Explore the signaling mechanisms of acquired resistance to tyrosine kinase inhibitors in AML

探讨急性髓系白血病获得性耐药的信号机制 摘要 急性髓系白血病(AML)是一种恶性血液病，是急性髓系白血病最常见的类型 成人中的白血病。白血病靶向治疗取得更大成功的一个主要障碍是耐药性。 急性髓系白血病耐药的机制尚不清楚。Flt3是一种细胞因子受体， 属于受体酪氨酸激酶(RTK)III类。激活FMS样酪氨酸激酶3的突变 (Flt3)现在被认为是AML中最常见的分子异常，Flt3ITD突变是 在近30%的急性髓系白血病患者中发现。奎扎替尼(AC220)是一种有效的、选择性的第二代抑制剂 和Flt3的关系。正在进行临床试验，用于治疗复发或难治性Flt3ITD阳性和阴性的AML 患者和AS维持治疗。值得注意的是，这些临床试验显示了非常有希望的结果。 然而，通过早期的临床研究也发现了对AC220的耐药性。 为了了解AC220耐药的潜在机制，我们采取了一种公正的方法 使用一个新的CRISPR池文库来筛选其功能丧失导致对AC220产生抗性的新基因。 在我们的筛选中，我们确定了RTK信号的细胞内抑制物SPRY3和典型的Wnt基因GSK3 信号拮抗剂，并证明了下游RTK/RAS/ERK和Wnt信号AS的重新激活 Flt3抑制剂耐药的主要机制。此外，我们还证实了我们在小学阶段的发现。 急性髓系白血病患者样本。我们证明了SPRY3和GSK3A的表达水平显著 AC220耐药的AML样本和SPRY3缺失的原代AML细胞对AC220耐药。 此外，我们用一种有效的MAPK抑制剂和β-3处理了SPRY3和GSK3基因敲除的急性髓细胞白血病细胞。 结果表明，两种抑制剂均能使AML细胞对AC220重新增敏。有趣的是， 我们发现，在定位了SPRY3的GSK3基因敲除的AML细胞中，SPRY3的表达显著降低 在抗性途径中位于GSK3下游。 在这个提案中，我们假设Sprouty(SPRY)和GSK3在对 急性髓系白血病中的酪氨酸激酶抑制剂。受SPRY3和GSK3调控的RAS/MEK/ERK和Wnt通路 对急性髓系白血病获得性耐药起重要作用。接下来，我们将进行一系列全面的研究 探索SpRY3和GSK3在AMLS中的新下游效应/相互作用伙伴及其分子 它们的作用机制。此外，我们将研究将我们的发现转化为新的 临床疗法。 综上所述，我们的研究确定了新的基因，其功能丧失会导致对选择性Flt3的抗性 抑制剂，并揭示了潜在的机制，从而提供了对信号通路的新见解 与急性髓系白血病获得性耐药有关。所学到的知识可能会导致更多 急性髓系白血病的有效综合治疗途径。

Explore the signaling mechanisms of acquired resistance to tyrosine kinase inhibitors in AML Abstract Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. One major obstacle to greater success with target therapy of leukemia is drug resistance. The mechanisms underlying drug resistance in AML are poorly understood. FLT3 is a cytokine receptor which belongs to the receptor tyrosine kinase (RTK) class III. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are now recognized as the most common molecular abnormality in AML and FLT3ITD mutations are found in nearly 30% of AML patients. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3ITD positive and negative AML patients and as maintenance therapy. Remarkably, those clinical trials have showed very promising result. However, drug resistance to AC220 has also been reported through the early clinical studies. To understand the underlying mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR pooled library to screen new genes whose loss of function confers resistance to AC220. In our screen, we identified SPRY3, an intracellular inhibitor of RTK signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated that re-activation of downstream RTK/Ras/ERK and Wnt signaling as major mechanisms of resistance to the FLT3 inhibitor. Furthermore, we also confirmed our findings in primary AML patient samples. We demonstrated that the expression level of SPRY3 and GSK3A is dramatically reduced in AC220 resistant AML samples and SPRY3 deleted primary AML cells are resistant to AC220. Additionally, we treated SPRY3 and GSK3 knockout AML cells with a potent MAP kinase inhibitor and β- catenin inhibitor respectively, demonstrated that both inhibitors re-sensitized AML cells to AC220. Intriguingly, we found that expression of SPRY3 is greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. In this proposal, we hypothesize that Sprouty (SPRY) and GSK3 play critical roles in the response to tyrosine kinase inhibitor in AML. The Ras/MEK/ERK and Wnt pathways regulated by SPRY3 and GSK3 are important for the acquired drug resistance in AML. Next, we will perform a series comprehensive study to explore novel downstream effectors/ interacting partners of SPRY3 and GSK3 in AMLs and the molecular mechanisms of their action. Furthermore, we will examine the possibility to translate our findings into new clinical therapies. Taken together, our study identified novel genes whose loss of function confers resistance to a selective FLT3 inhibitor and revealed the underlying mechanism, thereby providing new insight into signaling pathways that contribute to the acquired resistance in AML. The knowledge learned may lead to the development of more efficient combined therapeutic avenues for AML.