Development of resistance to type II JAK2 inhibition in MPN depends on AXL kinase and is targetable. Homo sapiens

Purpose: Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Since clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. Methods: MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC-/RNA-sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. Results: MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NSG mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Since inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL-MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL-MAPK. Conclusions: We report on a novel mechanism of AXL-MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.

研究目的：骨髓增殖性肿瘤（Myeloproliferative neoplasms, MPN）可异常调控JAK2（Janus激酶2）信号通路。由于临床使用的JAK2抑制剂仅具备有限的疾病修饰效果，诸如CHZ868这类可稳定非活性JAK2并减少MPN克隆的II型JAK2抑制剂正逐渐受到学界关注。本研究旨在探究MPN细胞是否会对II型JAK2抑制产生逃逸机制。
研究方法：将MPN细胞持续暴露于CHZ868环境中。采用磷酸化蛋白质组学分析、ATAC测序（Assay for Transposase-Accessible Chromatin sequencing）与RNA测序技术，对MPN细胞获得性II型JAK2抑制耐药性的特征进行解析，并针对MPN细胞与小鼠模型中的候选耐药介导因子开展靶向验证。
研究结果：MPN细胞经CHZ868处理后，半最大抑制浓度（half maximal inhibitory concentration, IC50）升高且凋亡水平降低，提示其获得了JAK2抑制耐药性。在超过2500个差异磷酸化位点中，丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）通路的激活最为显著，而JAK2-STAT3/5通路仍处于抑制状态。组蛋白结合占有率的改变促进了激活蛋白1（Activator Protein 1, AP-1）与GATA结合基序的暴露，该改变与AXL激酶表达上调及RAS靶基因谱富集显著相关。敲低AXL可使MPN细胞恢复对CHZ868的敏感性；联合使用bemcentinib或gilteritinib进行JAK2/AXL双抑制，可将IC50恢复至敏感细胞的水平。尽管在NSG小鼠（NOD-scid IL2rg null小鼠）中，即使给予JAK2抑制剂，耐药细胞仍可诱导肿瘤生长，但JAK2/AXL联合抑制可显著阻滞肿瘤进展。鉴于丝裂原活化蛋白激酶通路激酶抑制剂（如MEK抑制剂）已在其他恶性肿瘤中获批临床应用，本研究进一步评估了曲美替尼（trametinib）介导的JAK2/MAPK联合抑制对AXL-MAPK诱导的耐药性的干预效果。结果显示，该联合方案可阻滞肿瘤生长，效果与JAK2/AXL联合抑制相当；在系统性细胞系来源的小鼠模型中，骨髓浸润程度显著降低，进一步支持了AXL-MAPK通路在耐药机制中的核心依赖性。
研究结论：本研究揭示了AXL-MAPK通路驱动的MPN细胞逃逸II型JAK2抑制的全新机制，该机制可通过多个靶点进行干预。本研究凸显了AXL作为获得性耐药介质的关键作用，抑制AXL可增强JAK2抑制疗法在MPN治疗中的持续有效性。