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: 7965938
• 负责人: Louis Staudt
• 金额: $ 80.05万
• 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/7965938
• 关键词: Antigen Receptors; Apoptotic; B-Lymphocytes; BCL10 gene; Bar Codes; Base Pairing; Binding; Cancer Biology; Cell Line; Cells; Classification; Complex; Dependence; Dependency; Feedback; Gene Expression; Genes; Genetic; Genetic Screening; Genomics; Heel; Human Cell Line; IRF4 gene; Laboratories; Libraries; Lymphocyte; Lymphocyte Activation; Lymphoid; Lymphoma; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methodology; Molecular Abnormality; Molecular Target; Monitor; Multiple Myeloma; Mutate; NF-kappa B; National Institute of Allergy and Infectious Disease; Oncogenes; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Population; RNA Interference; Receptor Activation; Receptor Signaling; Regulation; Regulatory Pathway; Retroviral Vector; Role; Serine; Signal Pathway; Signal Transduction; Stimulus; Structure of germinal center of lymph node; System; T-Cell Receptor; T-Lymphocyte; Taxonomy; Therapeutic; Toxic effect; Work; addiction; base; cancer cell; cancer type; casein kinase; design; large cell Diffuse non-Hodgkin' s lymphoma; loss of function; mutant; new therapeutic target; novel; reconstitution; research study; small hairpin RNA; 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 (CK1α) that were toxic for ABC but not GCB DLBCL cell lines. Since this phenotype resembled the toxicity profile of CARD11 shRNAs, we suspected that CK1α might be a new component of the CBM signaling pathway. Indeed, CK1α knockdown decreased IKK kinase activity and NF-kB target gene expression, and CK1α colocalized with CARD11 in ABC DLBCL cell lines. Fortuitously, Mike Lenardo's laboratory in NIAID separately identified CK1α as a binding partner of CARD11 in a mass spectrometry-based screen. Collaborative experiments revealed the essential role of CK1α in T cell receptor activation of the NF-kB pathway. Biochemically, CK1α was required for the recruitment of IKK to the CBM complex. Interestingly, T cells reconstituted with a CK1α mutant that was defective in kinase activity provided a greater NF-kB stimulus than did wild type CK1α, demonstrating that CK1α has both positive and negative influences on the CBM pathway. The negative effect of CK1α was due to its phosphorylation of CARD11 on serines in its "linker" domain, causing CARD11 to be destabilized. This negative feedback loop involving CK1α was analogous to the negative regulation of the CBM complex caused by inhibitory phosphorylation of BCL10 by IKK. CK1α 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.

Staudt实验室已经对代表各种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。在最近的RNA干扰屏幕中，我们鉴定了几个靶向酪蛋白激酶1a1（CK1＆＃945;）的shRNA，它们对ABC有毒，但没有GCB DLBCL细胞系。由于这种表型类似于Card11 shrnas的毒性特征，因此我们怀疑CK1＆＃945;可能是CBM信号通路的新组件。确实，CK1＆＃945;敲低降低了IKK激酶活性和NF-KB靶基因表达，而CK1＆＃945;与ABC DLBCL细胞系中的Card11共定位。 幸运的是，Niaid的Mike Lenardo的实验室分别确定了CK1＆＃945; 作为基于质谱的屏幕中Card11的结合伙伴。协作实验揭示了CK1＆＃945;在NF-KB途径的T细胞受体激活中。在生化上，CK1＆＃945;是将IKK招募到CBM综合体所必需的。有趣的是，T细胞用CK1＆＃945;与野生型CK1相比，激酶活性有缺陷的突变体提供了更大的NF-KB刺激，证明CK1＆＃945;对CBM途径具有积极和负面影响。 CK1＆＃945;是由于其在其“接头”域中的丝氨酸上的Card11磷酸化，从而导致Card11不稳定。涉及CK1＆＃945;的负反馈循环;类似于由IKK对BCL10抑制性磷酸化引起的CBM复合物的负调节。 CK1＆＃945;从这项研究中浮现为CBM的新组成部分，证明了无偏的RNA干扰屏幕的力量，以发现细胞信号传导的被忽视方面。最近的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。 RNAi的IRF4敲低对10种不同的骨髓瘤细胞系有毒，代表了该癌症的许多已知遗传亚型。 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。