Identifying/Targeting Mechanisms of Lymphomagenesis Driven by CREBBP Inactivation

摘要 滤泡性淋巴瘤(FL)是生发中心B(GCB)细胞的一种无痛但无法治愈的恶性肿瘤。 第二常见的非霍奇金淋巴瘤(NHL)。编码赖氨酸的CREBBP的突变 乙酰转移酶(KAT)蛋白存在于约60%的FL中，并在疾病发展过程中早期出现。我们发现 CREBBP在小鼠模型中的条件性敲除(KO)，并可与Bcl2的过度表达协同驱动B- 细胞性淋巴瘤，但这些肿瘤与FL有关键区别。此外，FL肿瘤具有优势 CREBBP Kat结构域内的错义突变，以及罕见的无义/移码突变 它们是由KO建模的。因此，我们模拟了最频繁的CREBBP KAT结构域突变R1446C， 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)具有更深刻的表观遗传效应。H3K27Ac降低的地区 在生发中心B细胞中，富集了通常被CREBBP和BCL6结合的基因座，这表明 H3K27Ac的缺失可能与CREBBP不能对抗依赖于BCL6的HDAC3活性有关。值得注意的是， Bcl6也招募EZH2，在CREBBP-R1446C细胞中H3K27Ac的丢失伴随着 EZH2催化的H3K27me3标记，表明通过丢失H3K27Ac而使增强子失活 通过添加H3K27me3使增强器退役。此外，CREBBP突变表型 可以通过HDAC3或EZH2抑制来部分挽救，并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结构域突变抑制了组蛋白 通过显性抑制机制进行乙酰化，以及CREBBP和CREBBP之间表观遗传串扰 EZH2可能在调节B细胞淋巴瘤的表观遗传格局中发挥重要作用。我们已经延长了 根据这些观察，通过对主要的CREBBP突变进行生化和结构分析 热点和观察到的R1446C/H等位基因与下一个最常见的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起中介作用 与乙酰基供体CoA部分的相互作用，而Y1482和Y1503介导与 乙酰基。虽然R1446突变保持一定的催化活性，但Y1482和Y1503突变是 催化性死亡。因此，这些突变可能会产生不同的功能后果。在这 建议，我们的目标是(I)表征CREBBP Kat结构域的显性表观遗传抑制机制 Kat结构域热点突变之间的突变和功能差异，以及(Ii)定义了Kat结构域热点突变的作用 B细胞淋巴瘤中CREBBP和EZH2之间的表观遗传串扰及其对 目标特工。

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.