AML 相关的间充质基质细胞支持 AML 细胞的生长，这是激活 Notch 信号传导的结果，这可以通过地塞米松靶向,Blood

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AML 相关的间充质基质细胞支持 AML 细胞的生长，这是激活 Notch 信号传导的结果，这可以通过地塞米松靶向
Blood ( IF 21.0 ) Pub Date : 2020-11-05 , DOI: 10.1182/blood-2020-134177
Helal Mohammed Mohammed Ahmed Ahmed ₁ , Yahya Saleh Al-Matary ₂ , Subbaiah Chary Nimmagadda ₃ , Maren Fiori ₄ , Pradeep Kumar Patnana ₃ , Daria Frank ₃ , Lanying Wei ₅ , Judith Schütte ₃ , Tobias May ₆ , Joachim R. Goethert ₄ , Thomas Schroeder ₇ , Ulrike Buttkereit ₄ , Christian Recher ₈ , Manuel Rodríguez-Paredes ₉ , Thorsten Koenig ₁₀ , Wolfgang Hartmann ₁₁ , Jan Tuckermann ₁₂ , Bertram Opalka ₄ , Georg Lenz ₃ , Cyrus Khandanpour ₃

Affiliation

Department of Medicine A, University Hospital Muenster, Münster, Germany
Clinic of Dermatology, University Hospital Essen, Essen, Germany
Department of Medicine A, University Hospital Muenster, Muenster, Germany
University Hospital Essen, Essen, Germany
Institute of Medical Informatics, Muenster University, Muenster, Germany
InSCREENeX GmbH, Brunswick, Germany
University Hospital Duesseldorf, Duesseldorf, DEU
Clinical Hematology, ins, Toulouse, France
German Cancer Research Center, Heidelberg, Germany
Institute for Molecular Tumor Biology, University of Münster, Muenster, Germany
Pathology department, University Hospital Muesnter, Muenster, Germany
Institute for Molecular Endocrinology of Animals, Ulm, Germany

简介：急性髓性白血病（AML）是一种以预后不良为特征的血液系统恶性肿瘤。与骨髓间充质基质细胞 (BM-MSC) 的相互作用支持白血病细胞逃避化疗诱导的细胞凋亡。特别令人感兴趣的是，对小鼠和白血病前期综合征患者的 MSC 进行大规模平行转录组测序，发现炎症信号在诱导基因毒性应激、白血病发生和临床结果方面具有重要意义。最近，地塞米松 (Dex) 是一种抗炎药，在 AML 患者中表现出抗白血病活性并改善了临床结果。这些观察结果激发了我们进一步研究白血病-BM 生态位相关信号并确定支持白血病细胞和 BM-MSC 相互作用的信号级联。方法：诊断时来自 AML 患者的 BM-MSC，单独或与人 AML 细胞系（Kasumi-1 和 M-07e）一起培养，直接接触或通过 transwells 分离。在移植实验之后，通过微阵列分析分析BM-MSC。基因集富集分析 (GSEA) 用于识别在 AML MSC 中过度代表的标志性基因集。使用体外（细胞增殖和凋亡）和体内（白血病发展、药物反应和存活）分析研究了 BM-MSC 和 AML 细胞相互作用的功能特征。结果：为了在功能上评估 BM-MSC 的作用，我们将来自 AML (AML-MSC) 或非白血病 (non-AML-MSC) 患者的 MSC 与 Kasumi-1 或 M-07e 细胞共培养。AML-MSCs 通过改变从 G0/G1 到 S 期的细胞周期分布，显着支持 AML 细胞的增殖（30.5% vs 51.3%，p& lt;0.01)。引人注目的是，当细胞被 transwell 分离时，细胞周期曲线没有改变。一致地，在体内，MLL-AF9 白血病细胞（用 MLL-AF9 癌基因转导的 Lin 细胞）和 AML-MSC 仅在共同移植时死于白血病（40 天对 143 天，p < 0.05）。为了进一步了解白血病细胞诱导的 MSC 变化，我们在从二次移植的白血病（AML-MSC，n=3）和健康（HD-MSC，n=3）小鼠中分选出来的 MSCs 中进行了基因表达谱分析。GSEA 分析确定，与对照相比，AML-MSC 中的标志性缺口信号传导显着过多（p＜0.005，NES：1.7）。可以在 AML-MSC 的 mRNA 和蛋白质水平上验证 Notch-1 和 -2 表达的增加。为了在功能上确定 Notch 激活在 MSC 中的贡献，我们在鼠基质细胞系 MS-5 (Notch-ICN) 中异位表达 Notch 细胞内结构域 (Notch-ICN)。强调 Notch 信号在 AML-MSC 相互作用中的作用，Notch-ICN 表达显着支持 MLL-AF9 白血病细胞和 MSCs 的增殖。Notch 信号传导在调节炎症反应中的作用先前已被报道。Dex 是一种抗炎皮质类固醇，可增强 AML 患者的临床结果。因此，我们研究了是否可以使用 Dex 靶向 Notch 依赖性 AML-MSC 相互作用。有趣的是，Dex 治疗影响了 Notch1 的表达（52%；p<0.005）和白血病细胞的增殖（63%；p<0.001）。最后，我们通过治疗移植了 MLL-AF9 白血病细胞的小鼠来评估 Dex 的治疗效果。有趣的是，与接受赋形剂治疗的白血病小鼠相比，用 Dex 治疗的小鼠表现出显着延长的存活期（73 天对 21 天，p＜0.01）并且具有较低百分比的原始细胞（50%，p＜0.01）和 MSC。因此，我们的数据确定了 Dex 介导的 AML-MSC 相互作用的消除部分是通过影响 Notch 信号传导来介导的。结论：我们的研究表明 AML 和非 AML-MSC 之间存在明显的功能差异，并且直接 AML-MSC 相互作用是影响小鼠细胞增殖、白血病发生和存活的不可或缺的因素。我们进一步确定并证实了 Notch 信号在 AML-MSC 相互作用中的作用。在治疗上，Dex 消除了 AML-MSC 相互作用，降低了 AML 细胞活力和白血病小鼠的存活率。需要进一步研究以更好地了解 MSC 中的异常 Notch 信号传导如何促进 AML 的发展，以及单独使用 Dex 或与 Notch 抑制剂联合使用是否可以避免 AML 患者的化学抗性和复发。披露 Lenz：拜耳：咨询、酬金、研究基金、演讲局；诺华：咨询；贴水：研究经费；罗氏：咨询、酬金、研究经费、发言人局；阿斯利康：咨询、酬金、研究经费；Celgene：顾问、酬金、发言人局；Morphosys：咨询、酬金、研究经费；AQUINOX：研究经费；吉利德：咨询、酬金、研究经费、发言人局；Janssen：顾问、酬金、研究经费、发言人局；Verastem：研究经费；电池管理系统：咨询。Khandanpour：阿斯利康：研究经费；赛诺菲：咨询；安进：咨询；杨森：咨询；新基（Celgene）：咨询。

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AML Associated Mesenchymal Stroma Cells Support Growth of AML Cells As a Result of Activated Notch Signaling and This Can be Targeted By Dexamethasone

Introduction: Acute myeloid leukemia (AML) is a hematological malignancy characterized by poor prognosis. Interaction with bone marrow mesenchymal stromal cells (BM-MSC) supports leukemic cells to evade chemotherapy-induced apoptosis. Of particular interest, massive parallel transcriptome sequencing of MSCs from mice and patients with preleukemic syndromes identified inflammatory signaling in inducing genotoxic stress, leukemogenesis, and clinical outcome. Recently, Dexamethasone (Dex), an anti-inflammatory drug, demonstrated antileukemic activity and improved clinical outcome in AML patients. These observations intrigued us to further investigate leukemia-BM niche-associated signaling and to identify signaling cascades supporting leukemic cells and BM-MSC interaction. Methods: BM-MSC from AML patients at diagnosis, or in remission were cultured alone or together with human AML cell lines (Kasumi-1 and M-07e) in direct contact or separated by transwells. Following transplantation experiments, BM-MSC were analyzed by microarray analysis. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML MSC. The functional characterization of BM-MSC and AML cell interaction was investigated using in vitro (cell proliferation and apoptosis) and in vivo (leukemia development, drug response, and survival) analysis. Results: To functionally evaluate the role of BM-MSC, we co-cultured MSCs from AML (AML-MSC) or non-leukemic (non-AML-MSC) patients with Kasumi-1 or M-07e cells. AML-MSCs significantly supported proliferation of AML cells by altering cell cycle distribution from G0/G1 to S phase (30.5 vs 51.3%, p<0.01). Strikingly, the cell cycle profile was unchanged when the cells were separated by a transwell. Consistently, in vivo,MLL-AF9 leukemic cells (Lin-cells transduced with an MLL-AF9 oncogene) and AML-MSCs succumbed to leukemia only when cotransplanted (40 vs 143 days, p<0.05). To gain further insights into leukemic cell-induced changes in MSC, we performed gene expression profiling in MSCs sorted from secondary transplanted leukemic (AML-MSC, n=3) and healthy (HD-MSC, n=3) mice. GSEA analysis identified hallmark-Notch signaling to be significantly overrepresented in AML-MSC as compared to controls (p<0.005, NES: 1.7). Increased Notch-1 and -2 expression could be verified at both the mRNA and protein levels in AML-MSCs. To functionally determine the contribution of Notch activation in MSC, we ectopically expressed the Notch intracellular domain (Notch-ICN) in a murine stromal cell line, MS-5 (Notch-ICN). Underscoring a role for Notch signaling in AML-MSC interaction, Notch-ICN expression significantly supported the proliferation of both MLL-AF9 leukemic cells and MSCs. A role for Notch signaling in the modulation of the inflammatory response has been previously reported. Dex an anti-inflammatory corticosteroid enhanced clinical outcome in AML patients. We, therefore, investigated whether Notch-dependent AML-MSC interaction could be therapeutically targeted using Dex. Interestingly, Dex treatment affected Notch1 expression (52%; p<0.005) and proliferation of leukemia cells (63%; p<0.001). Finally, we evaluated the therapeutic efficacy of Dex by treating mice transplanted with MLL-AF9 leukemic cells. Interestingly, mice treated with Dex exhibited significantly prolonged survival (73 vs 21 days, p<0.01) and had a lower percentage of blasts (50%, p<0.01) and MSC compared to leukemic mice receiving vehicle treatment. Our data thus identified Dex mediated abrogation of AML-MSC interaction is in part mediated by affecting Notch signaling. Conclusion: Our study demonstrated distinct functional differences between AML and non-AML-MSC and direct AML-MSC interaction as an indispensable factor in influencing cell proliferation, leukemogenesis, and survival in mice. We further identified and confirmed the role of Notch signaling in AML-MSC interaction. Therapeutically, Dex abrogated AML-MSC interaction, decreasing AML cell viability, and leukemic mice survival. Further studies are warranted to better understand how aberrant Notch signaling in MSC contributes to AML development and whether the use of Dex alone or in combination with Notch inhibitors could circumvent chemoresistance and relapse in AML patients. Disclosures Lenz: Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy; Agios: Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria, Research Funding; AQUINOX: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; BMS: Consultancy. Khandanpour:Astra Zeneca: Research Funding; Sanofi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy.

更新日期：2020-11-05

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