Identifying/Targeting Mechanisms of Lymphomagenesis Driven by CREBBP Inactivation

摘要 滤泡性淋巴瘤（FL）是一种惰性但不可治愈的生殖中心B（GCB）细胞恶性肿瘤， 非霍奇金淋巴瘤（NHL）的第二常见形式。CREBBP的突变，其编码赖氨酸 乙酰转移酶（KAT）蛋白，发生在约60%的FL和疾病演变过程中出现的早期。我们发现 在小鼠模型中，Crebbp的条件性敲除（KO），并且可以与Bcl 2过表达协同驱动B- 细胞淋巴瘤，但这些肿瘤有关键的差异，FL。此外，FL肿瘤具有优势， CREBBP KAT结构域内的错义突变，并且很少携带无义/移码突变 由KO建模。因此，我们模拟了最常见的CREBBP KAT结构域突变R1446 C， 使用CREBBP野生型（WT）淋巴瘤细胞系的CRISPR编辑，发现CREBBP-R1446 C 突变具有比双等位基因敲除（KO）更深刻的表观遗传效应。H3 K27 Ac还原的区域 在生发中心B细胞中富集了通常被CREBBP和BCL 6结合的基因座，这表明 H3 K27 Ac的缺失可能与CREBBP不能对抗BCL 6依赖性HDAC 3活性有关。值得注意的是， BCL 6还募集EZH 2，CREBBP-R1446 C细胞中H3 K27 Ac的丧失伴随着EZH 2的增加。 EZH 2-催化的H3 K27 me 3标记，表明通过H3 K27 Ac的损失而导致的增强子失活之后， 通过添加H3 K27 me 3使增强剂退役。此外，CREBBP突变表型 HDAC 3或EZH 2抑制可部分挽救，并且通过HDAC 3或EZH 2抑制的组合进一步增强。 HDAC 3和EZH 2抑制剂。这些结果表明CREBBP KAT结构域突变抑制组蛋白表达， 通过显性抑制机制乙酰化，以及CREBBP和 EZH 2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。我们延长 通过对CREBBP的主要突变进行生物化学和结构分析， 热点和观察到的R1446 C/H等位基因和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介导了CREBBP的活性。 Y1482和Y1503介导与乙酰供体的CoA部分乙酰辅酶A的相互作用，而Y1482和Y1503介导与乙酰供体的CoA部分乙酰辅酶A的相互作用。 乙酰基。虽然R1446突变保持了一些催化活性，但Y1482和Y1503突变是不稳定的。 催化死亡。因此，这些突变可能具有不同的功能后果。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 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EZH 2之间的表观遗传串扰，以及对 目标代理人

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.