Identifying/Targeting Mechanisms of Lymphomagenesis Driven by CREBBP Inactivation

CREBBP 失活驱动的淋巴瘤发生的识别/靶向机制

基本信息

批准号：
10614012
负责人：
Michael Richard Green
金额：
$ 35.9万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-04 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10614012
关键词：
Acetyl Coenzyme A Acetylation Acetyltransferase Alleles Amino Acids B-Cell Development B-Cell Lymphomas B-Lymphocytes BCL6 gene Binding Biochemical Biology CREBBP gene Cell Line Cell Survival Cells Chromatin Chromatin Remodeling Factor Clustered Regularly Interspaced Short Palindromic Repeats Coenzyme A Compensation Complex DNA Damage Darkness Deacetylation Development Disease EP300 gene EZH2 gene Enhancers Enzymes Epigenetic Process Etiology Event Evolution Failure Follicular Dendritic Cells Follicular Lymphoma Frameshift Mutation Gene Expression Gene Mutation Genes HDAC3 gene Helper-Inducer T-Lymphocyte Histone Acetylation Histones Immunoglobulins Indolent Knock-out Light Link Lymphoma cell Lymphomagenesis Lysine Malignant Neoplasms Maps Mediating Methylation Missense Mutation Modeling Molecular Mutation Non-Hodgkin&apos s Lymphoma Pathway interactions Patient-Focused Outcomes Phenotype Play Polycomb Proliferating Proteins Recurrence Recycling Repression Repressor Proteins Role Structure of germinal center of lymph node Tail Therapeutic Transgenic Mice combinatorial conditional knockout efficacy evaluation epigenetic regulation epigenome genetic corepressor histone acetyltransferase improved in vitro Assay inhibitor molecular phenotype mouse model mutant mutational status neglect novel overexpression patient derived xenograft model prevent programs recruit response targeted agent transcription factor tumor

项目摘要

ABSTRACT Follicular lymphoma (FL) is an indolent but incurable malignancy of germinal center B (GCB)-cells, and is the second most common form of non-Hodgkin lymphoma (NHL). Mutations of CREBBP, which encodes a lysine acetyltransferase (KAT) protein, occur in ~60% of FL and arise early during disease evolution. We found that conditional knock-out (KO) of Crebbp in murine models and can cooperate with Bcl2 over-expression to drive B- cell lymphoma, but that these tumors had key differences to FL. Furthermore, FL tumors have a predominance of missense mutations within the CREBBP KAT domain and infrequently harbor nonsense/frameshift mutations that are modeled by KO. We therefore modeled the most frequent CREBBP KAT domain mutation, R1446C, using CRISPR editing of a CREBBP wild-type (WT) lymphoma cell line and found that CREBBP-R1446C mutation has a more profound epigenetic effect than biallelic knock-out (KO). Regions with reduced H3K27Ac were enriched for loci that are normally bound by both CREBBP and BCL6 in germinal center B-cells, suggesting that the loss H3K27Ac may be linked to failure of CREBBP to oppose BCL6-dependent HDAC3 activity. Notably, BCL6 also recruits EZH2 and loss of H3K27Ac in CREBBP-R1446C cells was accompanied by a gain of the EZH2-catalyzed H3K27me3 mark, suggesting that enhancer inactivation via loss of H3K27Ac is followed by enhancer decommissioning through addition of H3K27me3. Furthermore, the CREBBP mutation phenotype could be partially rescued by either HDAC3 or EZH2 inhibition, and was further enhanced by combination of HDAC3 and EZH2 inhibitors. These results suggest that CREBBP KAT domain mutations suppress histone acetylation through a dominant repressive mechanism, and that epigenetic crosstalk between CREBBP and EZH2 may play a prominent role in regulating the epigenetic landscape of B-cell lymphoma. We have extended upon these observations by performing biochemical and structural analysis of major CREBBP mutational hotspots and observed key differences between the R1446C/H alleles and the next most common hotspots at Y1482 and Y1503. These amino acids all reside within the catalytic pocket of CREBBP, but R1446 mediates interaction with the CoA portion of the acetyl donor, acetyl-CoA, while Y1482 and Y1503 mediate interaction with the acetyl group. While R1446 mutations maintain some catalytic activity, the Y1482 and Y1503 mutations are catalytically dead. Therefore, these mutations are likely to have different functional consequences. In this proposal, we aim to (i) characterize the mechanism of dominant epigenetic repression by CREBBP KAT domain mutations and differences in function between KAT domain hotspot mutations, and (ii) define the role of epigenetic crosstalk between CREBBP and EZH2 in B-cell lymphoma, and the consequences for response to targeted agents.

抽象的滤泡性淋巴瘤 (FL) 是一种惰性但无法治愈的生发中心 B (GCB) 细胞恶性肿瘤，是第二种最常见的非霍奇金淋巴瘤 (NHL)。 CREBBP 突变，编码赖氨酸乙酰转移酶 (KAT) 蛋白出现在约 60% 的 FL 中，并在疾病演变的早期出现。我们发现在小鼠模型中条件性敲除（KO）Crebbp，并可与 Bcl2 过表达配合驱动 B- 细胞淋巴瘤，但这些肿瘤与 FL 有关键区别。此外，FL肿瘤占主导地位 CREBBP KAT 结构域内存在错义突变，并且很少含有无义/移码突变是由 KO 建模的。因此，我们对最常见的 CREBBP KAT 结构域突变 R1446C 进行了建模，使用 CRISPR 编辑 CREBBP 野生型 (WT) 淋巴瘤细胞系，发现 CREBBP-R1446C 突变比双等位基因敲除（KO）具有更深远的表观遗传效应。 H3K27Ac 减少的区域富集了生发中心 B 细胞中通常与 CREBBP 和 BCL6 结合的位点，这表明 H3K27Ac 的缺失可能与 CREBBP 无法对抗 BCL6 依赖性 HDAC3 活性有关。尤其， BCL6 还招募 EZH2，CREBBP-R1446C 细胞中 H3K27Ac 的缺失伴随着 EZH2 的增加 EZH2 催化的 H3K27me3 标记，表明增强子通过 H3K27Ac 丢失而失活，随后是通过添加 H3K27me3 增强子停用。此外，CREBBP突变表型可以通过 HDAC3 或 EZH2 抑制来部分挽救，并且通过联合使用进一步增强 HDAC3 和 EZH2 抑制剂。这些结果表明 CREBBP KAT 结构域突变抑制组蛋白通过显性抑制机制乙酰化，以及 CREBBP 和 CREBBP 之间的表观遗传串扰 EZH2 可能在调节 B 细胞淋巴瘤的表观遗传景观中发挥重要作用。我们已经延长了根据这些观察结果，对主要 CREBBP 突变进行生化和结构分析热点并观察到 R1446C/H 等位基因与下一个最常见热点之间的关键差异 Y1482 和 Y1503。这些氨基酸都位于 CREBBP 的催化口袋内，但 R1446 介导与乙酰基供体乙酰辅酶 A 的 CoA 部分相互作用，而 Y1482 和 Y1503 介导与乙酰基。虽然 R1446 突变保持了一定的催化活性，但 Y1482 和 Y1503 突变催化死亡。因此，这些突变可能会产生不同的功能后果。在这个建议，我们的目标是 (i) 表征 CREBBP KAT 结构域显性表观遗传抑制的机制突变和 KAT 结构域热点突变之间的功能差异，以及 (ii) 定义 B 细胞淋巴瘤中 CREBBP 和 EZH2 之间的表观遗传串扰，以及对治疗反应的影响针对性的代理。