Sf3b1-K700E 的表达加速了 Del(13q) 小鼠模型中慢性淋巴细胞白血病的发展,Blood

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Sf3b1-K700E 的表达加速了 Del(13q) 小鼠模型中慢性淋巴细胞白血病的发展
Blood ( IF 21.0 ) Pub Date : 2020-11-05 , DOI: 10.1182/blood-2020-139096
Bo Zhang ₁ , Prajish Iyer ₂ , Meiling Jin ₃ , Elisa Ten Hacken ₄ , Zachary Cartun ₄ , Kevyn Hart ₃ , Laura Z. Rassenti ₅ , Thomas J. Kipps ₆ , Donna S. Neuberg ₇ , Ruben D. Carrasco ₈ , Catherine J. Wu ₉ , Lili Wang ₃

Affiliation

RNA 剪接因子 SF3B1 是慢性淋巴细胞白血病 (CLL) 中最常发生突变的基因之一，但仅在鼠 B 细胞中表达这种突变不会导致 CLL。这种基因突变通常是亚克隆的，并且与较差的存活率有关。这种突变如何影响 CLL 进展仍然难以捉摸。由于 SF3B1 突变经常与染色体 13q 缺失 (del(13q)) 共同发生，并且缺失 del(13q) 的最小删除区 (MDR) 的小鼠发展为惰性 CLL，因此我们询问 Sf3b1 突变的共表达是否可以在这种小鼠模型中加速 CLL 的发作。如果是这样，Sf3b1 突变如何在机制上促成 CLL。为此，我们首先将携带条件性敲除等位基因 Sf3b1-K700E 的小鼠和条件性敲除 MDR 的小鼠进行杂交。然后，我们用 CD19-Cre 小鼠培育后代，以产生具有（DM）或不具有（MDR-MT）杂合 Sf3b1-K700E 的 MDR 的 B 细胞特异性纯合缺失的小鼠群组。我们通过流式细胞术追踪外周血中的循环 B220+CD5+ CLL 样细胞来监测 CLL 的发作，从 6 个月大开始到 24 个月大结束。通过流式细胞术和免疫组织化学证实，我们在 24%（25 只中的 6 只）的 DM 和 7.4%（27 只中的 2 只）的脾脏、骨髓和淋巴结中检测到 CLL 样疾病，并且在脾脏、骨髓和淋巴结中存在疾病。DM 小鼠中 CLL 频率增加表明 Sf3b1-K700E 可以加速 CLL（Pearson Chi-Square 2-sided，p=0.098）。为了阐明 Sf3b1 突变如何导致 CLL 外显率增加，我们对来自患有和不患有 CLL 的 DM 小鼠的脾 B 细胞进行了整合的 RNA 测序 (RNA-seq) 和 TMT 蛋白质组学分析。我们发现，当我们将 DM-CLL 细胞与其 DM B 细胞对应物进行比较时，参与 MYC、细胞周期检查点和 mTORC1 途径的基因在 RNA 和蛋白质水平上都被上调和富集，表明这些细胞过程与 CLL 的发病有关. 为了进一步确定 Sf3b1-K700E 介导的可变剪接在激活这些途径中的作用，我们首先通过对 RNA-seq 数据的计算分析确定了候选剪接异构体（nfatc1、braf、depdc5、tsc2），然后在独立的样本队列（n = 3，）。关于这些亚型究竟如何影响 CLL 的功能注释正在进行中。重要的，与正常 B 细胞相比，我们还观察到具有 SF3B1 突变和 del(13q) 的人类 CLL 细胞中 mTORC1 通路的基因上调。我们接下来询问 DM CLL 细胞是否对体外 mTORC1 通路的抑制和 RNA 剪接抑制敏感。我们将 DM B 和 CLL 细胞单独或联合使用 Temsirolimus（Tem，mTORC1 抑制剂）或 H3B8800（H3B，SF3B1 抑制剂）24 小时，然后用 CellTiter-Glo 测定法测量细胞活力。与 DMSO 对照相比，Tem 和 H3B 单一处理均显着抑制 DM CLL 细胞的存活，但不抑制 DM B 细胞的存活（所有组与对照，非配对 t 检验，p＜0.01）。此外，当 1nM 的 H3B 与 Tem 处理组合时，在 DM CLL 细胞中观察到了累加效应（IC50：1.2nM 对 135.2uM，未配对 t 检验 p＜0.001）。然后，我们使用移植有 DM CLL 细胞的 NSG 小鼠在体内测试了这两种药物的作用。与单一治疗组或无药物治疗组（所有组与梳，p≤0.001）。此外，与对照相比，联合治疗增加了移植有 CLL 细胞的 NSG 小鼠的存活率（中位存活率：对照对梳状细胞 15 对 34 天，对数秩 p＜0.001）。重要的是，当我们将具有 del(13q) 和 sf3b1 突变（DM-CLL，n=3）或单独使用 del(13q)（n=2）或正常 B 细胞（n=4）的人类 CLL 细胞暴露于在体外联合治疗中，DM-CLL 细胞对治疗表现出最高的敏感性（DM-CLL 对比所有组，p<0.05），表明 SF3B1 突变可能通过调节 RNA 剪接和 mTORC1 途径加速具有 del(13q) 的 CLL。我们的研究表明，Sf3b1-K700E 的表达可以通过选择性 RNA 剪接和 mTORC1 激活加速基于 MDR 缺失小鼠模型的 CLL 的发展。这一发现支持在具有 SF3B1 突变和 del(13q) 的 CLL 患者亚群中使用 mTORC1 抑制剂和 RNA 剪接抑制剂。披露 Kipps：吉利德：实体董事会或咨询委员会、发言人局的成员；基因泰克/罗氏：成为实体董事会或咨询委员会、发言人局的成员；Celgene：酬金，研究经费；Ascerta/AstraZeneca、Celgene、Genentech/F. 霍夫曼-拉罗氏，吉利德，Janssen、Loxo Oncology、Octernal Therapeutics、Pharmacyclics/AbbVie、TG Therapeutics、VelosBio 和 Verastem：成为实体董事会或咨询委员会的成员；Pharmacyclics/ AbbVie、乳腺癌研究基金会、MD Anderson 癌症中心、Oncternal Therapeutics, Inc.、专业研究中心 (SCOR) - 白血病和淋巴瘤协会 (LLS)、加州再生医学研究所 (CIRM)：实体的会员资格董事会或咨询委员会、研究基金、发言人局；VelosBio：研究经费；Oncternal Therapeutics, Inc.：其他：Cirmtuzumab 由 Thomas J. Kipps 在 Thomas J. Kipps 实验室开发，并由加利福尼亚大学授权给 Oncternal Therapeutics, Inc.，后者为 Thomas J. Kipps 提供股票期权和研究资金实验室，研究经费。纽伯格：Celgene：研究经费；Madrigak Pharmaceuticals：上市公司的现任股东；药环学：研究经费。吴：BionTech：现任上市公司股东；药环学：研究经费。

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Expression of Sf3b1-K700E accelerates the Development of Chronic Lymphocytic Leukemia in a Del(13q) Murine Model

RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL), but expression of this mutation alone in murine B cells does not result in CLL. This gene mutation is often subclonal and associated with poor survival. How this mutation impacts CLL progression remains elusive. Since SF3B1 mutation frequently co-occurs with chromosome 13q deletion (del(13q)), and mice with deletion of the Minimal Deleted Region (MDR) of del(13q) develop indolent CLL, we therefore asked whether co-expression of Sf3b1 mutation can accelerate the onset of CLL in this murine model. If so, how does Sf3b1 mutation mechanistically contribute to CLL. To this end, we first crossed mice carrying conditional knock in allele Sf3b1-K700E and mice with conditional knockout of MDR. We then bred the offspring with CD19-Cre mice to generate cohorts of mice which have B cell-specific homozygous deletion of MDR with (DM) or without (MDR-MT) heterozygous Sf3b1-K700E. We monitored the onset of CLL by tracking of circulating B220+CD5+ CLL-like cells from peripheral blood with flow cytometry, starting at the age of 6-months and ending by 24-months. We detected CLL-like disease in 24% (6 of 25) of DM and 7.4% (2 of 27) of MDR-MT mice with disease presence in the spleen, bone marrow and lymph node, confirmed by flow cytometry and immunohistochemistry. The increased frequency of CLL in DM mice indicated that Sf3b1-K700E could accelerate CLL (Pearson Chi-Square 2-sided, p=0.098). To elucidate how Sf3b1 mutation contributes to increased CLL penetrance, we performed integrated RNA sequencing (RNA-seq) and TMT proteomics analysis with splenic B cells derived from DM mice with and without CLL. We found that genes involved in MYC, cell cycle checkpoints and mTORC1 pathways are upregulated and enriched at both the RNA and protein levels when we compared DM-CLL cells to their DM B cell counterparts, indicating these cellular processes are involved in the onset of CLL. To further define the role of Sf3b1-K700E mediated alternative splicing in the activation of these pathways, we first identified candidate splicing isoforms (nfatc1, braf, depdc5, tsc2) through computational analysis of RNA-seq data and then validated the isoforms in an independent cohort of samples (n=3,). Functional annotation of how exactly these isoforms impact CLL is ongoing. Importantly, we also observed gene upregulation of mTORC1 pathway in human CLL cells with SF3B1 mutation and del(13q) when compared with normal B cells. We next asked whether DM CLL cells are sensitive to inhibition of mTORC1 pathway and RNA splicing inhibition in vitro. We exposed DM B and CLL cells to either Temsirolimus (Tem, mTORC1 inhibitor), or H3B8800 (H3B, SF3B1 inhibitor) alone or in combination for 24 hours and then measured the cell viability with CellTiter-Glo assay. When compared to DMSO control, both Tem and H3B single treatments significantly inhibited the survival of DM CLL cells, but not DM B cells (all groups vs control, unpaired t test, p<0.01). Furthermore, an additive effect was observed in DM CLL cells when 1nM of H3B was combined with Tem treatment (IC50: 1.2nM vs. 135.2uM, unpaired t test p<0.001). We then tested the effects of both drugs in vivo using NSG mice engrafted with DM CLL cells. Mice treated with combination of Tem (15mg/kg, i.p, 5 days) and H3B (4mg/kg, gavage, 5 days) had a lower CLL burden in peripheral blood in comparison to either the single treatment or no drug treatment group (all groups vs. comb, p≤0.001). Furthermore, the combination treatment increased the survival of NSG mice engrafted with CLL cells compared to control (median survival: control vs. comb 15 vs. 34 days, log rank p<0.001). Importantly, when we exposed human CLL cells with both del(13q) and sf3b1 mutation (DM-CLL, n=3), or with del(13q) alone (n=2), or normal B cells (n=4) to the combination treatment in vitro, DM-CLL cells showed the highest sensitivity to the treatment (DM-CLL vs. all groups, p<0.05), suggesting that SF3B1 mutation may accelerate CLL with del(13q) through modulating RNA splicing and mTORC1 pathway. Our study demonstrates that expression of Sf3b1-K700E could accelerate the development of CLL based on MDR deleted murine model through alternative RNA splicing and mTORC1 activation. This finding supports the use of an mTORC1 inhibitor together with RNA splicing inhibitor in the subset of CLL patients with both SF3B1 mutation and del(13q). Disclosures Kipps: Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding. Neuberg:Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

更新日期：2020-11-05

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