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

Janus激酶(JAK)介导造血细胞的细胞因子和激素反应。JAK2基因是衰老造血系统和血液系统肿瘤中最常见的突变基因之一。值得注意的是，JAK突变构成激活下游信号，并作为骨髓增生性肿瘤(MPN)的驱动突变。出乎意料的是，JAK2突变的细胞并不完全依赖JAK活性：虽然JAK抑制剂有效地减少了细胞因子反应和骨髓增殖，但它们的临床应用对整体疾病负担或持久性克隆的进化有微妙的影响。这促使我们研究JAK抑制剂持久性进化的机制。在该提案的第一个资助期，我们应用了深入的磷酸蛋白质组谱分析，以确定参与mRNA加工的蛋白质为JAK2靶标，并辅之以基于RNA干扰(RNAi)的功能验证。其中，多效性蛋白YBX1的失活使JAK抑制剂持久性细胞对凋亡敏感。YBX1的失活导致RNA错误剪接，内含子保留丰富，细胞外信号调节激酶(ERK)信号的转录和翻译后控制中断。YBX1-ERK轴的失活与药物上的JAK抑制联合诱导JAK2依赖的小鼠和人类细胞凋亡，并导致体内恶性克隆的消退。总而言之，我们确定了YBX1在持久JAK2克隆的进化和维护中的关键作用。ERK信号被确认为一个易于处理的下游疗效调节因子表明了一种新的策略来影响JAK2突变肿瘤的疾病持久性。根据我们建议的工作计划，我们将研究JAK2突变细胞中差异YBX1磷酸化的机制后果。利用YBX1保守氨基酸残基的磷酸化突变体，我们将研究YBX1的功能后果、细胞分布和结合能力的变化以及YBX1差异磷酸化的转录后果。YBX1失活对ERK信号的影响将通过细胞内流式细胞术在细胞系和原代人类细胞中进行研究。YBX1缺失的转录和转录后后果将通过染色质和RNA免疫沉淀、RNA编辑分析和整体转录组分析来评估。到目前为止，还没有YBX1的特异性药物抑制剂。我们的目标是通过基因组编辑识别功能相关的YBX1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针对骨髓增生性肿瘤中持久的JAK突变克隆的治疗。

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.