Center of Research and Innovation of Myeloproliferative Neoplasms (CRIMM), Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.
DENOTHE Excellence Center, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
Blood Adv. 2021 Apr 27;5(8):2184-2195. doi: 10.1182/bloodadvances.2020003291.
Calreticulin (CALR), an endoplasmic reticulum-associated chaperone, is frequently mutated in myeloproliferative neoplasms (MPNs). Mutated CALR promotes downstream JAK2/STAT5 signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Here, we provide evidence of a novel mechanism contributing to CALR-mutated MPNs, represented by abnormal activation of the interleukin 6 (IL-6)-signaling pathway. We found that UT7 and UT7/mpl cells, engineered by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to express the CALR type 1-like (DEL) mutation, acquired cytokine independence and were primed to the megakaryocyte (Mk) lineage. Levels of IL-6 messenger RNA (mRNA), extracellular-released IL-6, membrane-associated glycoprotein 130 (gp130), and IL-6 receptor (IL-6R), phosphorylated JAK1 and STAT3 (p-JAK1 and p-STAT3), and IL-6 promoter region occupancy by STAT3 all resulted in increased CALR DEL cells in the absence of MPL stimulation. Wild-type, but not mutated, CALR physically interacted with gp130 and IL-6R, downregulating their expression on the cell membrane. Agents targeting gp130 (SC-144), IL-6R (tocilizumab [TCZ]), and cell-released IL-6 reduced proliferation of CALR DEL as well as CALR knockout cells, supporting a mutated CALR loss-of-function model. CD34+ cells from CALR-mutated patients showed increased levels of IL-6 mRNA and p-STAT3, and colony-forming unit-Mk growth was inhibited by either SC144 or TCZ, as well as an IL-6 antibody, supporting cell-autonomous activation of the IL-6 pathway. Targeting IL-6 signaling also reduced colony formation by CD34+ cells of JAK2V617F-mutated patients. The combination of TCZ and ruxolitinib was synergistic at very low nanomolar concentrations. Overall, our results suggest that target inhibition of IL-6 signaling may have therapeutic potential in CALR, and possibly JAK2V617F, mutated MPNs.
钙网织蛋白（CALR）是一种内质网相关伴侣蛋白，在骨髓增殖性肿瘤（MPN）中经常发生突变。突变的 CALR 通过与血小板生成素受体（MPL）相互作用并激活 MPL，促进下游 JAK2/STAT5 信号传导。在这里，我们提供了一个新的机制的证据，有助于 CALR 突变的 MPN，表现为白细胞介素 6（IL-6）信号通路的异常激活。我们发现，通过聚类规则间隔短回文重复（CRISPR）/CRISPR 相关蛋白 9（Cas9）工程改造的 UT7 和 UT7/mpl 细胞，表达 CALR 1 型样（DEL）突变，获得细胞因子独立性，并向巨核细胞（Mk）谱系分化。在没有 MPL 刺激的情况下，白细胞介素 6 信使 RNA（mRNA）、细胞外释放的白细胞介素 6（IL-6）、膜结合糖蛋白 130（gp130）和白细胞介素 6 受体（IL-6R）、磷酸化 JAK1 和 STAT3（p-JAK1 和 p-STAT3）以及 STAT3 对白细胞介素 6 启动子区域的占有率均导致 CALR DEL 细胞增加。野生型而非突变型 CALR 与 gp130 和 IL-6R 物理相互作用，下调其在细胞膜上的表达。靶向 gp130（SC-144）、IL-6R（托珠单抗[TCZ]）和细胞释放的 IL-6 减少了 CALR DEL 以及 CALR 敲除细胞的增殖，支持突变型 CALR 功能丧失模型。来自 CALR 突变患者的 CD34+细胞显示出更高水平的白细胞介素 6 mRNA 和 p-STAT3，并且 SC144 或 TCZ 以及白细胞介素 6 抗体抑制集落形成单位-Mk 生长，支持 IL-6 通路的细胞自主激活。靶向 IL-6 信号也降低了 JAK2V617F 突变患者的 CD34+细胞的集落形成。TCZ 和鲁索利替尼的组合在非常低的纳摩尔浓度下具有协同作用。总的来说，我们的结果表明，抑制白细胞介素 6 信号可能具有治疗 CALR 和可能的 JAK2V617F 突变 MPN 的潜力。
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