Functional genomic screens for molecular targets in cancer

功能基因组筛选癌症分子靶点

• 批准号: 10926110
• 负责人: Louis Staudt
• 金额: $ 144.57万
• 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/10926110
• 关键词: 9p24; Adaptor Signaling Protein; Address; Adopted; Adult T-Cell Leukemia/Lymphoma; Affect; Antigen Presentation; Antigen Receptors; Antigens; Apoptosis; Apoptotic; Attenuated; B-Cell Antigen Receptor; B-Cell Lymphomas; B-Lymphocytes; BCL10 gene; Bar Codes; Burkitt Lymphoma; CD22 gene; CRISPR screen; CRISPR/Cas technology; Cancer Biology; Cell Cycle Progression; Cell Line; Cell membrane; Cells; Chromosomes; Chronic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Complex; DNA Resequencing; Dependence; Development; Dose; Drops; Drug Targeting; Drug resistance; Drug usage; Endoplasmic Reticulum; Epigenetic Process; Essential Genes; Event; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomic approach; Genomics; Growth; Guide RNA; Hodgkin Disease; Homeostasis; Human Cell Line; IRAK1 gene; IRF4 gene; ITAM; Immune; Immune Evasion; Immunoglobulin M; Interleukin-13; JAK2 gene; Knowledge; LYN gene; Laboratories; Libraries; Lymphocyte; Lymphocyte Activation; Lymphoid; Lymphoma; Lymphoma cell; MHC Class I Genes; Malignant Neoplasms; Malignant lymphoid neoplasm; Measures; Mediastinal; Mediating; Methodology; Modeling; Molecular; Molecular Abnormality; Molecular Target; Mucosa- associated lymphoid tissue lymphoma translocation protein-1; Multiple Myeloma; Mutate; Mutation; NF-kappa B; NFKB Activation Pathway; Oncogenes; Oncogenic; PIK3CG gene; PLCG2 gene; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Plasma Cells; Proliferating; Protein Glycosylation; Protein Isoforms; Protein Kinase; Protein Tyrosine Kinase; Proteins; Proteomics; RNA; RNA Interference; RNA interference screen; Receptor Signaling; Recurrence; Regulatory Pathway; Resistance; Retroviral Vector; SYK gene; Signal Pathway; Signal Transduction; Structure of germinal center of lymph node; System; T cell factor 3; TCF3 gene; Taxonomy; Therapeutic; Therapeutically Targetable; Toll-like receptors; Toxic effect; Tumor Suppressor Proteins; Tyrosine; Up-Regulation; Work; Xenograft procedure; activated B cell like; autocrine; cancer cell; cancer survival; cancer type; cyclin D3; drug efficacy; endonuclease; functional genomics; genome-wide; genomic locus; glycosylation; inhibitor; kinase inhibitor; knock-down; large cell Diffuse non-Hodgkin' s lymphoma; lenalidomide; link protein; loss of function; molecular targeted therapies; mutant; new therapeutic target; novel; pharmacologic; protein kinase C beta; small hairpin RNA; src-Family Kinases; therapy resistant; transcription factor; ubiquitin-protein ligase

The Staudt laboratory has conducted RNA interference and CRISPR 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. Our RNA interference 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. Ibrutinib, a clinically available kinase inhibitor of BTK kills ABC DLBCL cells by blocking their chronic active BCR signaling. Our RNA interference screens additionally revealed 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 associated 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. 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 apoptosis of Burkitt lymphoma cell lines and induced regression of established Burkitt lymphoma xenografts. An 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. 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. Indeed, we showed that the efficacy of the drug lenalidomide in myeloma is due in large measure to its ability to decrease IRF4 expression. In adult T cell leukemia/lymphoma, our RNAi screens uncovered a dependence on a transcriptional network controlled by BATF and IRF4 that is therapeutically targetable with BET protein inhibitors. Most recently, we have adopted the Cas9/CRISPR technology, which is an alternative way to inactivate genes. In this system, a short guide RNA (sgRNA) is used to direct the Cas9 endonuclease precise genomic locations. If the sgRNA targets the beginning of the coding region of a gene, the gene can be inactivated as a result of repeated cutting and deletion events. We have used bar-coded lentiviral libraries expressing sgRNAs to identify essential genes in lymphoid malignancies. We unexpectedly discovered that TLR9 is required for the survival of ABC DLBCL cells. We used this knowledge to demonstrate, quite unexpectedly, that TLR9 associates with IgM in these lymphoma cells. Using quantitative proteomics we defined a MyD88-TLR9-BCR (My-T-BCR) supercomplex that coordinates all pro-survival NF-kB signaling in these ABC DLBCL cells. We have recently used "drug modifier" CRISPR screens to address the molecular pathways that cause resistance to drugs targeting oncogenic signaling in DLBCL. This effort has revealed new mechanisms of therapeutic resistance to BTK inhibitors in DLBCL. One mode of resistance is genetic inactivation of the tumor suppressor KLHL14, which we showed is an E3 ubiquitin ligase that increases the turnover of components of the B cell receptor in the endoplasmic reticulum. Another resistance mode relies on the epigenetic upregulation of RAC2, which can activate PLCG2 in lieu of BTK, thereby maintaining downstream NF-kB activity. In collaboration with Jon Yewdell, we used CRISPR screens to identify positive and negative regulators of MHC class I and class II expression in DLBCL, which uncovered novel mechanisms of immune evasion and identified mechanisms by which one could potentiate antigen presentation by DLBCL cells. Most recently, we devised genome-wide CRISPR-Cas9 screens to identify regulators of IRF4, a direct transcriptional target of NF-kB and an indicator of proximal BCR signaling in ABC DLBCL. Unexpectedly, inactivation of N-linked protein glycosylation by the oligosaccharyltransferase-B (OST-B) complex reduced IRF4 expression. OST-B inhibition of BCR glycosylation reduced BCR clustering and internalization while promoting its association with CD22, which attenuated PI3 kinase and NF-kB activitation. By directly interfering with proximal BCR signaling, OST-B inactivation killed models of ABC and GCB DLBCL, supporting the development of selective OST-B inhibitors for the treatment of these aggressive cancers.

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 干扰和 CRISPR 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 通路的组成型激活。我们的 RNA 干扰筛选使我们能够定义 B 细胞受体 (BCR) 信号的慢性活性形式，该信号在具有野生型 CARD11 的 ABC DLBCL 中激活 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。依鲁替尼是一种临床可用的 BTK 激酶抑制剂，通过阻断 ABC DLBCL 细胞的慢性活性 BCR 信号传导来杀死它们。我们的 RNA 干扰筛选还揭示了 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，靶向 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。这导致我们发现 MYD88 中反复发生的突变会产生自发激活 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 上的复发扩增子。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。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的药理学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。 RNAi 筛选发现多发性骨髓瘤细胞对 IRF4 的关键依赖性，IRF4 是淋巴细胞激活和浆细胞分化所需的淋巴限制性转录因子。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。这些结果确立了 IRF4 作为这种致命癌症的重要新治疗靶点。事实上，我们表明来那度胺在骨髓瘤中的功效在很大程度上归因于其降低 IRF4 表达的能力。在成人 T 细胞白血病/淋巴瘤中，我们的 RNAi 筛选发现了对由 BATF 和 IRF4 控制的转录网络的依赖性，该网络可通过 BET 蛋白抑制剂进行治疗。最近，我们采用了Cas9/CRISPR技术，这是另一种灭活基因的方法。在此系统中，短向导RNA (sgRNA) 用于指导Cas9 核酸内切酶精确的基因组位置。如果 sgRNA 靶向基因编码区的开头，则该基因可能会因重复切割和缺失事件而失活。我们使用表达 sgRNA 的条形码慢病毒文库来识别淋巴恶性肿瘤中的必需基因。我们意外地发现TLR9是ABC DLBCL细胞存活所必需的。我们利用这一知识出乎意料地证明，TLR9 与这些淋巴瘤细胞中的 IgM 相关。使用定量蛋白质组学，我们定义了 MyD88-TLR9-BCR (My-T-BCR) 超级复合物，它协调这些 ABC DLBCL 细胞中所有促生存 NF-kB 信号传导。我们最近使用“药物修饰剂”CRISPR 筛选来解决导致 DLBCL 中针对致癌信号传导的药物产生耐药性的分子途径。这项工作揭示了 DLBCL 对 BTK 抑制剂治疗耐药的新机制。一种耐药模式是肿瘤抑制因子 KLHL14 的基因失活，我们证明它是一种 E3 泛素连接酶，可增加内质网中 B 细胞受体成分的周转。另一种耐药模式依赖于RAC2的表观遗传上调，它可以激活PLCG2代替BTK，从而维持下游NF-kB活性。我们与 Jon Yewdell 合作，使用 CRISPR 筛选来鉴定 DLBCL 中 MHC I 类和 II 类表达的正向和负向调节因子，揭示了免疫逃避的新机制，并确定了增强 DLBCL 细胞抗原呈递的机制。最近，我们设计了全基因组 CRISPR-Cas9 筛选来识别 IRF4 的调节因子，IRF4 是 NF-kB 的直接转录靶标，也是 ABC DLBCL 中近端 BCR 信号传导的指标。出乎意料的是，寡糖转移酶-B (OST-B) 复合物对 N 连接蛋白糖基化的灭活降低了 IRF4 的表达。 OST-B 对 BCR 糖基化的抑制减少了 BCR 聚集和内化，同时促进其与 CD22 的结合，从而减弱了 PI3 激酶和 NF-kB 的激活。通过直接干扰近端 BCR 信号传导，OST-B 失活杀死了 ABC 和 GCB DLBCL 模型，支持开发选择性 OST-B 抑制剂来治疗这些侵袭性癌症。

项目成果

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2.

OCT2 调节正常 B 细胞分化和恶性 B 细胞存活。

• 发表时间: 2016-04-05
• 影响因子: 11.1
• 作者: Hodson, Daniel J;Shaffer, Arthur L;Xiao, Wenming;Wright, George W;Schmitz, Roland;Phelan, James D;Yang, Yandan;Webster, Daniel E;Rui, Lixin;Kohlhammer, Holger;Nakagawa, Masao;Waldmann, Thomas A;Staudt, Louis M
• 通讯作者: Staudt, Louis M

Compensatory IKKalpha activation of classical NF-kappaB signaling during IKKbeta inhibition identified by an RNA interference sensitization screen.

通过 RNA 干扰敏化筛选鉴定 IKKbeta 抑制过程中经典 NF-kappaB 信号传导的补偿性 IKKalpha 激活。

• 发表时间: 2008-12-30
• 影响因子: 11.1
• 作者: Lam, Lloyd T;Davis, R Eric;Ngo, Vu N;Lenz, Georg;Wright, George;Xu, Weihong;Zhao, Hong;Yu, Xin;Dang, Lenny;Staudt, Louis M
• 通讯作者: Staudt, Louis M

Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells.

转录抑制因子 Snail2 在结直肠癌细胞中致癌 RAS 转化中发挥关键作用。

• 发表时间: 2010-08-19
• 影响因子: 8
• 作者: Wang, Y;Ngo, V N;Marani, M;Yang, Y;Wright, G;Staudt, L M;Downward, J
• 通讯作者: Downward, J

Casein kinase 1alpha governs antigen-receptor-induced NF-kappaB activation and human lymphoma cell survival.

酪蛋白激酶 1alpha 控制抗原受体诱导的 NF-kappaB 激活和人淋巴瘤细胞存活。

• 发表时间: 2009-03-05
• 影响因子: 64.8
• 作者: Bidere, Nicolas;Ngo, Vu N.;Lee, Jeansun;Collins, Cailin;Zheng, Lixin;Wan, Fengyi;Davis, R. Eric;Lenz, Georg;Anderson, D. Eric;Arnoult, Damien;Vazquez, Aime;Sakai, Keiko;Zhang, Jun;Meng, Zhaojing;Veenstra, Timothy D.;Staudt, Louis M.;Lenardo, Michael J.
• 通讯作者: Lenardo, Michael J.