RNA interference-based screens for molecular targets in cancer

基于 RNA 干扰的癌症分子靶点筛选

• 批准号: 8552931
• 负责人: Louis Staudt
• 金额: $ 101.37万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552931
• 关键词: 1-Phosphatidylinositol 3-Kinase; 9p24; Affect; Antigen Receptors; Antigens; Apoptotic; B-Cell Lymphomas; B-Lymphocytes; BCL10 gene; Bar Codes; Base Pairing; Binding; Burkitt Lymphoma; Cancer Biology; Cell Cycle Progression; Cell Line; Cell Nucleus; Cell membrane; Cells; Chromatin Structure; Chromosomes; Chronic; Code; Complex; DNA Resequencing; Dasatinib; Dependence; Dependency; Drug Delivery Systems; Epigenetic Process; Essential Genes; Family; Feedback; Gene Expression; Genes; Genetic; Genetic Screening; Genomics; Heterochromatin; Histone H3; Histones; Hodgkin Disease; Homeostasis; Human Cell Line; IRAK1 gene; IRF4 gene; ITAM; Immune; Interleukin-13; JAK2 gene; LYN gene; Laboratories; Libraries; Lymphocyte; Lymphocyte Activation; Lymphoid; Lymphoma; Malignant Neoplasms; Malignant lymphoid neoplasm; Mass Spectrum Analysis; Mediastinal; Mediating; Methodology; Modeling; Molecular Abnormality; Molecular Target; Monitor; Multiple Myeloma; Mutate; Mutation; NF-kappa B; National Institute of Allergy and Infectious Disease; Oncogenes; Oncogenic; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Population; Protein Isoforms; Protein Kinase; Protein Tyrosine Kinase; Proteins; RNA; RNA Interference; Receptor Activation; Receptor Signaling; Receptors, Antigen, B-Cell; Recurrence; Regulation; Regulatory Pathway; Retroviral Vector; Role; SYK gene; Screening procedure; Serine; Signal Pathway; Signal Transduction; Stimulus; Structure of germinal center of lymph node; System; T-Cell Receptor; T-Lymphocyte; TCF3 gene; Tail; Taxonomy; Therapeutic; Toll-like receptors; Toxic effect; Tyrosine; Work; Xenograft procedure; adapter protein; addiction; autocrine; base; cancer cell; cancer type; casein kinase; chromatin modification; cyclin D3; design; genetic regulatory protein; heterochromatin-specific nonhistone chromosomal protein HP-1; killings; kinase inhibitor; large cell Diffuse non-Hodgkin' s lymphoma; loss of function; mutant; new therapeutic target; novel; prevent; reconstitution; research study; small hairpin RNA; success; synergism; transcription factor; vector

The Staudt laboratory has conducted RNA interference genetic screens for genes required for the proliferation and/or survival of human cell lines representing various subtypes of lymphoma and multiple myeloma. In diffuse large B cell lymphoma (DLBCL), previous work in the Staudt laboratory demonstrated that the anti-apoptotic NF-kB pathway is constitutively active in the activated B cell-like (ABC) subtypes of DLBCL but not the germinal center B cell-like (GCB) subtype of DLBCL, but the mechanisms underlying this abnormal signaling were enigmatic. The laboratory therefore conducted an RNAi screen in ABC and GCB DLBCL cell lines, searching for shRNAs that were selectively toxic for ABC DLBCL cells. This effort revealed that a signaling complex comprised of CARD11, MALT1, and BCL10 is required for the survival of ABC but not GCB DLBCL cell lines. In normal lymphocytes, this CARD11 complex engages the NF-kB pathway during antigen receptor signaling. The Staudt laboratory demonstrated that this signaling complex is responsible for the constitutive activation of the NF-kB pathway in ABC DLBCLs.In a recent RNA interference screen we identified several shRNAs targeting casein kinase 1A1 (CK1alpha) that were toxic for ABC but not GCB DLBCL cell lines. Since this phenotype resembled the toxicity profile of CARD11 shRNAs, we suspected that CK1alpha might be a new component of the CBM signaling pathway. Indeed, CK1alpha knockdown decreased IKK kinase activity and NF-kB target gene expression, and CK1alpha colocalized with CARD11 in ABC DLBCL cell lines. Fortuitously, Mike Lenardo's laboratory in NIAID separately identified CK1alpha as a binding partner of CARD11 in a mass spectrometry-based screen. Collaborative experiments revealed the essential role of CK1alpha in T cell receptor activation of the NF-kB pathway. Biochemically, CK1alpha was required for the recruitment of IKK to the CBM complex. Interestingly, T cells reconstituted with a CK1alpha mutant that was defective in kinase activity provided a greater NF-kB stimulus than did wild type CK1alpha, demonstrating that CK1alpha has both positive and negative influences on the CBM pathway. The negative effect of CK1alpha was due to its phosphorylation of CARD11 on serines in its "linker" domain, causing CARD11 to be destabilized. This negative feedback loop involving CK1alpha was analogous to the negative regulation of the CBM complex caused by inhibitory phosphorylation of BCL10 by IKK. CK1alpha emerged from this study as a new component of the CBM, demonstrating the power of unbiased RNA interference screens to uncover overlooked aspects of cellular signaling.A recent RNAi screen uncovered a crucial dependency of multiple myeloma cells on IRF4, a lymphoid-restricted transcriptional factor that is required for both lymphocyte activation and for plasmacytic differentiation. IRF4 knockdown by RNAi was toxic to 10 different myeloma cell lines representing many of the known genetic subtypes of this cancer. Of note, IRF4 is not translocated, amplified or mutated in most cases of multiple myeloma, and thus the dependency of myeloma cells on IRF4 exemplifies a new concept in cancer biology known as non-oncogene addiction. These results establish IRF4 as an important new therapeutic target in this lethal cancer.Most recently, our Achilles heel screens allowed us to define a chronic active form of B cell receptor (BCR) signaling that activates NF-kB in ABC DLBCLs with wild type CARD11. Such ABC DLBCLs die upon knockdown of BCR signaling components, including subunits of the B cell receptor itself. ABC DLBCLs have prominent clusters of the BCR in the plasma membrane, similar to antigen-stimulated normal B cells. Cancer gene resequencing revealed that over one fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR. The most common mutations, present in 18% of ABC DLBCLs, involved a single tyrosine of the BCR signaling subunit, CD79B. These mutations affect the critical ITAM signaling motif, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. Importantly, the BCR pathway offers a wealth of targets that can be exploited therapeutically, including several protein kinases (SRC-family kinases, SYK, BTK, PKCbeta) as well as PI(3) kinase. Dasatinib, a clinically available kinase inhibitor that targets BTK and SRC-family kinases, kills ABC DLBCL cells by blocking their chronic active BCR signaling.An important new initiative in the past year was the construction of a new RNAi library that targets each gene with 12 shRNAs. This approach allows us to identify effective shRNAs that do not have appreciable off-target effects. This library targets all protein kinases, PI(3) kinase pathway component, and other regulatory proteins relevant to lymphoid malignancies. Using this new library we have embarked on an ambitious project to screen more than 50 cell line models of various lymphoid malignancies, searching for pathways that are selectively required for the proliferation and survival of particular cancer subtypes.A recent success was the identification of the MYD88 signaling pathway as essential for the survival of ABC DLBCL cells. MYD88 is a key adapter protein in the signaling pathway downstream of Toll-like receptors in innate immune cells. The RNAi screen identified shRNAs targeting MYD88 and its assocatiated kinase IRAK1 as toxic for ABC DLBCL cells but not for cell line models of other lymphoma subtypes. This led us to discover recurrent mutations in MYD88 that create mutant isoforms that spontaneously activate the NF-kB pathway and are oncogenic.We investigated a recurrent amplicon in primary mediastinal large B cell lymphoma (PMBL) and Hodgkin lymphoma on chromosome 9p24 using RNAi screens. We uncovered three essential genes using an RNAi screen: JAK2, JMJD2C, and RANBP6. We showed that the kinase activity of JAK2 is activated in these lymphomas by autocrine IL-13 signaling. Surprisingly, JAK2 cooperated with JMJD2C in promoting survival of these lymphoma cells. JMJD2C is a histone H3K9 demethylase, which activates gene expression by removing this histone mark, thereby preventing the recruitment of the heterochromatin protein HP-1 alpha. We traced the synergism between JAK2 and JMJD2C to cooperative epigenetic modification of chromatin. JAK2 acts in the nucleus of these lymphoma cells to phosphorylate the histone H3 tail on tyrosine 41, which also blocks recruitment of HP-1 alpha. A major target of epigenetic modication by JAK2 and JMJD2C is MYC, but in addition, these two proteins modify the chromatin structure of several hundred protein-coding genes. Importantly, drugs that target JAK2 kill these lymphoma cells.RNA interference screening has proven highly successful in unraveling the key survival pathways in Burkitt lymphoma. We discovered that knockdown of the transcription factor TCF3 was lethal to Burkitt lymphoma cell lines, but not to cell line models of other aggressive lymphomas. By RNA resequencing, we found that TCF3 and its negative regulator are mutated in 70% of cases of sporadic Burkitt lymphoma, leading to TCF3 dependency. Our RNA interference screens also demonstrated that about two thirds of Burkitt lymphoma cell lines depend upon tonic signaling from the B cell receptor (BCR). Further, we discovered that TCF3 amplifies this tonic BCR signaling. Finally, we discovered that Burkitt lymphoma cell lines require cyclin D3/CDK6 for cell cycle progression. RNA resequencing revealed oncogenic activating mutations in cyclin D3 in 38% of cases. Surprisingly, pharmacologic inhibition of CDK6 causes apoptotis of Burkitt lymphoma cell lines and induced regression of established Burkitt lymphoma xenografts.

Staudt实验室对代表淋巴瘤和多发性骨髓瘤各种亚型的人类细胞系增殖和/或存活所需的基因进行了RNA干扰遗传筛选。 在弥漫性大 B 细胞淋巴瘤 (DLBCL) 中，Staudt 实验室之前的工作表明，抗凋亡 NF-kB 通路在 DLBCL 的活化 B 细胞样 (ABC) 亚型中具有组成性活性，但在生发中心 B 细胞中则没有。类似 DLBCL 的 (GCB) 亚型，但这种异常信号传导背后的机制是神秘的。 因此，该实验室在 ABC 和 GCB DLBCL 细胞系中进行了 RNAi 筛选，寻找对 ABC DLBCL 细胞具有选择性毒性的 shRNA。 这项工作表明，ABC 细胞系的存活需要由 CARD11、MALT1 和 BCL10 组成的信号复合物，但 GCB DLBCL 细胞系则不需要。 在正常淋巴细胞中，这种 CARD11 复合物在抗原受体信号传导过程中参与 NF-kB 通路。 Staudt 实验室证明，该信号复合物负责 ABC DLBCL 中 NF-kB 通路的组成性激活。在最近的 RNA 干扰筛选中，我们发现了几种靶向酪蛋白激酶 1A1 (CK1alpha) 的 shRNA，它们对 ABC 有毒，但对 GCB DLBCL 没有毒性。细胞系。 由于这种表型与 CARD11 shRNA 的毒性特征相似，我们怀疑 CK1α 可能是 CBM 信号通路的新组成部分。 事实上，CK1α 敲低降低了 IKK 激酶活性和 NF-kB 靶基因表达，并且 CK1α 在 ABC DLBCL 细胞系中与 CARD11 共定位。 幸运的是，NIAID 的 Mike Lenardo 实验室在基于质谱的筛选中单独将 CK1alpha 鉴定为 CARD11 的结合伴侣。 合作实验揭示了 CK1α 在 NF-kB 通路 T 细胞受体激活中的重要作用。 从生化角度来看，CK1α 是 IKK 募集至 CBM 复合体所必需的。 有趣的是，用激酶活性缺陷的 CK1α 突变体重建的 T 细胞比野生型 CK1α 提供了更大的 NF-kB 刺激，这表明 CK1α 对 CBM 途径既有积极影响，也有消极影响。 CK1alpha 的负面影响是由于它对 CARD11 的“连接器”结构域中的丝氨酸进行磷酸化，导致 CARD11 不稳定。 这种涉及 CK1α 的负反馈回路类似于 IKK 对 BCL10 的抑制性磷酸化引起的 CBM 复合物的负调节。 这项研究中出现的 CK1alpha 作为 CBM 的新组成部分，证明了无偏 RNA 干扰筛选能够发现细胞信号传导中被忽视的方面。最近的一项 RNAi 筛选发现了多发性骨髓瘤细胞对 IRF4（一种淋巴限制性转录因子）的关键依赖性这是淋巴细胞激活和浆细胞分化所必需的。 RNAi 敲低 IRF4 对 10 种不同的骨髓瘤细胞系具有毒性，这些细胞系代表了这种癌症的许多已知遗传亚型。 值得注意的是，IRF4 在大多数多发性骨髓瘤病例中不会发生易位、扩增或突变，因此骨髓瘤细胞对 IRF4 的依赖性体现了癌症生物学中称为非癌基因成瘾的新概念。 这些结果使 IRF4 成为这种致命癌症的重要新治疗靶点。最近，我们的致命弱点筛选使我们能够定义 B 细胞受体 (BCR) 信号传导的慢性活性形式，该信号传导具有野生型 CARD11 的 ABC DLBCL 中的 NF-kB 。 这种 ABC DLBCL 在 BCR 信号传导成分（包括 B 细胞受体本身的亚基）被敲除后就会死亡。 ABC DLBCL 的质膜上有明显的 BCR 簇，类似于抗原刺激的正常 B 细胞。 癌症基因重测序显示，超过五分之一的 ABC DLBCL 在 BCR 的 CD79B 或 CD79A 亚基中存在突变。 最常见的突变存在于 18% 的 ABC DLBCL 中，涉及 BCR 信号亚基 CD79B 的单个酪氨酸。 这些突变影响关键的 ITAM 信号基序，产生避免 LYN 酪氨酸激酶负向自动调节的 BCR。 重要的是，BCR 途径提供了大量可用于治疗的靶点，包括多种蛋白激酶（SRC 家族激酶、SYK、BTK、PKCbeta）以及 PI(3) 激酶。 达沙替尼是一种临床可用的激酶抑制剂，针对 BTK 和 SRC 家族激酶，通过阻断 ABC DLBCL 细胞的慢性活性 BCR 信号传导来杀死它们。过去一年的一项重要新举措是构建了一个新的 RNAi 文库，该文库针对每个基因具有 12 shRNA。 这种方法使我们能够识别没有明显脱靶效应的有效 shRNA。 该库针对所有蛋白激酶、PI(3) 激酶通路成分以及与淋巴恶性肿瘤相关的其他调节蛋白。 利用这个新的库，我们开始了一个雄心勃勃的项目，筛选 50 多种不同淋巴恶性肿瘤的细胞系模型，寻找特定癌症亚型的增殖和生存选择性所需的途径。最近的成功是鉴定了 MYD88信号通路对于 ABC DLBCL 细胞的生存至关重要。 MYD88 是先天免疫细胞中 Toll 样受体信号通路下游的关键衔接蛋白。 RNAi 筛选发现，针对 MYD88 及其相关激酶 IRAK1 的 shRNA 对 ABC DLBCL 细胞有毒，但对其他淋巴瘤亚型的细胞系模型无毒性。 这使我们发现了 MYD88 中的反复突变，这些突变产生了自发激活 NF-kB 通路并具有致癌性的突变亚型。我们使用 RNAi 筛选研究了原发性纵隔大 B 细胞淋巴瘤 (PMBL) 和霍奇金淋巴瘤染色体 9p24 上的反复扩增子。 我们使用 RNAi 筛选发现了三个必需基因：JAK2、JMJD2C 和 RANBP6。 我们发现 JAK2 的激酶活性在这些淋巴瘤中通过自分泌 IL-13 信号传导被激活。 令人惊讶的是，JAK2 与 JMJD2C 合作促进这些淋巴瘤细胞的存活。 JMJD2C 是一种组蛋白 H3K9 去甲基酶，它通过去除该组蛋白标记来激活基因表达，从而阻止异染色质蛋白 HP-1 α 的募集。 我们将 JAK2 和 JMJD2C 之间的协同作用追溯到染色质的协同表观遗传修饰。 JAK2 在这些淋巴瘤细胞的细胞核中发挥作用，使酪氨酸 41 上的组蛋白 H3 尾部磷酸化，这也会阻止 HP-1 α 的募集。 JAK2 和 JMJD2C 表观遗传修饰的主要目标是 MYC，但此外，这两种蛋白还修饰数百个蛋白编码基因的染色质结构。 重要的是，针对 JAK2 的药物可以杀死这些淋巴瘤细胞。事实证明，RNA 干扰筛选在揭示伯基特淋巴瘤的关键生存途径方面非常成功。 我们发现转录因子 TCF3 的敲低对伯基特淋巴瘤细胞系是致命的，但对其他侵袭性淋巴瘤的细胞系模型则不然。 通过RNA重测序，我们发现70%的散发性伯基特淋巴瘤病例中TCF3及其负调节因子发生突变，导致TCF3依赖性。 我们的 RNA 干扰筛选还表明，约三分之二的伯基特淋巴瘤细胞系依赖于 B 细胞受体 (BCR) 的强直信号传导。 此外，我们发现 TCF3 放大了这种强直性 BCR 信号传导。 最后，我们发现伯基特淋巴瘤细胞系需要细胞周期蛋白 D3/CDK6 来促进细胞周期进展。 RNA 重测序显示 38% 的病例中细胞周期蛋白 D3 存在致癌激活突变。 令人惊讶的是，CDK6的药物抑制导致伯基特淋巴瘤细胞系凋亡并诱导已建立的伯基特淋巴瘤异种移植物的消退。