Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr 30, 52074, Aachen, Germany.
Institute for Computational Genomics, Faculty of Medicine, RWTH Aachen University, Aachen, Germany.
J Hematol Oncol. 2019 Apr 2;12(1):36. doi: 10.1186/s13045-019-0722-9.
Interferon alpha (IFNa) monotherapy is recommended as the standard therapy in polycythemia vera (PV) but not in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa efficacy in JAK2V617F- vs. BCR-ABL-positive cells.
Gene expression microarrays and RT-qPCR of PV vs. CML patient PBMCs and CD34+ cells and of the murine cell line 32D expressing JAK2V617F or BCR-ABL were used to analyze and compare interferon-stimulated gene (ISG) expression. Furthermore, using CRISPR/Cas9n technology, targeted disruption of STAT1 or STAT2, respectively, was performed in 32D-BCR-ABL and 32D-JAK2V617F cells to evaluate the role of these transcription factors for IFNa efficacy. The knockout cell lines were reconstituted with STAT1, STAT2, STAT1Y701F, or STAT2Y689F to analyze the importance of wild-type and phosphomutant STATs for the IFNa response. ChIP-seq and ChIP were performed to correlate histone marks with ISG expression.
Microarray analysis and RT-qPCR revealed significant upregulation of ISGs in 32D-JAK2V617F but downregulation in 32D-BCR-ABL cells, and these effects were reversed by tyrosine kinase inhibitor (TKI) treatment. Similar expression patterns were confirmed in human cell lines, primary PV and CML patient PBMCs and CD34+ cells, demonstrating that these effects are operational in patients. IFNa treatment increased Stat1, Stat2, and Irf9 mRNA as well as pY-STAT1 in all cell lines; however, viability was specifically decreased in 32D-JAK2V617F. STAT1 or STAT2 knockout and reconstitution with wild-type or phospho-deficient STAT mutants demonstrated the necessity of STAT2 for IFNa-induced STAT1 phosphorylation in BCR-ABL- but not in JAK2V617F-expressing cells. STAT1 was essential for IFNa activity in both BCR-ABL- and JAK2V617F-positive cells. Furthermore, ChIP experiments demonstrate higher repressive and lower active chromatin marks at the promoters of ISGs in BCR-ABL-expressing cells.
JAK2V617F but not BCR-ABL sensitizes MPN cells to interferon, and this effect was dependent on STAT1. Moreover, STAT2 is a survival factor in BCR-ABL- and JAK2V617F-positive cells but an IFNa-sensitizing factor solely in 32D-JAK2V617F cells by upregulation of STAT1 expression.
干扰素 α（IFNa）单药治疗被推荐为真性红细胞增多症（PV）的标准治疗方法，但不适用于慢性髓系白血病（CML）。在这里，我们研究了 IFNa 在 JAK2V617F-与 BCR-ABL 阳性细胞中的疗效机制。
通过基因表达微阵列和 RT-qPCR 分析比较 PV 与 CML 患者 PBMCs 和 CD34+细胞以及表达 JAK2V617F 或 BCR-ABL 的小鼠细胞系 32D 中的干扰素刺激基因（ISG）表达。此外，使用 CRISPR/Cas9n 技术分别对 32D-BCR-ABL 和 32D-JAK2V617F 细胞中的 STAT1 或 STAT2 进行靶向敲除，以评估这些转录因子对 IFNa 疗效的作用。用 STAT1、STAT2、STAT1Y701F 或 STAT2Y689F 重建敲除细胞系，以分析野生型和磷酸化突变型 STATs 对 IFNa 反应的重要性。进行 ChIP-seq 和 ChIP 以将组蛋白标记与 ISG 表达相关联。
微阵列分析和 RT-qPCR 显示 32D-JAK2V617F 中的 ISG 显著上调，而 32D-BCR-ABL 中的 ISG 下调，酪氨酸激酶抑制剂（TKI）治疗可逆转这些效应。在人类细胞系、原发性 PV 和 CML 患者 PBMCs 和 CD34+细胞中证实了类似的表达模式，表明这些效应在患者中起作用。IFNa 治疗可增加所有细胞系中 Stat1、Stat2 和 Irf9 mRNA 以及 pY-STAT1 的表达；然而，仅在 32D-JAK2V617F 中细胞活力特异性降低。STAT1 或 STAT2 敲除并用野生型或磷酸化缺陷 STAT 突变体重建表明，STAT2 是 BCR-ABL 中 IFNa 诱导的 STAT1 磷酸化所必需的，但在 JAK2V617F 表达细胞中则不是。STAT1 是 BCR-ABL 和 JAK2V617F 阳性细胞中 IFNa 活性所必需的。此外，ChIP 实验表明，在表达 BCR-ABL 的细胞中，ISG 启动子处的抑制性染色质标记更高，而活性染色质标记更低。
JAK2V617F 而非 BCR-ABL 使 MPN 细胞对干扰素敏感，这种作用依赖于 STAT1。此外，STAT2 是 BCR-ABL 和 JAK2V617F 阳性细胞中的存活因子，但在 32D-JAK2V617F 细胞中通过上调 STAT1 表达成为 IFNa 增敏因子。
