Effects of YBX1 on gene expression and translation in acute myeloid leukemia (AML)

恶性细胞在细胞毒或靶向治疗后的持久性是血液病患者不良结局的主要决定因素。尽管大多数急性髓系白血病(AML)患者在化疗后获得完全缓解，但其中很大一部分是由于残留的恶性细胞而复发的。在停止治疗后，这些持续克隆细胞在骨髓利基中具有相对于正常细胞的竞争优势，从而导致疾病的重新建立。迫切需要靶向策略，在不影响正常细胞的情况下，专门降低恶性细胞的竞争性。最近，我们的团队发现剪接因子YBX1是JAK2-激酶的下游效应因子，并发现了它在稳定细胞信号转导中的作用。值得注意的是，正如RNAi屏幕所示，JAK2突变的细胞主要并不依赖于YBX1。然而，YBX1的失活使JAK2突变的细胞对JAK抑制剂治疗敏感。相比之下，对阿喀琉斯项目(布罗德研究所)人类癌细胞系的公开可用CRISPR-Cas9相关性筛选数据集的分析表明，YBX1存在泛癌症相关性，这在造血癌中更为显著，在AML细胞系中最为明显。这些发现表明AML中存在相关的依赖和独特的机制。我们用排列的CRISPR-Cas9阴性选择筛选验证了AML细胞的依赖性，结果显示在感染4个不同的sgRNA后对YBX1有很强的依赖性。这种作用可以在几个AML细胞系和不同的AML体内模型中得到证实。YBX1失活后的转录组分析显示，参与翻译和延长的基因发生了放松调控。根据我们提出的工作计划，我们将在条件小鼠模型中确认YBX1与白血病干细胞的功能相关性。在人类AML(患者衍生异种移植；PDX)的临床前模型中，我们将专门评估与正常造血细胞的细胞竞争，这是在最小残留疾病情况下的治疗相关环境。这一方法将通过测试可能干扰YBX1功能的药理化合物来补充。此外，我们将通过多聚体图谱和整体蛋白质组分析来研究YBX1介导的(转录后)调控的机制后果。利用人类AML细胞的基因组编辑方法，我们的目标是识别依赖YBX1功能的相关分子。综上所述，我们的目标是在临床前模型中确认AML细胞对YBX1的相关依赖性，然后从机制方面进行研究，以确定相关的效应分子。因此，我们的实验方法可能有助于针对经常持续存在的白血病细胞的治疗靶点，这是AML复发的来源。

Persistence of malignant cells following cytotoxic or targeted therapy is a major determinant of adverse outcome in patients with hematologic cancers. Despite the fact that the majority of patients with acute myeloid leukemia (AML) achieve complete remission after chemotherapy, a large proportion of them relapse as a result of residual malignant cells. After discontinuation of treatment, these persistent clones have a competitive advantage over normal cells in the bone marrow niche leading to re-establishment of the disease. Targeting strategies that specifically reduce competitivity of malignant cells while leaving normal cells unaffected are highly needed. Recently, our group identified the splicing factor YBX1 as a downstream effector of JAK2-kinase and uncovered its function in stabilizing cell signaling. Of note, JAK2-mutated cells are not primarily dependent on YBX1, as indicated by an RNAi-screen. However, inactivation of YBX1 sensitized JAK2-mutated cells to JAK-inhibitor treatment.In contrast, analysis of publicly available CRISPR-Cas9 dependency screening datasets on human cancer cell lines of the Achilles Project (Broad Institute) indicated a pan-cancer dependency for YBX1 which was even more significant in hematopoietic cancers and most pronounced in AML cell lines. These findings indicate a relevant dependency and distinct mechanism in AML. We have validated the dependency of AML cells using an arrayed CRISPR-Cas9 based negative selection screen, which showed a strong dependency on Ybx1 following infection with 4 different sgRNAs. This effect could be confirmed in several AML cell lines and in different in vivo models of AML. Transcriptome analysis following YBX1 inactivation revealed deregulation of genes involved in translation and elongation.According to our proposed working program we will confirm the functional relevance of YBX1 for leukemia stem cells in conditional mouse models. In pre-clinical models of human AML (patient-derived xenograft; PDX) we will specifically assess for cell competition against normal hematopoietic cells, a therapeutically relevant setting in a minimal residual disease situation. This approach will be complemented by testing of pharmacologic compounds which may interfere with YBX1 function. Moreover, we will investigate the mechanistic consequences of YBX1-mediated (post-) transcriptional regulation by polysome profiling and global proteome analysis. Using genome editing approaches in human AML cells, we aim to identify relevant molecules that depend on YBX1 function.In summary, we aim to confirm the relevant dependency of AML cells on YBX1 in pre-clinical models followed by investigation of mechanistic aspects to identify relevant effector molecules. Our experimental approach may therefore facilitate therapeutic targeting of frequently persisting leukemic cells, the source of relapse in AML.