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

酪氨酸激酶的组成性活性突变体（JAK 2，点突变JAK 2 V617 F）可导致骨髓增生性肿瘤的发生。用JAK 2 V617 F转化血细胞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的药理学干预策略的受欢迎的靶点。我们想了解为什么HDAC抑制剂（HDACi）对JAK 2突变的白血病细胞毒性很大。根据最近的结果，HDAC是在具有复制应激和DNA损伤的细胞中激活检查点激酶的关键调节剂。此外，HDAC通过泛素-蛋白酶体系统对蛋白质降解产生重要影响。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JAK 2 V617 F的蛋白酶体降解，这导致生长促进癌基因的耗尽。我们的假设是，这一过程依赖于在蛋白质水平上诱导E3泛素连接酶SIAH 2。SIAH 2的这种积累似乎是通过HDAC 3作为看门人特异性调节的，这可能涉及通过乙酰化控制SIAH 2。因此，我们的目标是在分子上定义SIAH 2与JAK 2 V617 F的相互作用，JAK 2 V617 F的多聚泛素化，以及在各种细胞系统中通过乙酰化控制SIAH 2。我们的目的是澄清分子定义的SIAH降解决定子基序和蛋白质的SOCS家族参与酪氨酸磷酸化和非磷酸化JAK 2 V617 F和野生型JAK 2的泛素化和蛋白酶体降解。此外，我们想研究SIAH 2是否会使JAK 2 V617 F与其已知的相互作用伙伴E2泛素缀合酶UBCH 8不稳定。已发表的数据表明，DNA损伤后检查点激酶对SIAH 2的磷酸化使SIAH 2失活。然而，我们发现SIAH 2在HDACi诱导的复制应激/DNA损伤后仍然对JAK 2 V617 F具有活性。这一发现表明，我们首次描述了SIAH 2的HDAC依赖性控制。在这种情况下，我们想解决SIAH 2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的细胞系统来严格测试我们的理论。无偏蛋白质组学将定义SIAH 2依赖性蛋白质组。我们关于HDAC 3的作用及其对具有突变JAK 2的白血病细胞存活的抑制的分析将揭示HDAC 3是否确实是最适合的药理学靶结构以及SIAH 2的积累是否是HDAC i功效的药效学标志物。JAK 2 V617 F阳性白血病患者可能会从这些见解中受益。

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.