Functional characterization of cold-shock protein Ybx1 as a potential therapeutic target in Jak2V617F positive myeloproliferative neoplasia (MPN)

冷休克蛋白 Ybx1 作为 Jak2V617F 阳性骨髓增生性肿瘤 (MPN) 潜在治疗靶点的功能表征

• 批准号: 320028127
• 负责人: Professor Dr. Florian Heidel
• 金额: --
• 依托单位: Klinik für Hämatologie, Hämostaseologie, Onkologie und Stammzelltransplantation
• 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/320028127?language=en
• 关键词: Functional; characterization; cold; shock; protein

Janus kinases (JAK) mediate cytokine and hormone responses in hematopoietic cells. JAK2 is one of the most frequently mutated genes in the aging hematopoietic system and in hematopoietic cancers. Notably, JAK mutations constitutively activate downstream signaling and act as driver mutations of myeloproliferative neoplasms (MPN). Unexpectedly, JAK2-mutated cells do not fully depend on JAK-activity: while JAK inhibitors effectively reduce cytokine response and myelo-proliferation, their clinical use has subtle effects on overall disease burden or evolution of persistent clones.This prompted us to investigate the mechanism underlying evolution of JAK inhibitor persistence. In the first funding period of this proposal we applied in-depth phospho-proteome profiling to identify proteins involved in the mRNA processing as JAK2 targets, complemented by RNA interference (RNAi)-based functional validation. Among those, inactivation of the pleiotropic protein YBX1 sensitized the JAK-inhibitor persistent cells to apoptosis. Inactivation of YBX1 results in RNA mis-splicing, retained intron enrichment and disruption of the transcriptional and post-translational control of extracellular signal-regulated kinase (ERK) signaling. Inactivation of the YBX1-ERK axis in combination with pharmacological JAK-inhibition induces apoptosis in JAK2-dependent murine and human cells and leads to in vivo regression of the malignant clone. Collectively, we identified a role of YBX1 critical for the evolution and maintenance of persistent JAK2 clones. Identification of ERK-signaling as a tractable downstream regulator of therapeutic efficacy indicates a novel strategy to influence disease persistence in JAK2-mutated neoplasms.According to our proposed working program we will investigate the mechanistic consequences of differential YBX1-phosphorylation in JAK2-mutated cells. Using phospho-mutants of YBX1 at conserved amino acid residues, we will investigate functional consequences, changes in cellular distribution and binding capacity and transcriptional consequences of differential YBX1 phosphorylation. Effects of YBX1 inactivation on ERK-signaling will be studied by intracellular flow cytometry in cell lines and primary human cells. Transcriptional and post-transcriptional consequences of YBX1 deletion will be assessed by Chromatin- and RNA-immunoprecipitation, analysis of RNA-editing and global transcriptome analyses. So far, specific pharmacologic inhibitors of YBX1 are not available. We aim to identify functionally relevant YBX1 protein domains by genome editing to facilitate development of specific compounds in future projects. Our experimental approach may therefore facilitate therapeutic targeting of the persistent JAK-mutated clone in myeloproliferative neoplasia.

Janus激酶（JAK）介导造血细胞中的细胞因子和激素应答。JAK 2是衰老造血系统和造血系统癌症中最常见的突变基因之一。值得注意的是，JAK突变组成型激活下游信号传导，并作为骨髓增生性肿瘤（MPN）的驱动突变。出乎意料的是，JAK 2突变的细胞并不完全依赖于JAK活性：虽然JAK抑制剂有效地减少细胞因子反应和骨髓增殖，但其临床应用对整体疾病负担或持续克隆的演变有微妙的影响。在该提案的第一个资助期内，我们应用了深入的磷酸化蛋白质组分析来识别参与mRNA加工的蛋白质作为JAK 2靶标，并辅以基于RNA干扰（RNAi）的功能验证。其中，多效蛋白YBX 1的失活使JAK抑制剂持续细胞对凋亡敏感。YBX 1的失活导致RNA错误剪接、保留的内含子富集以及细胞外信号调节激酶（ERK）信号传导的转录和翻译后控制的破坏。YBX 1-ERK轴的失活与药理学JAK抑制相结合诱导JAK 2依赖性鼠和人细胞的凋亡，并导致恶性克隆的体内消退。总的来说，我们确定了YBX 1对持久性JAK 2克隆的进化和维持至关重要的作用。ERK信号转导作为治疗效果的易处理的下游调节剂的鉴定表明了一种新的策略，以影响JAK 2突变的肿瘤中的疾病持续性。根据我们提出的工作计划，我们将研究JAK 2突变的细胞中差异YBX 1磷酸化的机制后果。使用磷酸突变的YBX 1在保守的氨基酸残基，我们将研究功能的后果，在细胞分布和结合能力的变化和转录的差异YBX 1磷酸化的后果。将通过细胞系和原代人细胞中的细胞内流式细胞术研究YBX 1失活对ERK信号传导的影响。将通过染色质和RNA免疫沉淀、RNA编辑分析和全局转录组分析评估YBX 1缺失的转录和转录后后果。到目前为止，还没有YBX 1的特异性药理学抑制剂。我们的目标是通过基因组编辑来识别功能相关的YBX 1蛋白结构域，以促进未来项目中特定化合物的开发。因此，我们的实验方法可能有助于骨髓增生性肿瘤中持续JAK突变克隆的治疗靶向。