Functional genomic screens for molecular targets in cancer

功能基因组筛选癌症分子靶点

• 批准号: 10702452
• 负责人: Louis Staudt
• 金额: $ 145.04万
• 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/10702452
• 关键词: 1-Phosphatidylinositol 3-Kinase; 9p24; Adaptor Signaling Protein; Address; Adopted; Adult T-Cell Leukemia/Lymphoma; Affect; Antigen Presentation; Antigen Receptors; Antigens; Apoptosis; Apoptotic; B-Cell Activation; B-Cell Antigen Receptor; B-Cell Lymphomas; B-Lymphocytes; BCL10 gene; Bar Codes; Burkitt Lymphoma; 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; 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; Location; 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; Oncogenes; Oncogenic; PLCG2 gene; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; 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; Toll-like receptors; Toxic effect; Tumor Suppressor Proteins; Tyrosine; Up-Regulation; Work; Xenograft procedure; autocrine; base; cancer cell; cancer survival; cancer type; cyclin D3; drug efficacy; endonuclease; functional genomics; inhibitor; kinase inhibitor; knock-down; large cell Diffuse non-Hodgkin' s lymphoma; lenalidomide; loss of function; molecular targeted therapies; mutant; new therapeutic target; novel; small hairpin RNA; src-Family Kinases; therapeutic target; 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.

Staudt实验室对代表各种亚型淋巴瘤和多发性骨髓瘤亚型的人类细胞系的增殖和/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，先前在Staudt实验室中的工作表明，抗凋亡的NF-KB途径在DLBCL的活化B细胞样（ABC）亚型中具有组成性活性，但不是dlbcl的生发性中心（GGCB）dlbcl的dlbcl，但这种机制的构成，但是因此，实验室在ABC和GCB DLBCL细胞系中进行了RNAi筛选，寻找对ABC DLBCL细胞有选择性毒性的SHRNA。这项工作表明，ABC的存活需要Card11，Malt1和Bcl10组成的信号传导复合物，而不是GCB DLBCL细胞系的生存。在正常的淋巴细胞中，该Card11复合物在抗原受体信号传导过程中参与NF-KB途径。 Staudt实验室表明，该信号传导复合物负责ABC DLBCL中NF-KB途径的组成型激活。我们的RNA干扰屏幕使我们能够定义B细胞受体（BCR）信号的慢性活性形式，该信号通过使用野生型Card11激活ABC DLBCL中的NF-KB。这种ABC DLBCL在敲低BCR信号传导成分时死亡，包括B细胞受体本身的亚基。 ABC DLBCL在质膜中具有BCR的突出簇，类似于抗原刺激的正常B细胞​​。癌症基因重新定位表明，超过五分之一的ABC DLBCL在BCR的CD79B或CD79A亚基中具有突变。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。这些突变会影响关键的ITAM信号基序，从而产生BCR，从而避免Lyn酪氨酸激酶负面调节。重要的是，BCR途径提供了许多可以通过治疗剥削的靶标，包括几种蛋白激酶（SRC-家庭激酶，SYK，BTK，PKCBETA）以及PI（3）激酶。 Ibrutinib是一种临床上可用的BTK激酶抑制剂，通过阻断其慢性活性BCR信号来杀死ABC DLBCL细胞。我们的RNA干扰筛选还揭示了MyD88信号通路对于ABC DLBCL细胞的存活至关重要。 MyD88是固有免疫细胞中Toll样受体下游的信号通路中的关键衔接蛋白。 RNAi筛选鉴定出靶向MyD88及其相关的激酶IRAK1对ABC DLBCL细胞有毒，但对其他淋巴瘤亚型的细胞系模型没有毒性。这使我们发现了MyD88中的复发突变，这些突变产生了突变的同工型，从而自发激活NF-KB途径并具有致癌性。我们使用RNAi筛选研究了原发性纵隔大B细胞淋巴瘤（PMBL）和Hodgkin淋巴瘤中的复发性扩增子。我们使用RNAi屏幕（JAK2，JMJD2C和RANBP6）发现了三个必需基因。我们表明，自分泌IL-13信号传导在这些淋巴瘤中激活JAK2的激酶活性。令人惊讶的是，JAK2与JMJD2C合作，促进了这些淋巴瘤细胞的存活。事实证明，RNA干扰筛查在揭开伯基特淋巴瘤的关键存活途径方面非常成功。我们发现转录因子TCF3的敲低对Burkitt淋巴瘤细胞系致命，但对其他侵袭性淋巴瘤的细胞系模型却不是致命的。通过RNA重新陈述，我们发现TCF3及其阴性调节剂在70％的零星Burkitt淋巴瘤中突变，从而导致TCF3依赖性。我们的RNA干扰筛选还表明，大约三分之二的伯基特淋巴瘤细胞系取决于B细胞受体（BCR）的滋补信号。此外，我们发现TCF3放大了这种补品BCR信号。最后，我们发现Burkitt淋巴瘤细胞系需要细胞周期的细胞周期进程。 RNA重新定价显示在38％的病例中，细胞周期蛋白D3的致癌激活突变。令人惊讶的是，CDK6的药理抑制会导致伯基特淋巴瘤细胞系的细胞凋亡，并诱导已建立的伯基特淋巴瘤异种移植物的消退。 RNAi筛查发现了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必需的淋巴限制的转录因子。值得注意的是，在大多数多发性骨髓瘤的情况下，IRF4不会被易位，扩增或突变，因此骨髓瘤细胞对IRF4的依赖性在癌症生物学中的新概念被称为非癌基因成瘾。这些结果将IRF4作为这种致命癌症的重要新治疗靶点。实际上，我们表明该药物为髓类药物在骨髓瘤中的疗效很大程度上是由于其降低IRF4表达的能力。在成年T细胞白血病/淋巴瘤中，我们的RNAi筛查发现了对由BATF和IRF4控制的转录网络的依赖性，该转录网络在治疗上可用于BET蛋白抑制剂。 最近，我们采用了CAS9/CRISPR技术，这是使基因失活的另一种方法。在该系统中，使用简短的指南RNA（SGRNA）来指导Cas9核酸内切酶精确的基因组位置。如果SGRNA靶向基因的编码区域的开头，则基因可能因重复切割和缺失事件而被灭活。我们已经使用了表达SGRNA的条形编码的慢病毒文库来鉴定淋巴恶性肿瘤中的必要基因。我们出乎意料地发现，ABC DLBCL细胞存活需要TLR9。我们使用这些知识来表明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）超复合物，该超复合物可以在这些ABC DLBCL细胞中协调所有促生物存在的NF-KB信号传导。我们最近使用了“药物修饰” CRISPR筛选来解决对靶向DLBCL致癌信号的抗性的分子途径。这项工作揭示了DLBCL中对BTK抑制剂的治疗性抗性的新机制。一种耐药的方式是抑制肿瘤KLHL14的遗传失活，我们表明的是E3泛素连接酶，可增加内质网中B细胞受体的成分的周转。另一种电阻模式取决于Rac2的表观遗传上调，后者可以激活PLCG2代替BTK，从而维持下游NF-KB活性。与乔恩·尤德尔（Jon Yewdell）合作，我们使用CRISPR屏幕来识别DLBCL中MHC I类和II类表达的阳性和阴性调节剂，这些调节剂发现了免疫逃避的新型机制，并确定了可以通过DLBCL细胞增强抗原表现的机制。