-PCR,和酶联免疫吸附试验，以确定索拉非尼耐药的潜在驱动因素。FLT3信号被索拉非尼或奎扎替尼抑制，通过使用拮抗剂或通过敲除抑制SPHK1。通过细胞计数试剂盒-8，PI染色，在FLT3突变和野生型AML细胞系中评估细胞生长和凋亡，和膜联蛋白-V/7AAD测定。采用蛋白质印迹和免疫荧光分析，通过使用SPHK1过表达和外源S1P的拯救实验来探索潜在的分子机制。以及S1P2，β-连环蛋白的抑制剂，PP2A,和GSK3β。异种移植鼠模型,患者样本,和公开可用的数据进行了分析，以证实我们的体外结果。
结果：我们证明长期索拉非尼治疗可上调SPHK1/1-磷酸鞘氨醇（S1P）信号传导，它又通过S1P2受体积极调节β-连环蛋白信号传导以抵消TKI介导的对FLT3突变的AML细胞的抑制。SPHK1的遗传或药理学抑制在体外有效增强了TKI介导的FLT3突变的AML细胞的增殖抑制和凋亡诱导。SPHK1敲低增强索拉非尼功效并改善AML异种移植小鼠的存活率。机械上,靶向SPHK1/S1P/S1P2信号与FLT3TKIs协同作用，通过激活蛋白磷酸酶2A（PP2A）-糖原合酶激酶3β（GSK3β）途径来抑制β-catenin活性。
结论：这些发现确立了鞘脂代谢酶SPHK1作为TKI敏感性的调节因子，并表明将SPHK1抑制与TKIs结合可能是治疗FLT3突变的AML的有效方法。
方法：我们进行了脂质组学分析，RNA-seq,qRT-PCR,和酶联免疫吸附试验，以确定索拉非尼耐药的潜在驱动因素。FLT3信号被索拉非尼或奎扎替尼抑制，通过使用拮抗剂或通过敲除抑制SPHK1。通过细胞计数试剂盒-8，PI染色，在FLT3突变和野生型AML细胞系中评估细胞生长和凋亡，和膜联蛋白-V/7AAD测定。采用蛋白质印迹和免疫荧光分析，通过使用SPHK1过表达和外源S1P的拯救实验来探索潜在的分子机制。以及S1P2，β-连环蛋白的抑制剂，PP2A,和GSK3β。异种移植鼠模型,患者样本,和公开可用的数据进行了分析，以证实我们的体外结果。
结果：我们证明长期索拉非尼治疗可上调SPHK1/1-磷酸鞘氨醇（S1P）信号传导，它又通过S1P2受体积极调节β-连环蛋白信号传导以抵消TKI介导的对FLT3突变的AML细胞的抑制。SPHK1的遗传或药理学抑制在体外有效增强了TKI介导的FLT3突变的AML细胞的增殖抑制和凋亡诱导。SPHK1敲低增强索拉非尼功效并改善AML异种移植小鼠的存活率。机械上,靶向SPHK1/S1P/S1P2信号与FLT3TKIs协同作用，通过激活蛋白磷酸酶2A（PP2A）-糖原合酶激酶3β（GSK3β）途径来抑制β-catenin活性。
结论：这些发现确立了鞘脂代谢酶SPHK1作为TKI敏感性的调节因子，并表明将SPHK1抑制与TKIs结合可能是治疗FLT3突变的AML的有效方法。
