Regulation of FLT3 Signaling in Leukemia

总结 信号蛋白的时空分布受翻译后调控 修改（PTM）。PTM如磷酸化、泛素化或脂质修饰决定蛋白质活性 并获得基质，从而获得细胞结果。信号通路的精确控制对于 正常的造血和异常的信号传导导致造血干细胞的恶性转化， 祖细胞（HSPC）。这项应用是基于我们的新发现，FLT 3（FMS样酪氨酸激酶3） 被棕榈酰化，破坏致癌FLT 3突变体的棕榈酰化会改变其亚细胞定位， 重新连接下游信号并促进白血病进展。FLT 3内的内部串联重复 （FLT 3-ITD）是急性髓性白血病（AML）中最常见的突变之一，与不良的免疫反应相关。 预后而野生型FLT 3受体酪氨酸激酶在质膜被激活， PI 3 K/AKT和RAS/MAPK信号传导，FLT 3-ITD驻留在内质网（ER）中并触发 组成型STAT 5磷酸化。这种异常FLT 3-ITD亚细胞定位的潜在机制或 其对白血病发生影响仍然知之甚少。我们发现，FLT 3-ITD是S-棕榈酰化的， ZDHHC 6酰基转移酶。棕榈酰化的破坏将FLT 3-ITD重定向至质膜并重新连接 它的下游信号通过激活AKT和ERK途径除了STAT 5。因此，废除 通过ZDHHC 6消耗的FLT 3-ITD棕榈酰化促进FLT 3-ITD表面表达，信号传导， 增加异种移植小鼠模型中的白血病进展。此外，我们证明FLT 3 蛋白质在原代人AML细胞中被棕榈酰化。FLT 3-ITD棕榈酰化的稳定化 药理学抑制去棕榈酰化与FLT 3酪氨酸激酶抑制剂（TKI）gilteritinib协同作用， 消除原代FLT 3-ITD+ AML细胞的生长。这项资助的中心目标是定义分子 通过棕榈酰化调节致癌FLT 3信号转导的基础，并探讨其生理和 在骨髓恶性肿瘤中的功能意义。我们建议定义ZDHHC 6在FLT 3-ITD中的作用 在骨髓增生性肿瘤（MPN）和AML的小鼠模型中，我们还将确定 修饰FLT 3-ITD定位的FLT 3-ITD脱棕榈酰酶，和使用组合方法的活性 有针对性和公正的化学生物学，分子生物学和遗传学。此外，我们会探讨 在原代人FLT 3-ITD+ AML中靶向FLT 3-ITD脱棕榈酰化的治疗潜力。我们将 使用原代人AML细胞研究FLT 3-ITD脱棕榈酰酶抑制是否增强对TKI的应答 以及患者来源的异种移植（PDX）模型。这些发现提供了新的见解脂质- FLT 3-ITD信号传导的依赖性区室化，并建议靶向去棕榈酰化作为新的 治疗FLT 3-ITD+白血病的治疗策略。

Summary The temporal and spatial distribution of signaling proteins is dynamically regulated by post-translational modifications (PTMs). PTMs such as phosphorylation, ubiquitination, or lipid modification dictate protein activities and access to substrates, thereby cellular outcomes. The precise control of signaling pathways is critical to normal hematopoiesis and aberrant signaling leads to malignant transformation of hematopoietic stem and progenitor cells (HSPCs). This application is based on our novel finding that FLT3 (FMS-like tyrosine kinase 3) is palmitoylated and disrupting palmitoylation of oncogenic FLT3 mutants changes their subcellular localization, rewires downstream signaling, and promotes leukemic progression. Internal tandem duplication within FLT3 (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and correlates with poor prognosis. While wildtype FLT3 receptor tyrosine kinase is activated at the plasma membrane to transduce PI3K/AKT and RAS/MAPK signaling, FLT3-ITD resides in the endoplasmic reticulum (ER) and triggers constitutive STAT5 phosphorylation. Mechanisms underlying this aberrant FLT3-ITD subcellular localization or its impact on leukemogenesis remain poorly understood. We discovered that FLT3-ITD is S-palmitoylated by the ZDHHC6 acyltransferase. Disruption of palmitoylation redirects FLT3-ITD to the plasma membrane and rewires its downstream signaling by activating AKT and ERK pathways in addition to STAT5. Consequently, abrogation of FLT3-ITD palmitoylation via ZDHHC6 depletion promotes FLT3-ITD surface expression, signaling, and increased leukemic progression in xenotransplanted mouse models. Furthermore, we demonstrate that FLT3 proteins are palmitoylated in primary human AML cells. Stabilization of FLT3-ITD palmitoylation by pharmacological inhibition of depalmitoylation synergizes with FLT3 tyrosine kinase inhibitor (TKI) gilteritinib in abrogating the growth of primary FLT3-ITD+ AML cells. The central goal of this grant is to define the molecular basis underlying the regulation of oncogenic FLT3 signaling by palmitoylation and explore its physiological and functional significance in myeloid malignancies. We propose to define roles of ZDHHC6 in FLT3-ITD palmitoylation in vivo in mouse models of myeloproliferative neoplasm (MPN) and AML. We will also identify depalmitoylase(s) for FLT3-ITD that modify FLT3-ITD localization, and activity using combinatorial approaches of targeted and unbiased chemical biology, molecular biology, and genetics. Moreover, we will explore the therapeutic potential of targeting FLT3-ITD depalmitoylation in primary human FLT3-ITD+ AMLs. We will investigate if inhibition of FLT3-ITD depalmitoylase enhances responses to TKI using primary human AML cells as well as patient-derived xenotransplant (PDX) models. These findings provide novel insights into lipid- dependent compartmentalization of FLT3-ITD signaling and suggest targeting depalmitoylation as a new therapeutic strategy to treat FLT3-ITD+ leukemias.