Gorantla Sivahari P, Oelschläger Lorenz, Prince Gerin, Osius Jasmin, Kolluri Suresh Babu, Maluje Yamil, Fähnrich Anke, Ernst Nancy, Barbosa Gulde Alanis, Ludwig Ralf Joachim, Gemoll Timo, Fliedner Stephanie, Walter Wencke, Haferlach Torsten, Gebauer Niklas, Busch Hauke, Duyster Justus, von Bubnoff Nikolas
Department of Hematology and Oncology, University Medical Center Schleswig-Holstein, and University Cancer Center Schleswig-Holstein, Lübeck, Germany.
Institute of Experimental Dermatology, University of Luebeck, Luebeck, Germany.
Leukemia. 2025 Apr 23. doi: 10.1038/s41375-025-02594-7.
Myelofibrosis (MF) in 50% of cases is driven by an activating JAK2 mutation, mostly V617F. Ruxolinitib is approved for the treatment of MF. Responses to ruxolitinib in MF are of limited duration. Unexpectedly, treatment of JAK2-V617F expressing cells with ruxolitinib causes paradoxical hyperphosphorylation of JAK2 at activation loop Tyr1007/Tyr1008. The significance of ruxolitinib-induced JAK2 hyperphosphorylation is not well understood. We found that a ruxolitinib-resistant JAK2 variant (V617F + L983F) and a kinase dead mutant (JAK2-V617F + K882R) did not show paradoxical hyperphosphorylation after ruxolitinib treatment indicating that it is an intrinsic mechanism. Antibodies against pTyr1007/1008 failed to immunoprecipitate native JAK2-V617F in the presence of ruxolitinib, although JAK2-V617F was hyperphosphorylated at these sites, suggesting that in the presence of ruxolitinib the JAK2 activation loop is buried within the kinase domain. This stabilization of the activation loop conformation resulted in the protection of pTyr1007/1008 sites from phosphatases. Mutation of Arg975 and Lys999 to Ala reduced the phosphorylation at both Tyr1007/Tyr1008 residues, and notably, ruxolitinib treatment did not lead to JAK2 hyperphosphorylation. Importantly, hyperphosphorylated JAK2 after ruxolitinib dissociation displayed excess rebound activation of STAT5 target gene PIM kinase. Our results suggest a novel mode of kinase regulation by modulating kinase activity through conformational changes induced by ruxolitinib.Subject categories: JAK2-V617F, Ruxolitinib, JAK2 hyperphosphorylation, Phosphatases action, PIM kinases.
50%的骨髓纤维化（MF）病例由激活的JAK2突变驱动，主要是V617F。芦可替尼已被批准用于治疗MF。MF患者对芦可替尼的反应持续时间有限。出乎意料的是，用芦可替尼处理表达JAK2-V617F的细胞会导致JAK2在激活环Tyr1007/Tyr1008处出现反常的过度磷酸化。芦可替尼诱导的JAK2过度磷酸化的意义尚不完全清楚。我们发现，一种对芦可替尼耐药的JAK2变体（V617F + L983F）和一种激酶失活突变体（JAK2-V617F + K882R）在芦可替尼处理后未表现出反常的过度磷酸化，这表明这是一种内在机制。尽管JAK2-V617F在这些位点发生了过度磷酸化，但针对pTyr1007/1008的抗体在芦可替尼存在的情况下未能免疫沉淀天然的JAK2-V617F，这表明在芦可替尼存在的情况下，JAK2激活环被埋在激酶结构域内。激活环构象的这种稳定导致pTyr1007/1008位点免受磷酸酶的作用。将Arg975和Lys999突变为Ala会降低Tyr1007/Tyr1008两个残基的磷酸化，值得注意的是，芦可替尼处理不会导致JAK2过度磷酸化。重要的是，芦可替尼解离后过度磷酸化的JAK2表现出STAT5靶基因PIM激酶的过度反弹激活。我们的结果提示了一种通过芦可替尼诱导的构象变化调节激酶活性来调控激酶的新模式。主题分类：JAK2-V617F、芦可替尼、JAK2过度磷酸化、磷酸酶作用、PIM激酶。
