mTORC2信号在FLT3-ITD突变的急性髓细胞白血病中的功能研究

Internal tandem duplication of FLT3 (Fms-like tyrosine kinase 3) was associated with poor prognosis in patients with acute myeloid leukemia (AML), therefore, it has been known as an attractive drug target for AML since a decade. However, tyrosine kinase inhibitor (TKI) targeting aberrant activation of FLT3 only showed modest clinical response without improving overall survival rate as well as relapsed rate. Meanwhile, TKI resistance emerges as a main problem to further development of TKI to treat AML patients. Of note, one of the most important resistance mechanisms of FLT3-ITD AML cells to TKI is activation of compensatory survival pathways. In our preliminary studies, we have observed mTOR signaling was constitutively activated in FLT3-ITD transformed hematopoietic cells. As we expected, TKI treatment decreased phosphorylation of 4E-BP which is a major substrate of mTORC1, indicating mTORC1 signaling rely on FLT3-ITD signaling. However, we did not observe significant change of total phosphorylated mTOR expression following treatment of FLT3-TKI. Importantly, mTOR is comprised of two complexes: mTORC1 and mTORC2. mTORC1 controls protein synthesis via its substrate 4E-BP1. mTORC2 has been shown to be able to phosphorylate AKT and SGK1 as their upstream regulator. Phosphorylation of SGK1 is completely dependent on the activation of mTORC2. Interestingly, FLT3-TKI showed no effect on SGK1 phosphorylation, suggesting mTORC2 conferred constant activation of mTOR signaling instead of m TORC1. These preliminary results provide us the rationale to further explore the role of mTORC2 in FLT3-ITD+AML cells. We would like to answer the question of whether mTORC2 plays an important role of primary resistance of FLT3-ITD AML cells to FLT3-TKI. In this proposal, we plan to test our hypothesis through performing studies both in vitro and in vivo. To accomplish this proposed study will provide a strong basis for further development of a new therapeutics of TKI in combination with mTORC2 inhibition in AML patients harboring FLT3-ITD.

急性髓细胞白血病(AML)患者中FLT3内部重复序列突变(FLT3-ITD)与不良预后密切相关，但FLT3抑制剂(FLT3-TKI)的临床疗效有限。不依赖FLT3的替代通路活化是FLT3-TKI原发耐药的重要机制。我们前期研究发现mTOR可被异常活化的FLT3激活。FLT3-TKI可明显抑制mTOR复合物1（mTORC1）下游调控蛋白合成的4E-BP磷酸化并减少蛋白合成，提示mTORC1活性依赖于FLT3信号。但我们未能观察到FLT3-TKI下调总mTOR蛋白磷酸化。有趣的是我们同时发现直接反映mTORC2活性的下游分子SGK1蛋白的磷酸化在FLT3-TKI作用后的细胞中仍持续激活。提示FLT3-TKI未能抑制的mTOR活性由mTORC2引起，而不是mTORC1。因此本课题拟通过体内外实验探明mTORC2在FLT3-ITD+AML细胞中的功能，及其是否参与FLT3-TKI原发耐药。

成人AML患者中FLT3-ITD突变与不良预后密切相关。然而多种FLT3-TKI的临床疗效非常有限。不依赖FLT3信号的旁路活化参与FLT3-TKI耐药机制。我们在前期工作中发现高浓度FLT3-TKI亦未能抑制直接反映mTORC2活性的SGK1 Ser422位点磷酸化。因此我们推 测FLT3-TKI作用后残留的mTOR活性更可能来源于mTORC2。mTORC2持续激活很可能是FLT3-TKI原 发耐药的机制之一。本课题首次研究了FLT3-ITD+AML细胞中mTORC2信号活化水平与体外FLT3-TKI细胞毒效应的关系。研究表明，FLT3抑制剂可部分下调总mTOR蛋白表达，但不能下调mTOC2信号通路中Rictor、磷酸化SGK表达，从而导致残留AKT磷酸化活性。进而我们构建了稳定转染Rictor-shRNA的MV4-11细胞，并在蛋白和mRNA水平验证了干扰RNA对Rictor蛋白活性的下调。细胞增殖效应分析及凋亡检测均提示FLT3抑制剂AC220能更明显杀伤稳定转染Rictor-shRNA的MV4-11细胞。为了在体内研究中证实上述结果，我们构建了FLT3-ITD AML小鼠模型，并给药处理小鼠。结果显示，FLT3抑制剂联合mTORC1/C2抑制剂AZD8055与单药治疗比较，能更明显地减少AML小鼠体内白血病细胞负荷。该研究结果为急性髓系白血病靶向治疗提供了新的理论依据，且具有一定的应用前景。