Oncogenic mechanisms and molecular targets in myeloma

骨髓瘤的致癌机制和分子靶点

• 批准号: 10014505
• 负责人: Louis Staudt
• 金额: $ 28.37万
• 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/10014505
• 关键词: Affect; Apoptosis; Autophagocytosis; B-Cell Activation; B-Lymphocytes; BCL2 gene; Bad protein; Binding; Biological Assay; Bone Marrow; CASP10 gene; CRISPR screen; CRISPR/Cas technology; Cell Cycle; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Characteristics; Chromosomal Duplication; Chromosomal translocation; Cleaved cell; Complex; Data; Defect; Deletion Mutation; Dependence; Disease; Essential Genes; Event; Extracellular Signal Regulated Kinases; Family member; Gene Expression Profile; Gene Expression Profiling; Gene-Modified; Generations; Genes; Genetic; Genetic Screening; Genetic Transcription; Heavy-Chain Immunoglobulins; IKBKB; IRF4 gene; Immunoglobulin G; Immunoglobulin M; Investigation; Laboratories; Lesion; Libraries; Ligation; Lymphoid; Lymphoma; MEK inhibition; MEKs; MYC gene; Malignant Neoplasms; Malignant lymphoid neoplasm; Mature B-Lymphocyte; Mediating; Messenger RNA; Metabolism; Molecular; Molecular Abnormality; Molecular Target; Monoclonal gammopathy of uncertain significance; Multiple Myeloma; Mus; Mutation; NF-kappa B; Oncogenes; Oncogenic; Pathway interactions; Patients; Peptide Hydrolases; Pharmacology; Phosphotransferases; Plasma Cells; Premalignant; Protein Isoforms; Proteins; Proteomics; Proto-Oncogenes; RNA Interference; RNA interference screen; Recurrence; Regulatory Pathway; Retroviral Vector; Sampling; Signal Pathway; Signal Transduction; Somatic Mutation; Subgroup; TNF receptor-associated factor 3; TNFRSF5 gene; Therapeutic; Therapeutic Agents; Transact; Transcription Coactivator; Transcription Factor AP-1; Waldenstrom Macroglobulinemia; Work; base; cancer cell; chromatin immunoprecipitation; experimental study; functional genomics; genome-wide; genomic data; inhibitor/antagonist; interest; knock-down; large cell Diffuse non-Hodgkin' s lymphoma; lenalidomide; loss of function; mutant; new therapeutic target; novel therapeutics; overexpression; promoter; protein protein interaction; research clinical testing; side effect; small hairpin RNA; small molecule; therapeutic target; therapy development; transcription factor; treatment response; whole genome

The NF-kB pathway has become a particular focus of the Staudt laboratory due to its recurrent involvement in various lymphoid cancers. The laboratory demonstrated that multiple myeloma has frequent engagement of the NF-kB pathway due to diverse genetic abnormalities in regulators of the pathway, including amplification or translocation of NIK, deletion or somatic mutation of TRAF3, deletion of the locus encoding c-IAP1 and c-IAP2 deletion, deletion of the CYLD, and overexpression of CD40, NFkB1. The laboratory demonstrated that NIK overexpression and TRAF3 inactivation were responsible for constitutive activation of the classical NF-kB pathway in the multiple myeloma. Inhibition of IkappaB kinase beta, the critical kinase in the classical NF-kB pathway, was lethal to many myeloma cell lines. The laboratory developed a gene expression signature of NF-kB pathway activation in multiple myeloma and showed that the majority of primary myeloma cases have NF-kB activation in the malignant cells and proposed that this pathway is a promising new target for therapy of myeloma. To identify further therapeutic targets in multiple myeloma, the laboratory conducted an RNA interference-based genetic screen for genes required for the proliferation and survival of myeloma cells. This loss-of-function screen utilized a library of retroviral vectors expressing small hairpin RNAs (shRNAs), which mediate RNA interference. shRNAs targeting IRF4 were toxic for multiple myeloma cell lines but not to lymphoma cell lines. IRF4 is a lymphoid-restricted transcription factor that is required for B cell activation and for differentiation of mature B cells into plasma cells. IRF4 inactivation was toxic to 10 different myeloma cell lines representing most of the recurrent genetic subtypes of myeloma. Notably, IRF4 is not genetically abnormal in most myeloma cases. Therefore, the dependence of myeloma cells on IRF4 is an prime example of non-oncogene addiction, a phenomenon wherein cancer cells become dependent upon normal cellular proteins for their survival. To understand the molecular basis for this non-oncogene addiction, the Staudt laboratory combined gene expression profiling and genome-wide chromatin immunoprecipitation to determine the target genes activated by IRF4. Among the 35 genes that were directly activated by IRF4 were genes that encode regulators of the cell cycle, metabolism and energy, general transcription, cell death, and plasma cell function. Some of these targets are highly expressed in normal activated B cells while other are instead expressed highly in normal plasma cells. This indicates that myelomas are addicted to an aberrant genetic network regulated by IRF4. Of special interest was the proto-oncogene MYC, which is frequently overexpresed in multiple myeloma due to chromosomal translocation or amplification. The Staudt laboratory demonstrated that IRF4 directly transactivates MYC and MYC in turn transactives IRF4, thereby forming a positive autoregulatory loop. Consistent with this concept, myelomas have higher expression of both MYC and IRF4 than normal plasma cells. IRF4 emerges from these experiments as an attractive new therapeutic target with potential in all forms of multiple myeloma, regardless of underlying genetic abnormality. Since IRF4 deficient mice have discrete defects in B cell activation and plasma cell generation, therapeutic targeting of IRF4 would be predicted to have defined and manageable on-target side effects. Indeed, recent investigations by our laboratory and others have revealed that lenalidomide decreases expression of IRF4, and this likely explains its efficacy in treating multiple myeloma. Modulating aberrant transcription of oncogenes is a relatively unexplored opportunity in cancer therapeutics. In 10% of multiple myelomas, the initiating oncogenic event is translocation of MAF, a transcriptional activator of key target genes such as cyclinD2. Our prior work showed that MAF is upregulated in an additional 30% of MM cases, albeit by an unknown mechanism. We recently discovered a common mechanism inducing MAF transcription in both instances. The second mode of MAF transcription occurred in myelomas with MMSET translocation, and these cases overexpressed MAF target genes. MMSET knockdown decreased MAF transcription and cell viability. A small molecule screen found an inhibitor of MEK, which activates ERK-MAP kinases, reduced MAF mRNA in cells representing MMSET or MAF subgroups. ERK activates transcription of FOS, one subunit of the AP-1 transcription factor. By chromatin immunoprecipitation, FOS bound the MAF promoter, and MEK inhibition decreased this interaction. MEK inhibition selectively induced apoptosis in MAF-expressing myelomas, and FOS inactivation was similarly toxic. Re-expression of MAF rescued cells from death induced by MMSET depletion, MEK inhibition, or FOS inactivation. The data presented herein demonstrate that the MEK-ERK pathway regulates MAF transcription, providing molecular rationale for clinical evaluation of MEK inhibitors in MAF-expressing myeloma. Recent RNA interference screens in multiple myeloma revealed caspase-10 as an essential gene that blocks an autophagic form of cell death in multiple myeloma. In other settings caspase-10 is a fully active protease that initiates apoptosis. In multiple myeloma, caspase-10 forms a complex with c-FlipL, creating a partially active protease. The reason multiple myeloma cells have this unusual caspase-10 isoform is that the genes encoding caspase-10 and c-FlipL are both direct targets of IRF4. When caspase-10 is inhibited genetically or pharmacologically, autophagy is induced, followed by a non-apoptotic form of cell death. The mechanism of caspase-10 action in multiple myeloma relates to its ability to cleave and inactivate BCLAF1. BCLAF1 was originally identified as a BCL2-binding protein. We discovered that it potently induces autophagic cell death when ectopically expressed in cells. It does so by blocking the ability of BCL2 and related family members from sequestering beclin, a key inducer of autophagy. Thus, by inactivating BCLAF1, caspase-10 promotes the binding of beclin to BCL2, thereby blocking autophagy. These studies suggest that caspase-10 inhibitors might be developed for the therapy of multiple myeloma. Recently we identified recurrent mutations affecting MyD88 in the ABC subtype of diffuse large B cell lymphomas. One predominant mutation, L265P, was also discovered to be highly prevalent in a plasmacytic disorder known as Waldenstrom's macroglobulinemia (WM). These mutations are rare or absent in multiple myeloma. Myeloma is preceded by a pre-malignant disorder known as monoclonal gammopathy of undetermined significance (MGUS) that is characterized by an excess of plasmacytic cells in the bone marrow without other disease manifestations. Together with Ola Landgren, we identified the MYD88 L265P mutation in some patients with MGUS. However, these samples utilized the IgM immunoglobulin heavy chain whereas more typical MGUS samples utilized IgG. Since IgM is the characteristic isotype of WM, we propose that these MYD88 mutant lesions are actually precursors of WM rather than multiple myeloma. Current projects involve the use of whole-genome CRISPR-Cas9 screens for essential genes in multiple myeloma and for genes that modify the response of therapeutic agents in this disease. We are also using SILAC quantitative proteomics and proximity ligation assays to define functional protein-protein interactions that regulate oncogenic signaling in myeloma.

NF-KB途径已成为Staudt实验室的特殊重点，因为它经常参与各种淋巴癌。该实验室表明，由于该途径调节器中多种遗传异常，多发性骨髓瘤经常参与NF-KB途径，包括NIK的扩增或易位TRAF3的扩增或易位，c-iap1和c-iap2 deletion of cyf cyf cyf cyf cyf cyf cyf cyfrest和cyflk的deletion traf3的扩增或易位。该实验室表明，NIK过表达和TRAF3失活是导致多发性骨髓瘤中经典NF-KB途径的组成型激活。 Ikappab激酶β的抑制作用是经典NF-KB途径中的临界激酶，对许多骨髓瘤细胞系致命。实验室在多发性骨髓瘤中开发了NF-KB途径激活的基因表达特征，并表明大多数原发性骨髓瘤病例在恶性细胞中具有NF-KB激活，并提出该途径是骨髓瘤治疗的有希望的新靶标。为了鉴定多发性骨髓瘤的进一步治疗靶标，实验室对基于RNA干扰的遗传筛选了骨髓瘤细胞的增殖和存活所需的基因。这种功能丧失的屏幕利用了表达小发夹RNA（SHRNA）的逆转录病毒载体库，该库介导RNA干扰。靶向IRF4的SHRNA对多发性骨髓瘤细胞系有毒，但对淋巴瘤细胞系有毒。 IRF4是B细胞激活和成熟B细胞分化为浆细胞所必需的淋巴限制转录因子。 IRF4失活对10种不同的骨髓瘤细胞系有毒，代表大多数骨髓瘤的复发亚型。值得注意的是，在大多数骨髓瘤病例中，IRF4在遗传上并不异常。因此，骨髓瘤细胞对IRF4的依赖性是非癌基因成瘾的一个主要例子，这是一种现象，其中癌细胞依赖于正常细胞蛋白的生存。为了了解这种非癌基因成瘾的分子基础，Staudt实验室结合了基因表达分析和全基因组染色质免疫沉淀，以确定由IRF4激活的靶基因。在由IRF4直接激活的35个基因中，是编码细胞周期调节因子的基因，代谢和能量，一般转录，细胞死亡和浆细胞功能。其中一些靶标在正常活化的B细胞中高度表达，而其他靶标则在正常的浆细胞中表达高度表达。这表明骨髓瘤沉迷于由IRF4调节的异常遗传网络。特别感兴趣的是原癌基因MYC，由于染色体易位或扩增，它经常在多发性骨髓瘤中过度表现。 Staudt实验室表明，IRF4直接反复激活MYC和MYC反过来transactives IRF4，从而形成了阳性的自动调节环。与此概念一致，MYC和IRF4的表达比正常的浆细胞更高。 IRF4从这些实验中出现是一个有吸引力的新治疗靶标，无论是潜在的多发性骨髓瘤，无论基本的遗传异常如何。由于IRF4缺乏小鼠在B细胞激活和浆细胞产生中具有离散的缺陷，因此将预测IRF4的治疗靶向定义且易于管理的靶向副作用。确实，我们的实验室和其他人最近的研究表明，Lenalidomide降低了IRF4的表达，这可能解释了其在治疗多发性骨髓瘤方面的功效。调节癌基因的异常转录是癌症治疗中相对未开发的机会。在10％的多发性骨髓瘤中，启动致癌事件是MAF的易位，MAF是关键靶基因（例如Cyclind2）的转录激活因子。我们先前的工作表明，MAF在另外30％的MM病例中被上调，尽管是未知机制。最近，我们发现了在这两种情况下诱导MAF转录的常见机制。 MAF转录的第二种模式发生在带有MMSET易位的骨髓瘤中，这些情况过表达MAF靶基因。 MMSET敲低降低了MAF转录和细胞活力。一个小分子筛选发现了MEK的抑制剂，该抑制剂激活ERK-MAP激酶，减少了代表MMSET或MAF亚组的细胞中的MAF mRNA。 ERK激活FOS的转录，AP-1转录因子的一个亚基。通过染色质免疫沉淀，FOS结合MAF启动子，MEK抑制减少了这种相互作用。 MEK抑制在表达MAF的骨髓瘤中有选择性诱导的凋亡，而FOS失活同样有毒。 MAF的重新表达使细胞从MMSET耗竭，MEK抑制或FOS失活引起的死亡中。本文提供的数据表明，MEK-ERK途径调节MAF转录，为表达MAF表达骨髓瘤的MEK抑制剂的临床评估提供了分子原理。最近在多发性骨髓瘤中的RNA干扰筛选显示caspase-10是一种必不可少的基因，它阻断了多发性骨髓瘤中细胞死亡的一种自噬形式。在其他环境中，caspase-10是一种完全活跃的蛋白酶，可引发凋亡。在多发性骨髓瘤中，caspase-10与C-FLIPL形成复合物，形成部分活性的蛋白酶。多发性骨髓瘤细胞具有这种不寻常的caspase-10同工型的原因是编码caspase-10和c-Flipl的基因都是IRF4的直接靶标。当caspase-10在遗传或药理上抑制时，会诱导自噬，然后诱导无凋亡的细胞死亡形式。多发性骨髓瘤中caspase-10作用的机制与其裂解和失活的BCLAF1的能力有关。 BCLAF1最初被鉴定为Bcl2结合蛋白。我们发现，当异位在细胞中表达时，它有力诱导自噬细胞死亡。这样做是通过阻止BCL2及相关家庭成员的能力，从隔离的Beclin（自动噬菌体的关键诱导者）中。因此，通过灭活BCLAF1，caspase-10促进了Beclin与Bcl2的结合，从而阻止了自噬。这些研究表明，CASPASE-10抑制剂可能是为多发性骨髓瘤的治疗而开发的。最近，我们确定了在弥漫性大B细胞淋巴瘤的ABC亚型中影响MyD88的复发突变。在称为Waldensstrom的大型球蛋白血症（WM）的浆细胞障碍中，也发现一种主要的突变L265p高度流行。这些突变在多发性骨髓瘤中很少见或不存在。骨髓瘤之前是一种恶性疾病，称为单克隆肾上腺病，具有不确定的意义（MGU），其特征是骨髓中没有其他疾病表现的骨囊性细胞过多。我们与Ola Landgren一起确定了一些MGU患者的MYD88 L265P突变。但是，这些样品利用了IgM免疫球蛋白重链，而更典型的MGU样品使用了IgG。由于IgM是WM的特征同种型，因此我们建议这些MyD88突变病变实际上是WM的前体，而不是多发性骨髓瘤。当前的项目涉及将全基因组CRISPR-CAS9筛选用于多发性骨髓瘤的必需基因以及修改该疾病治疗剂反应的基因。我们还使用SILAC定量蛋白质组学和接近连接测定法来定义调节骨髓瘤中致癌信号传导的功能性蛋白质蛋白相互作用。