Synthetic lethality in leukemic stem cells in juvenile myelomonocytic leukemia

项目摘要 幼年性粒单核细胞白血病(JMML)是一种儿童早期的克隆性血液系统恶性肿瘤， 治疗选择。复发仍然是治疗失败的主要原因，很可能是由于持续的 白血病干细胞(LSCs)，一小部分自我更新的前体细胞，导致大部分肿瘤的产生 细胞。这种肿瘤细胞的储存库负责长期维持白血病的生长，也是一种 耐药性的主要来源。因此，一种新的治疗方法着眼于独特的特点 为了针对JMML的根本原因，需要LSC的漏洞。然而，它仍然是一个关键 挑战如何根除这些白血病前体细胞。JMML是由各种基因突变引起的 在参与RAS通路的信号蛋白中，包括蛋白酪氨酸磷酸酶Ptpn11(Shp2)， RAS途径的正调控因子最常发生突变。这些突变导致的 Shp2和Ptpn11突变的JMML患者在所有亚型中预后最差 JMML。我们之前创造了一组带有Ptpn11E76K突变的条件性敲入小鼠，这是最常见的 在JMML中发现活跃的Ptpn11突变。小鼠Ptpn11E76K/+突变的诱导 Mx1-Cre+)导致了JMML样骨髓增生性肿瘤(MPN)，提示了一种 该突变在JMML发病机制中的作用。为了了解白血病的生物学特性 JMML中的前体细胞，我们最近在这个小鼠模型中检测了突变的造血干细胞(参考文献 转化为LSCs)，发现Stat5被过度激活。更重要的是，Ptpn11E76K/+LSCs似乎依赖于 由于Ptpn11E76K/+/Mx1-Cre+/Stat5fl/fl双突变体中Stat5的缺失，Stat5用于维护和自我更新 小鼠导致大量LSC细胞死亡，而Stat5基因敲除和Ptpn11E76K/+单独敲入的小鼠都没有 干细胞中的这种表型。因此，Ptpn11E76K/+/Mx1-Cre+/Stat5fl/fl双突变小鼠的LSC池 明显减少。进一步的机理研究表明，这是因为酪氨酸(Tyr705) 在Ptpn11E76K/+LSCs中，Shp2底物蛋白之一的STAT3的磷酸化水平降低了3-5倍 可能是由于高度活跃的Shp2 E76K突变体增强了去磷酸化作用。因此，STAT3活性减弱 代表了Ptpn11E76K/+LSCs的致命弱点，使它们容易受到Stat5耗尽诱导的细胞死亡的影响。 在Ptpn11激活的JMML干细胞中丢失Stat5导致的合成致死性引起了一个有趣的问题 可能，即，药物抑制Stat5可能有效地根除这一亚型的JMML中的LSCs。 我们计划验证这一假设，并通过追求以下两个目标来实现本项目的目标。1) 定义Stat5在维护与Ptpn11相关的JMML中的LSC中的作用。2)检验治疗效果 在Ptpn11(Shp2)激活的JMML中，临床上使用的抗精神病药物--Stat5抑制剂匹莫齐特。

Project Summary Juvenile myelomonocytic leukemia (JMML), a clonal hematological malignancy of early childhood, has limited therapeutic options. Relapse remains the main cause of treatment failure, most likely due to the persistence of leukemic stem cells (LSCs), a small population of self-renewing precursor cells that give rise to the bulk of tumor cells. This reservoir of tumor cells is responsible for long-term maintenance of leukemia growth and is also a major source of drug resistance. Therefore, a novel treatment approach focused on the unique characteristics and vulnerabilities of LSCs is needed in order to target this root cause of JMML. However, it remains a critical challenge how these leukemic precursor cells may be eradicated. JMML is caused by various genetic mutations in the signaling proteins involved in the Ras pathway, among which protein tyrosine phosphatase Ptpn11 (Shp2), a positive regulator of the Ras pathway, is most frequently mutated. These mutations cause greatly increased catalytic activity of Shp2, and JMML patients with Ptpn11 mutations have the worst prognosis in all subtypes of JMML. We previously created a line of conditional knock-in mice with the Ptpn11E76K mutation, the most common and active Ptpn11 mutation found in JMML. Induction of the Ptpn11E76K/+ mutation in these mice (Ptpn11E76K/+/ Mx1-Cre+) resulted in JMML-like myeloproliferative neoplasm (MPN) with full penetrance, suggesting a causative role of this mutation in the pathogenesis of JMML. In an effort to understand the biological properties of leukemic precursor cells in JMML, we recently examined mutated hematopoietic stem cells in this mouse model (referred to as LSCs) and found that Stat5 was hyper-activated. More importantly, Ptpn11E76K/+ LSCs appeared to rely on Stat5 for maintenance and self-renewal because deletion of Stat5 in Ptpn11E76K/+/Mx1-Cre+/Stat5fl/fl double mutant mice resulted in massive LSC cell death while neither Stat5 knockout nor Ptpn11E76K/+ knock-in alone mice had such phenotypes in stem cells. Consequently, the LSC pool in Ptpn11E76K/+/Mx1-Cre+/Stat5fl/fl double mutant mice was markedly decreased. Further mechanistic investigation revealed that this was because tyrosine (Tyr705) phosphorylation of Stat3, one of the Shp2 substrate proteins, was decreased by 3 to 5-fold in Ptpn11E76K/+ LSCs likely due to enhanced dephosphorylation by the hyperactive Shp2 E76K mutant. Thus, diminished Stat3 activity represents an Achilles’ heel of Ptpn11E76K/+ LSCs, making them vulnerable to Stat5 depletion-induced cell death. The synthetic lethality induced by loss of Stat5 in Ptpn11-activated JMML stem cells raises an intriguing possibility, i.e., pharmacological inhibition of Stat5 might be effective in eradicating LSCs in this subtype of JMML. We plan to test this hypothesis and accomplish the objective of this project by pursuing the following two aims. 1) To define the role of Stat5 in maintenance of LSCs in Ptpn11-associated JMML. 2) To test the therapeutic effects of the Stat5 inhibitor pimozide, a clinically used antipsychotic drug, in Ptpn11 (Shp2)-activated JMML.