目的：急性髓系白血病（Acute Myeloid Leukemia，AML）是一种高度异质性的血液系统恶性肿瘤，由髓系祖细胞的恶性克隆增殖引发，导致正常细胞分化受阻，并在骨髓内积聚大量异常细胞。AML的治愈性手段有限，长期预后不佳。其中，FMS样酪氨酸激酶3（FLT3）的激活性突变，尤其是内部串联重复突变（FLT3-ITD），是AML中最为常见的突变，与疾病进展、复发和不良预后密切相关。近年来，随着医学的发展和技术的进步，针对FLT3突变的小分子靶向药物相继问世，并在临床治疗中展现出较好的疗效。其中，吉瑞替尼（Gilteritinib）已被FDA批准用于临床治疗FLT3-ITD突变AML患者。然而，尽管患者在初期对治疗产生良好反应，但多数患者仍难以避免疾病复发及耐药的发生，从而严重影响其长期生存率。本研究拟通过逐步增加药物剂量的方法，在体外模拟耐药过程，构建FLT3-ITD突变型AML的Gilteritinib耐药模型，以深入探究其耐药机制。此外，本研究还利用激酶特异性CRISPR-Cas9文库进行筛选，以寻找潜在的联合用药靶点，为克服AML耐药提供新的理论依据和治疗策略。

方法：本研究中，我们首先通过长期低剂量诱导及剂量递增的方式，对携带FLT3-ITD突变的AML细胞进行连续刺激，成功构建了Gilteritinib耐药细胞系。为了探寻潜在的分子治疗靶点，我们利用激酶文库在构建的Gilteritinib耐药细胞中进行了CRISPR-Cas9筛选。通过高通量功能敲降评估了已知激酶对Gilteritinib耐药的影响，并筛选出对Gilteritinib具有协同作用的候选靶点。随后，我们通过增殖竞争、细胞活力、凋亡及周期检查验证了候选靶点的可靠性。结合基因敲降与小分子抑制剂干预策略，探讨候选靶点对吉瑞替尼敏感性的调控机制。同时，通过免疫共沉淀串联质谱（Co-IP-MS）技术深入解析FLT3与关键靶点之间的相互作用。最后，利用AML异种移植小鼠模型，在体内评估联合用药对Gilteritinib耐药细胞的抗肿瘤效果。

结果：

1.全外显子测序结果显示，在经过长期Gilteritinib刺激后，Gilteritinib耐药细胞中出现了FLT3-ITD-F691L“守门人”突变，该突变会导致蛋白构象改变，降低Gilteritinib与FLT3的结合效率，从而导致耐药性的产生。此外，多种其他基因突变也会对药物敏感性有潜在影响。

2.CRISPR-Cas9筛选结果显示，p38丝裂原活化蛋白激酶（MAPK）信号通路在Gilteritinib耐药细胞中起到关键调控作用。敲降p38基因或使用特异性p38

抑制剂，可显著恢复耐药细胞对吉瑞替尼的敏感性，抑制细胞增殖和周期进展，并显著诱导细胞凋亡。然而，对同属MAPK家族的ERK1/2通路的干预则未观察到显著的协同效应。

3.体内异种移植小鼠模型实验显示，Gilteritinib联合p38抑制剂的治疗组较单药组表现出更强的肿瘤抑制效果，提示联合用药可有效克服耐药细胞的药物耐受性。

4.机制研究进一步显示，Gilteritinib处理后，耐药细胞中FLT3与p38之间的结合较对照细胞明显增强，伴随p38激活水平提高，这表明p38信号通路可能作为Gilteritinib耐药的重要调控元件，促进了AML细胞的存活和增殖。

结论：本研究成功构建了对Gilteritinib耐药的FLT3-ITD突变AML细胞模型，借助激酶特异性CRISPR-Cas9筛选策略，鉴定出p38为潜在联合治疗靶点。体内外实验结果显示，Gilteritinib与p38抑制剂联合使用，能够克服耐药细胞的药物耐受性，显著增强抗肿瘤效果。这一发现为FLT3-ITD突变的AML耐药患者提供了一种具有临床转化潜力的联合治疗策略，为改善AML患者预后提供精准治疗新方案。

Objective: Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy driven by the malignant clonal proliferation of myeloid progenitor cells, resulting in an arrest of normal cellular differentiation and the accumulation of abnormal cells within the bone marrow. Curative treatment options for AML remain limited, and long-term outcomes are generally poor. Among the genetic lesions implicated, activating mutations in FMS-like tyrosine kinase 3 (FLT3), most notably the internal tandem duplication (FLT3-ITD), represent the most common aberrations in AML, closely correlating with disease progression, relapse, and adverse prognosis. In recent years, advances in medical science and technology have led to the development of small-molecule targeted therapies against FLT3 mutations, which have shown promising clinical efficacy. Gilteritinib, in particular, has been demonstrated to confer a measurable clinical benefit. However, clinical investigations reveal that although a subset of patients initially exhibits a favorable response to treatment, the majority ultimately succumb to relapse and acquired drug resistance, severely impacting long-term survival rates. In this study, we have established an in vitro model of Gilteritinib resistance in FLT3-ITD-mutated AML by employing a stepwise escalation of drug dosage to mimic the resistance acquisition process. This model was designed to facilitate an in-depth exploration of the underlying mechanisms conferring drug resistance. Additionally, a kinase-specific CRISPR-Cas9 library was utilized to perform a targeted screening for potential combinatorial therapeutic targets. Our findings aim to offer novel mechanistic insights and to propose alternative treatment strategies to effectively overcome drug resistance in AML.

Method: In this study, we first established a Gilteritinib-resistant cell line by continuously exposing FLT3-ITD mutant AML cells to long-term low-dose treatment followed by stepwise dose escalation. In an effort to uncover potential molecular therapeutic targets, we subsequently employed a kinase-focused CRISPR-Cas9 screen in the Gilteritinib-resistant cells. High-throughput loss-of-function assays evaluated the roles of known kinases in mediating Gilteritinib resistance and identified candidate targets that synergistically enhanced the drug’s activity. The robustness of these candidate targets was then validated through assays assessing proliferation competition, cell viability, apoptosis, and cell cycle dynamics. By integrating gene knockdown and small molecule inhibitor strategies, we dissected the regulatory mechanisms influencing Gilteritinib sensitivity at these candidate targets. Moreover, immunoprecipitation coupled with mass spectrometry (Co-IP-MS) was utilized to delineate the interactions between FLT3 and these key molecular entities. Finally, using an AML xenograft mouse model, we assessed the antitumor efficacy of the combined treatment regimen in vivo against Gilteritinib-resistant cells.

Results: 1. Whole-exome sequencing revealed that prolonged Gilteritinib exposure induced the emergence of a FLT3-ITD-F691L "gatekeeper" mutation in the resistant cells. This mutation induces a conformational change in the protein, thereby diminishing Gilteritinib’s binding affinity to FLT3 and driving the development of resistance. Additionally, a spectrum of other gene mutations may further modulate drug sensitivity. 2. CRISPR-Cas9 screening results demonstrated that the p38 mitogen-activated protein kinase (MAPK) pathway plays a critical regulatory role in Gilteritinib-resistant cells. Both p38 gene knockdown and treatment with a selective p38 inhibitor markedly restored the sensitivity of resistant cells to Gilteritinib, inhibiting cell proliferation and cell cycle progression, while significantly inducing apoptosis. In stark contrast, modulation of the ERK1/2 pathway, another arm of the MAPK family, failed to elicit any significant synergistic effect. 3. In an in vivo xenograft mouse model, the combination of Gilteritinib with a p38 inhibitor exhibited a significantly enhanced tumor-suppressive effect compared to monotherapy, suggesting that such combination treatment can effectively overcome resistance in refractory cells. 4. Further mechanistic studies revealed that Gilteritinib treatment markedly increased the binding between FLT3 and p38 in resistant cells, coupled with an elevated level of p38 activation, indicating that the p38 signaling pathway may serve as a critical regulatory element in mediating Gilteritinib resistance and promoting the survival and proliferation of AML cells.

Conclusions: In this study, we successfully developed a Gilteritinib-resistant FLT3-ITD AML cell model and, using a kinase-specific CRISPR-Cas9 screening strategy, identified p38 as a promising target for combination therapy. Both in vitro and in vivo experiments demonstrated that co-treatment with Gilteritinib and a p38 inhibitor can overcome drug resistance and significantly enhance antitumor efficacy. These findings offer a clinically translatable combined therapeutic strategy for patients with FLT3-ITD mutant AML who have developed resistance, thereby paving the way for a precision medicine approach to improve patient outcomes.