泛素结合酶 UBE2N 在髓系恶性肿瘤中的治疗靶向,Blood

当前位置： X-MOL 学术 › Blood › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

泛素结合酶 UBE2N 在髓系恶性肿瘤中的治疗靶向
Blood ( IF 21.0 ) Pub Date : 2018-11-29 , DOI: 10.1182/blood-2018-99-115359
Laura Barreyro ₁ , Avery Sampson ₁ , Kathleen Hueneman ₁ , Lyndsey C Bolanos ₁ , Mario Pujato ₂ , Somchai Chutipongtanate _{3,

4} , Michael Wyder ₃ , Wendy Haffey ₃ , Kwangmin Choi ₁ , Yaseswini Neelamraju ₅ , Molly A Smith _{1,

3} , Madeline Niederkorn _{1,

3} , Vighnesh Ramesh ₁ , Tomoya Muto ₁ , Eric O'Brien ₆ , Cem Meydan ₇ , Susanne Christie ₁ , Xiaona Liu ₁ , Francine Garret-Bakelman ₈ , Boris Bartholdy ₉ , Britta Will ₉ , Matthew T. Weirauch ₁₀ , Zartash Gul ₁₁ , Stephen Medlin ₁₁ , John Peter Perentesis ₁₂ , Kenneth D Greis ₃ , William Seibel ₆ , Daniel T. Starczynowski _{1,

13}

Affiliation

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Center for Autoimmune Genetics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Department of Cancer Biology, University of Cincinnati, Cincinnati, OH
Mahidol University, Bangkok, Thailand
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA
Division of Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY
Department of Medicine and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA
Cell Biology, Albert Einstein College of Medicine, Bronx, NY
Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Department of Internal Medicine, University of Cincinnati, Cincinnati, OH
Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH

摘要炎症和先天免疫信号通路在白血病干细胞和祖细胞中被激活，并有助于血液系统恶性肿瘤的发病机制，例如骨髓增生异常综合征 (MDS) 和急性髓细胞白血病 (AML)。UBE2N 是一种泛素 (Ub) 结合酶，可在对广泛的先天免疫信号通路的信号转导至关重要的底物上催化赖氨酸 63 (K63) 连接的 Ub 链。在这里，我们报告 UBE2N 通过介导致癌的先天免疫信号传导是白血病细胞功能所必需的，并确定了一种新的化学类小分子抑制剂，其靶向 UBE2N 酶活性。在用两种慢病毒表达的 shRNA 下调 UBE2N 后，MOLM-13 和 THP-1 细胞失去其克隆形成潜力并经历细胞死亡。与 AML 细胞不同，UBE2N 对于健康脐带血 CD34+ 细胞的集落形成和活力是可有可无的。UBE2N 的活性位点在 87 位包含一个半胱氨酸 (Cys)，这对于 Ub 与其底物的结合和转移至关重要。我们进行了基于计算机结构和体外细胞的筛选，以识别与 UBE2N 的活性位点对接并与 Cys-87 共价结合的小分子，作为抑制 Ub 转移至底物的方法。两种结构相关的候选物 UC-764864 和 UC-764865 成为 UBE2N 的抑制剂，因为它们在体外特异性地阻断了 E1-UBE2N 硫酯转移。用 UC-764864 和 UC-764865 治疗 MDS/AML 细胞系和患者来源的原代细胞可抑制先天免疫信号传导并在 MDS/AML 细胞系和原代细胞中诱导细胞毒性作用，同时在体外和体内保留健康的造血细胞。为了确定 UBE2N 抑制的分子基础，我们通过质谱法对泛素化底物的变化进行了全局 Ub 筛选，并通过 RNA-seq 在用载体对照或新衍生的 UBE2N 抑制剂处理的 MOLM-13 细胞中评估了基因表达的变化。用 UC-764864 处理的 MOLM-13 细胞的 RNA-seq 显示，抑制白血病细胞中的 UBE2N 靶向致癌先天免疫途径，包括 NF-kB 和 I 型干扰素信号网络。UC-764864 和 UC-764865 降低了 UBE2N 的泛素化状态，并通过主要减少与 K63 连接的 Ub 修饰来改变参与先天免疫信号传导和 DNA 损伤反应的蛋白质的泛素化。Ub 筛选鉴定的两种底物 DDB1 和 UBE2M 是 CUL4-CRBN E3 连接酶复合物的成分，也是抗白血病治疗来那度胺 (LEN) 的目标。LEN 在 del(5q) MDS 患者中显示出令人鼓舞的结果；然而，其作用仅限于 MDS 或 AML 的其他细胞遗传学亚型。因此，需要鉴定可以在更广泛的患者中增强或扩展 LEN 使用的分子靶点。因此，我们探讨了 LEN 和 UBE2N 抑制剂对 MDS/AML 细胞协同作用的可能性。与单独治疗相比，LEN 和 UC-764864 的组合，UC-764865 或 UBE2N shRNA 在体外显着抑制 MDS/AML 细胞系和患者样本的功能和活力。更引人注目的是，LEN 和 UBE2N 抑制剂的治疗会损害 MDS/AML 细胞，这些细胞对单独的 LEN 或 UBE2N 抑制剂治疗无效。这些发现表明，UBE2N 是将 LEN 的使用扩展到其他 MDS 或 AML 亚型的有希望的目标。总之，我们将 Ub 结合酶 UBE2N 作为 MDS/AML 中的靶标，并确定了靶向 UBE2N 并改变 Ub E3 连接酶功能的新型小分子抑制剂，这对 UBE2N 相关疾病（包括自身炎症和自身免疫性疾病）很重要，和血液系统恶性肿瘤。披露没有需要声明的相关利益冲突。

"点击查看英文标题和摘要"

Therapeutic Targeting of the Ubiquitin Conjugating Enzyme UBE2N in Myeloid Malignancies

Abstract Inflammatory and innate immune signaling pathways are activated in leukemic stem and progenitor cells and contribute to the pathogenesis of hematologic malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). UBE2N is a ubiquitin (Ub) conjugating enzyme that catalyzes lysine 63 (K63)-linked Ub chains on substrates that are critical for signal transduction of broad innate immune signaling pathways. Here we report that UBE2N is required for leukemic cell function by mediating oncogenic innate immune signaling, and identified a novel chemical class of small molecule inhibitors that target UBE2N enzymatic activity. Upon UBE2N downregulation with two lentivirally expressed shRNAs, MOLM-13 and THP-1 cells lose their clonogenic potential and undergo cell death. Unlike for AML cells, UBE2N was dispensable for colony formation and viability of healthy cord blood CD34+ cells. The active site of UBE2N contains a cysteine (Cys) at position 87, which is essential for binding and transfer of Ub to its substrates. We performed in silico structure- and in vitro cell-based screens to identify small molecules that dock to the active site of UBE2N and covalently bind to Cys-87, as an approach to inhibit Ub transfer to substrates. Two structurally-related candidates, UC-764864 and UC-764865, emerged as inhibitors of UBE2N, as they specifically blocked the E1-UBE2N thioester transfer in vitro. Treatment of MDS/AML cell lines and patient-derived primary cells with UC-764864 and UC-764865 suppressed innate immune signaling and induced cytotoxic effects in MDS/AML cell lines and primary cells while sparing healthy hematopoietic cells in vitro and in vivo. To identify the molecular basis of UBE2N inhibition, we performed a global Ub screen for changes in ubiquitinated substrates by mass spectrometry and evaluated changes in gene expression by RNA-seq in MOLM-13 cells treated with vehicle control or the newly derived UBE2N inhibitors. RNA-seq of MOLM-13 cells treated with UC-764864 revealed that inhibition of UBE2N in leukemic cells targets oncogenic innate immune pathways, including NF-kB and Type I interferon signaling networks. UC-764864 and UC-764865 reduced the ubiquitination status of UBE2N, and altered the ubiquitination of proteins involved in innate immune signaling and the DNA damage response by primarily reducing K63-linked Ub modifications. Two substrates identified by the Ub screen, DDB1 and UBE2M, are components of the CUL4-CRBN E3 ligase complex and a target of the anti-leukemic therapy, Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and UBE2N inhibitors on MDS/AML cells. As compared to individual treatments, the combination of LEN and UC-764864, UC-764865 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and UBE2N inhibitors impaired MDS/AML cells that are refractory to treatment of LEN or UBE2N inhibitors alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS or AML. In summary, we implicate the Ub conjugating enzyme UBE2N as a target in MDS/AML, and identified novel small molecule inhibitors that target UBE2N and modify the function of Ub E3 ligases that are important for UBE2N-associated diseases, including autoinflammatory and autoimmune disorders, and hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

更新日期：2018-11-29

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南