Lyn 激酶有助于成纤维细胞的重编程，促进慢性淋巴细胞白血病的进展,Blood

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Lyn 激酶有助于成纤维细胞的重编程，促进慢性淋巴细胞白血病的进展
Blood ( IF 21.0 ) Pub Date : 2019-11-13 , DOI: 10.1182/blood-2019-129299
Alexander Frederik Vom Stein ₁ , Maximilian Koch ₁ , Sebastian Reinarzt ₁ , Anna Lucas ₁ , Rocio Rebollido-Rios ₁ , Thomas Oellerich ₂ , Phuong Hien Nguyen ₁ , Michael Hallek ₃

Affiliation

慢性淋巴细胞白血病细胞的生长强烈依赖于滋养的微环境生态位，该生态位由多种双向相互作用特异性引发，以促进白血病归巢、增殖和进展。Src 家族激酶 Lyn 先前被我们的小组鉴定为这种促白血病生态位的形成和 CLL 细胞扩增的关键因素，使用 Eμ-TCL1 小鼠模型。为了将 Lyn 的促白血病功能归因于特定的细胞类型，通过致死照射 BL6J 小鼠并用 Lyn-WT 恢复其造血系统，在造血或非造血隔室中产生具有 Lyn 谱系特异性缺陷的嵌合小鼠或 Lyn-KO 干细胞。连续用TCL1+-恶性细胞异种移植小鼠。非造血室内的 Lyn 缺乏比造血室内的 Lyn 缺乏更能减缓白血病的扩张。与两种谱系特异性 Lyn 缺陷小鼠品系相比，完全 Lyn 缺陷小鼠表现出更显着的白血病扩增阻滞，这表明两种不同隔室对白血病扩增的累加效应。在关注非造血成纤维细胞旁观者细胞时，将原代人 CLL 细胞与 Lyn 缺陷小鼠胚胎成纤维细胞以及 Lyn-KO 人 HS5 细胞体外共培养，通过 CRISPR-Cas9 系统生成，并评估白血病细胞存活率随着时间的推移。与 WT 基质相比，所有 Lyn 缺陷的成纤维细胞对 CLL 细胞的摄食能力显着降低，表明 Lyn 在白血病相关成纤维细胞中的功能相关性。随后，对 HS5 细胞中与 Lyn-KO 相关的转录组、蛋白质组和磷酸化蛋白质组的改变进行了全面分析，揭示了基因和蛋白质表达模式的惊人广泛变化，这些变化似乎主要在转录水平受到调节。差异表达的基因通常与细胞外基质 (ECM)、细胞骨架或细胞因子相关。GO-term 富集分析还表明与 ECM 过程存在相关性。因此，我们假设 Lyn 缺乏可能诱导癌症相关成纤维细胞 (CAF) 样表型的转录变化，从而导致白血病喂养能力降低。在 Lyn-KO 成纤维细胞中验证了与这种降低的激活状态一致的几种 CAF 制造商的表达减少，以及所选靶基因的转录调节差异表达。其中，去泛素化酶 UCHL1 在 Lyn-KO HS5 细胞中减少最多，显示 mRNA 和蛋白质表达几乎完全丧失。将特定的 UCHL1 抑制剂（以对 CLL 细胞没有毒性影响的剂量）应用于 CLL 基质共培养导致显着阻碍饲养层效应并降低 CLL 细胞存活，这意味着微环境 UCHL1 与我们系统中的基质支持具有功能相关性。此外，在 Lyn-KO 基质中完全禁止由 WT 细胞中较高药物浓度诱导的基质细胞死亡，说明 Lyn 对调节 UCHL1 表达和功能的重要性。总之，我们提出 Lyn 激酶通过将基质成纤维细胞转录重编程为“CAF 样”表型，从而促进支持性微环境的形成，从而提高 CLL 细胞的活力。此外，UCHL1 可能是该激活过程的潜在药物介质。披露 Hallek：罗氏、吉利德科学公司、萌蒂制药、杨森、新基（Celgene）、Pharmacyclics、艾伯维（AbbVie）：酬金、研究基金、发言人局。我们提出 Lyn 激酶通过将基质成纤维细胞转录重编程为“CAF 样”表型，从而促进支持性微环境的形成，从而提高 CLL 细胞的活力。此外，UCHL1 可能是该激活过程的潜在药物介质。披露 Hallek：罗氏、吉利德科学公司、萌蒂制药、杨森、新基（Celgene）、Pharmacyclics、艾伯维（AbbVie）：酬金、研究基金、发言人局。我们提出 Lyn 激酶通过将基质成纤维细胞转录重编程为“CAF 样”表型，从而促进支持性微环境的形成，从而提高 CLL 细胞的活力。此外，UCHL1 可能是该激活过程的潜在药物介质。披露 Hallek：罗氏、吉利德科学公司、萌蒂制药、杨森、新基（Celgene）、Pharmacyclics、艾伯维（AbbVie）：酬金、研究基金、发言人局。

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Lyn Kinase Contributes to the Reprogramming of Fibroblasts Promoting Chronic Lymphocytic Leukemia Progression

Growth of chronic lymphocytic leukemia cells strongly depends on a nurturing microenvironmental niche that is specifically primed by diverse, bi-directional interactions to promote leukemic homing, proliferation and progression. The Src-family kinase Lyn was previously identified by our group as a key factor for the formation of this pro-leukemic niche and for the expansion of CLL cells, using the Eµ-TCL1 mouse model. In order to attribute the pro-leukemic function of Lyn to a specific cell type, chimeric mice with lineage-specific defects of Lyn within hematopoietic or non-hematopoietic compartments were generated by irradiating BL6J-mice lethally and restoring their hematopoietic system with Lyn-WT or Lyn-KO stem cells. Consecutively mice were xenotransplanted with TCL1+-malignant cells. Lyn deficiency within the non-hematopoietic compartment decelerated leukemic expansion to a higher degree than did Lyn deficiency within the hematopoietic compartment. Completely Lyn deficient mice showed a more prominent retardation of leukemic expansion compared with both lineage specific Lyn deficient mouse strains, suggesting an additive effect of the two distinct compartments for leukemic expansion. In focusing on the non-hematopoietic fibroblastic bystander cells, primary human CLL cells were cocultured in vitro with Lyn-deficient mouse embryonic fibroblasts as well as Lyn-KO human HS5 cells, generated via the CRISPR-Cas9 system, and leukemic cell survival was assessed over time. All Lyn-deficient fibroblasts showed a significantly reduced feeding capacity for CLL cells compared to WT stroma, indicating the functional relevance of Lyn in leukemia-associated fibroblasts. Subsequently, transcriptomic, proteomic and phosphoproteomic alterations related to Lyn-KO in HS5 cells were comprehensively analyzed, revealing a surprisingly extensive change in gene and protein expression pattern that appeared to be regulated mainly at the transcriptional level. The differentially expressed genes were remarkably often extracellular matrix (ECM)-, cytoskeleton- or cytokine-associated. GO-term enrichment analysis additionally suggested a correlation with ECM processes. Therefore, we hypothesized that Lyn-deficiency might induce transcriptional changes of the cancer-associated fibroblast (CAF)-like phenotype, thus leading to a reduction in leukemic feeding capacity. The diminished expression of several CAF-makers congruent with this reduced activation status was validated in Lyn-KO fibroblasts, as well as the transcriptionally regulated differential expression of chosen target genes. Amongst those, the deubiquitinating enzyme UCHL1 was most abundantly reduced in Lyn-KO HS5 cells, showing an almost complete loss of mRNA and protein expression. Application of a specific UCHL1-inhibitor -in a dose without toxic effects on CLL cells - to CLL-stroma coculture resulted in a significantly hampered feeder effect and reduced CLL cell survival, implying a functional relevance of microenvironmental UCHL1 for stromal support in our system. Additionally, stroma cell death induced by higher drug concentrations in WT cells was completely prohibited in Lyn-KO stroma, illustrating the importance of Lyn for regulating UCHL1 expression and function. In summary, we propose that the Lyn kinase contributes to the formation of a supportive microenvironment via the transcriptional reprogramming of stroma fibroblasts into a "CAF-like" phenotype, which echances viability of CLL cells. In addition, UCHL1 might be a potentially druggable mediator of this activation process. Disclosures Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.

更新日期：2019-11-13

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