Oncogenic mechanisms and molecular targets in lymphoma

淋巴瘤的致癌机制和分子靶点

• 批准号: 8763303
• 负责人: Louis Staudt
• 金额: $ 140.71万
• 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/8763303
• 关键词: 1-Phosphatidylinositol 3-Kinase; Adopted; Adverse effects; Affect; Africa; African Burkitt' s lymphoma; Amino Acids; Antigens; B-Lymphocytes; BCL10 gene; Biopsy; Burkitt Lymphoma; Candidate Disease Gene; Cell Cycle Progression; Cell Death; Cell membrane; Cells; ChIP-seq; Child; Chronic; Clinical; Clinical Trials; Combined Modality Therapy; Complex; Coupled; DNA Resequencing; Data; Dependence; Dependency; Developed Countries; Developing Countries; Development; Diagnosis; Drug Combinations; Drug Targeting; Dysmyelopoietic Syndromes; Elderly; Evaluation; Fostering; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Genetic Screening; Genomics; HIV; High Dose Chemotherapy; Homeostasis; Human; Human Herpesvirus 4; IRAK1 gene; IRAK4 gene; IRF4 gene; ITAM; In complete remission; Interferon Type I; Interferon-beta; Investigation; LYN gene; Laboratories; Leucine; Lymphoma; Malignant Neoplasms; Malignant lymphoid neoplasm; Methodology; Methods; Molecular; Molecular Target; Multiple Myeloma; Mutation; NF-kappa B; Newly Diagnosed; Oncogenes; Oncogenic; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacodynamics; Phase; Phase II Clinical Trials; Phenotype; Phosphotransferases; Play; Prior Therapy; Production; Proline; Protein Isoforms; Protein Tyrosine Kinase; RNA; RNA Interference; RNA Sequences; Receptor Signaling; Receptors, Antigen, B-Cell; Recurrence; Refractory; Regulatory Pathway; Relapse; Role; STAT3 gene; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Somatic Mutation; Structure; Structure of germinal center of lymph node; TCF3 gene; Toxicity due to chemotherapy; Tumor Suppressor Genes; Tyrosine; Validation; adapter protein; addiction; autocrine; base; cell killing; chemotherapy; comparative genomics; cyclin D3; functional genomics; improved; inhibitor/antagonist; killings; large cell Diffuse non-Hodgkin' s lymphoma; lenalidomide; mutant; new therapeutic target; novel; novel therapeutics; older patient; partial response; randomized trial; response; screening; small molecule; therapeutic development; transcription factor; tumor

Our laboratory has embarked on a new initiative to discover oncogenic somatic mutations in lymphoid malignancies by cancer gene resequencing. Previously, in an RNAi-based genetic screen, we discovered that a pathway involving CARD11, BCL10 and MALT1 was responsible for the constitutive NF-kB signaling in ABC DLBCL. In a clear validation of the RNAi-based genetic screen, our lab discovered recurrent somatic mutations in the CARD11 gene in ABC DLBCL tumor biopsies. All of the CARD11 mutations in DLBCL changed amino acids in one small domain that is predicted to adopt a coiled-coil structure. These CARD11 mutations created protein isoforms that constitutively engaged NF-kB signaling, apparently due to their ability to spontaneously form large cytosolic aggregates that colocalize with signaling proteins in the NF-kB pathway. 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. We have recently identified oncogenic signaling by the adapter protein MYD88 as the genetic basis for the JAK-STAT3 activation in ABC DLBCL. An RNAi screen in ABC DLBCL revealed the dependence of these cells on MYD88 and its downstream kinases IRAK1 and IRAK4. High-throughput RNA resequencing (RNA-seq) uncovered recurrent mutations in MYD88. MYD88 mutations are present in 39% of ABC DLBCLs, with 29% affecting changing one leucine in the MYD88 TIR domain to proline (L265P). The L265P mutant isoform spontaneously coordinates a signaling complexed involving IRAK1 and IRAK4, which turns on the NF-kB, JAK-STAT3 pathway, and type I interferon pathway. Small molecule inhibitors of IRAK4 kinase are selectively lethal to ABC DLBCL cells, offering new therapeutic prospects. ABC DLBCL cells are killed in a synergistic fashion by combined inhibition of the BCR and MYD88 pathways, suggesting the development of clinical trials combining drugs targeting both pathways. There are several new drugs entering early phase clinical trials that target the pathways we have implicated using our functional genomics methods. The B cell receptor signaling pathway affords many possible targets for the treatment of ABC DLBCL, notably BTK. We initiated a phase Ib clinical trial of a small molecule BTK inhibitor, termed ibrutinib, in patients with relapsed/refractory DLBCL, with gene expression profiling used for both diagnosis and pharmacodynamics. Thus far, ibrutinib monotherapy has induced many complete and partial responses in patients with ABC DLBCL, including those with primary refractory tumors that had never responded to any prior therapy. One patient has been in a sustained complete response for over 3 years, taking ibrutinib daily with no discernible side effects. Of note, ABC DLBCL tumors with and without CD79B mutations have responded, suggesting that BCR pathway addiction may be a prevalent feature in this lymphoma subtype. Based on these promising results, we have completed a multicenter phase 2 trial of ibrutinib in patients with relapsed/refractory DLBCL. Patients were assigned to the ABC or GCB subtypes by gene expression profiling and were given ibrutinib monotherapy. Ibrutinib produced a 41% response rate (complete + partial) in patients with relapsed/refractory ABC DLBCL, but only a 5% response rate in GCB DLBCL, as predicted by our laboratory investigations. Many of the responses have been durable, lasting from 6 months to over 1 year. Based on these promising results, ibrutinib is now being evaluated in newly diagnosed ABC DLBCL in combination with R-CHOP chemotherapy in a phase 3 randomized trial. We expect that ibrutinib will not be curative as a single agent in most patients with ABC DLBCL, leading to search for other agents that might synergize with ibrutinib in killing these lymphoma cells. Lenalidomide is a drug of relatively obscure action that has shown potent clinical activity in multiple myeloma and myelodysplastic syndromes. Lenalidomide has also had activity in early phase clinical trials against ABC DLBCL, prompting us to investigate its mode of action in this setting. Unexpectedly, we discovered that lenalidomide induces the secretion of interferon beta by the ABC DLBCL cells, and that this autocrine production is an important component of lenalidomide-induced cell death in ABC DLBCL. In addition, lenalidomide blocked B cell receptor signaling to NF-kB by decreasing expression of CARD11. Both of these phenotypes could be traced to the ability of lenalidomide to decrease expression of IRF4. By RNA interference and ChIP-seq analysis, we showed that IRF4 plays and essential survival role in ABC DLBCL. IRF4 is itself an NF-kB target gene, so that that agents that inhibit BCR signaling, such as ibrutinib, also decrease IRF4 expression. We found that combined treatment with lenalidomide and ibrutinib virtually eliminated IRF4 expression, leading to synergistic killing of ABC DLBCLs. These results support evaluation of this drug combination in clinical trials. Burkitt lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL (eBL) in developing countries, necessitating new strategies1. The normal germinal center B cell is the presumed cell of origin for both BL and diffuse large B cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may utilize different oncogenic pathways2. BL is subdivided into a sporadic subtype (sBL) that is diagnosed in developed countries, the EBV-associated endemic subtype (eBL), and an HIV-associated subtype (hivBL), but it is unclear whether these subtypes employ similar or divergent oncogenic mechanisms. Here we used high throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sBL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival PI(3) kinase pathway in BL, in part by augmenting tonic B cell receptor signaling. In 38% of sBL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL. In particular, current BL therapy entails high dose chemotherapy that cannot be delivered safely to elderly patients or to children in Africa with the endemic-form of BL. Potentially, combinations of targeted agents can be developed based on the molecular pathways that we have defined in BL that can be used safely and effectively in these settings.

我们的实验室已着手开展一项新举措，通过癌症基因重测序发现淋巴恶性肿瘤中的致癌体细胞突变。此前，在基于 RNAi 的遗传筛选中，我们发现涉及 CARD11、BCL10 和 MALT1 的通路负责 ABC DLBCL 中的组成型 NF-kB 信号传导。在对基于 RNAi 的遗传筛查的明确验证中，我们的实验室在 ABC DLBCL 肿瘤活检中发现了 CARD11 基因中反复出现的体细胞突变。 DLBCL 中的所有 CARD11 突变都改变了一个小结构域中的氨基酸，预计该结构域将采用卷曲螺旋结构。这些 CARD11 突变产生了组成性参与 NF-kB 信号传导的蛋白质亚型，这显然是由于它们能够自发形成与 NF-kB 通路中的信号蛋白共定位的大胞质聚集体。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，该信号传导使用野生型 CARD11 激活 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。我们最近发现接头蛋白 MYD88 的致癌信号传导是 ABC DLBCL 中 JAK-STAT3 激活的遗传基础。 ABC DLBCL 中的 RNAi 筛选揭示了这些细胞对 MYD88 及其下游激酶 IRAK1 和 IRAK4 的依赖性。高通量 RNA 重测序 (RNA-seq) 发现 MYD88 中反复出现的突变。 39% 的 ABC DLBCL 中存在 MYD88 突变，其中 29% 影响 MYD88 TIR 结构域中的一个亮氨酸变为脯氨酸 (L265P)。 L265P 突变异构体自发地协调涉及 IRAK1 和 IRAK4 的信号复合物，从而开启 NF-kB、JAK-STAT3 通路和 I 型干扰素通路。 IRAK4激酶的小分子抑制剂对ABC DLBCL细胞具有选择性致死作用，提供了新的治疗前景。 ABC DLBCL 细胞通过 BCR 和 MYD88 途径的联合抑制以协同方式被杀死，这表明针对这两种途径的联合药物的临床试验的发展。有几种新药进入早期临床试验，这些药物针对我们使用功能基因组学方法所涉及的途径。 B 细胞受体信号通路为 ABC DLBCL 的治疗提供了许多可能的靶点，尤其是 BTK。我们在复发/难治性 DLBCL 患者中启动了小分子 BTK 抑制剂依鲁替尼 (ibrutinib) 的 Ib 期临床试验，基因表达谱用于诊断和药效学。迄今为止，依鲁替尼单药疗法已在 ABC DLBCL 患者中诱导了许多完全和部分缓解，包括那些对先前任何治疗均无反应的原发性难治性肿瘤患者。一名患者每天服用依鲁替尼，持续完全缓解超过 3 年，没有明显的副作用。值得注意的是，具有和不具有 CD79B 突变的 ABC DLBCL 肿瘤都有反应，这表明 BCR 通路成瘾可能是这种淋巴瘤亚型的普遍特征。基于这些有希望的结果，我们已经完成了伊布替尼治疗复发/难治性 DLBCL 患者的多中心 2 期试验。根据基因表达谱将患者分为 ABC 或 GCB 亚型，并接受依鲁替尼单药治疗。正如我们实验室研究预测的那样，依鲁替尼对复发/难治性 ABC DLBCL 患者的缓解率（完全 + 部分）为 41%，但对 GCB DLBCL 患者的缓解率仅为 5%。许多反应都是持久的，持续时间从 6 个月到 1 年多。基于这些有希望的结果，目前正在一项 3 期随机试验中评估依鲁替尼与 R-CHOP 化疗联合治疗新诊断的 ABC DLBCL。我们预计依鲁替尼作为单一药物无法治愈大多数 ABC DLBCL 患者，因此需要寻找可能与依鲁替尼协同杀死这些淋巴瘤细胞的其他药物。来那度胺是一种作用相对模糊的药物，但已在多发性骨髓瘤和骨髓增生异常综合征中显示出有效的临床活性。来那度胺还在针对 ABC DLBCL 的早期临床试验中发挥了作用，促使我们研究其在这种情况下的作用方式。出乎意料的是，我们发现来那度胺诱导ABC DLBCL细胞分泌干扰素β，并且这种自分泌产生是来那度胺诱导的ABC DLBCL细胞死亡的重要组成部分。此外，来那度胺通过降低 CARD11 的表达来阻断 B 细胞受体向 NF-kB 发出的信号。这两种表型都可以追溯到来那度胺降低 IRF4 表达的能力。通过RNA干扰和ChIP-seq分析，我们表明IRF4在ABC DLBCL中发挥重要的生存作用。 IRF4 本身是 NF-kB 靶基因，因此抑制 BCR 信号传导的药物（例如依鲁替尼）也会降低 IRF4 表达。我们发现来那度胺和依鲁替尼联合治疗实际上消除了 IRF4 表达，从而协同杀死 ABC DLBCL。这些结果支持临床试验中对该药物组合的评估。伯基特淋巴瘤 (BL) 通常可以通过强化化疗治愈，但这种疗法的毒性使其无法在发展中国家的老年人和地方性 BL (eBL) 患者中使用，因此需要新的策略1。正常生发中心 B 细胞被认为是 BL 和弥漫性大 B 细胞淋巴瘤 (DLBCL) 的起源细胞，但基因表达分析表明这些恶性肿瘤可能利用不同的致癌途径2。 BL分为在发达国家诊断的散发亚型（sBL）、EBV相关地方病亚型（eBL）和HIV相关亚型（hivBL），但尚不清楚这些亚型是否采用相似或不同的致癌机制。在这里，我们使用高通量 RNA 测序和 RNA 干扰筛选来发现 BL 中与 MYC（这种癌症的定义癌基因）配合的重要调控途径。在 70% 的 sBL 病例中，影响转录因子 TCF3 (E2A) 或其负调节因子 ID3 的突变促进了 TCF3 依赖性。 TCF3 部分通过增强强直性 B 细胞受体信号传导，激活 BL 中的促生存 PI(3) 激酶通路。在 38% 的 sBL 病例中，致癌 CCND3 突变产生高度稳定的细胞周期蛋白 D3 亚型，驱动细胞周期进展。这些发现为改善 BL 患者的治疗提供了机会。特别是，目前的 BL 疗法需要高剂量化疗，无法安全地向患有 BL 流行的非洲老年患者或儿童提供治疗。潜在地，可以根据我们在 BL 中定义的分子途径开发靶向药物的组合，并可以在这些环境中安全有效地使用。