Delineating the novel HDAC3-SIAH2 signaling node and its impact on oncogenic JAK2 in leukemic cells

描述新型 HDAC3-SIAH2 信号节点及其对白血病细胞中致癌 JAK2 的影响

• 批准号: 445785155
• 负责人: Professor Dr. Oliver Holger Krämer
• 金额: --
• 依托单位: Institut für Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/445785155?language=en
• 关键词: Delineating; novel; HDAC3; SIAH2; signaling

Constitutively active mutants of the tyrosine kinase (JAK2, point mutation JAK2V617F) causally contribute to the development of myeloproliferative neoplasms. The transformation of blood cells with JAK2V617F triggers replication stress through increased cell proliferation and aberrant gene expression patterns. Epigenetic modulators of the histone deacetylase (HDAC) family are appreciated targets for novel pharmacological intervention strategies. We want to understand why HDAC inhibitors (HDACi) are very toxic for leukemic cells with mutant JAK2. According to recent results, HDACs are key regulators for the activation of checkpoint kinases in cells with replication stress and DNA damage. Moreover, HDACs critically influence protein degradation via the ubiquitin-proteasome-system. Consequently, HDACi can modulate the growth of tumor cells via these parameters. Our novel data suggest that HDACi accelerate the proteasomal degradation of JAK2V617F, which leads to a depletion of the growth-promoting oncogene. Our hypothesis is that this process relies on an induction of the E3 ubiquitin-ligase SIAH2 at the protein level. Such an accumulation of SIAH2 appears to be specifically regulated through HDAC3 as a gatekeeper and this might involve the control of SIAH2 by acetylation. Therefore, we aim to molecularly define the interaction of SIAH2 with JAK2V617F, the poly-ubiquitination of JAK2V617F, and a control of SIAH2 by acetylation in various cell systems. We aim to clarify whether molecularly defined SIAH degron motifs and proteins of the SOCS family are involved in the ubiquitination and proteasomal degradation of tyrosine-phosphorylated and unphosphorylated JAK2V617F and wild-type JAK2. Moreover, we want to investigate whether SIAH2 destabilizes JAK2V617F with its known interaction partner, the E2 ubiquitin-conjugase UBCH8. Published data show that the phosphorylation of SIAH2 by checkpoint kinases after DNA damage inactivates SIAH2. However, we find that SIAH2 remains active against JAK2V617F after HDACi-induced replication stress/DNA damage. This finding indicates that we describe for the first time an HDAC-dependent control of SIAH2. Within this context, we want to address whether SIAH2 targets checkpoint kinases. For our analyses, we want to follow hypotheses-driven approaches and we aim to create new CRISPR-Cas9-based cell systems to test our theories stringently. Unbiased proteomics will define the SIAH2-dependent proteome. Our analyses concerning the role of HDAC3 and its inhibition for the survival of leukemic cells with mutant JAK2 will reveal if HDAC3 is indeed a best suited pharmacological target structure and whether the accumulation of SIAH2 is a pharmacodynamic marker for the efficacy of HDACi. Patients with JAK2V617F-positive leukemia might profit from such insights.

酪氨酸激酶（JAK2，点突变JAK2V617F）的组成型活性突变可导致骨髓增殖性肿瘤的发生。携带JAK2V617F的血细胞转化通过增加细胞增殖和异常基因表达模式触发复制应激。组蛋白去乙酰化酶（HDAC）家族的表观遗传调节剂是新型药物干预策略的重要靶点。我们想了解为什么HDAC抑制剂（HDACi）对JAK2突变的白血病细胞有很大的毒性。根据最近的研究结果，hdac是复制应激和DNA损伤细胞中检查点激酶激活的关键调节因子。此外，hdac通过泛素-蛋白酶体系统严重影响蛋白质降解。因此，HDACi可以通过这些参数调节肿瘤细胞的生长。我们的新数据表明，HDACi加速了JAK2V617F的蛋白酶体降解，从而导致促生长癌基因的耗竭。我们的假设是，这一过程依赖于E3泛素连接酶SIAH2在蛋白质水平上的诱导。这种SIAH2的积累似乎是通过作为看门人的HDAC3特异性调节的，这可能涉及通过乙酰化控制SIAH2。因此，我们的目标是分子定义SIAH2与JAK2V617F的相互作用，JAK2V617F的多泛素化，以及在各种细胞系统中通过乙酰化控制SIAH2。我们的目的是阐明SOCS家族的SIAH降解基元和蛋白是否参与酪氨酸磷酸化和非磷酸化JAK2V617F和野生型JAK2的泛素化和蛋白酶体降解。此外，我们想要研究SIAH2是否会通过其已知的相互作用伙伴E2泛素偶联酶UBCH8破坏JAK2V617F的稳定性。已发表的数据表明，DNA损伤后检查点激酶对SIAH2的磷酸化使SIAH2失活。然而，我们发现SIAH2在hdac诱导的复制应激/DNA损伤后仍然对JAK2V617F具有活性。这一发现表明我们首次描述了SIAH2的hdac依赖性控制。在这种情况下，我们想解决SIAH2是否靶向检查点激酶。对于我们的分析，我们希望遵循假设驱动的方法，我们的目标是创建新的基于crispr - cas9的细胞系统，以严格测试我们的理论。无偏蛋白质组学将定义siah2依赖性蛋白质组。我们对HDAC3的作用及其对JAK2突变白血病细胞存活的抑制作用的分析将揭示HDAC3是否确实是最合适的药理学靶标结构，以及SIAH2的积累是否是HDACi疗效的药效学标志。jak2v617f阳性白血病患者可能会从这些发现中受益。