Hepatitis B virus integrations in KMT2B drive hepatocellular carcinoma

摘要 慢性乙肝病毒感染是导致肝细胞癌的主要原因。仅有乙肝病毒 占全球所有癌症地点癌症死亡总数的3%以上。乙肝病毒如何推广 癌症的发生机制还不完全清楚，导致晚期疾病的治疗选择有限。我们 先前发现，10%的乙肝病毒相关的肝细胞癌在KMT2B中携带有乙肝病毒的克隆性整合 癌细胞基因组中的基因座。所有这些整合位于KMT2B的外显子3和6之间 吉恩。KMT2B是KMT2A(也称为MLL1)唯一的同源基因。已经很好地确定了 影响KMT2A的染色体易位是婴儿混合血统白血病的主要驱动因素。乙肝病毒 肝细胞癌中KMT2B的整合可能反映了白血病中的KMT2A重排，但其功能尚未得到证实 学习。我们已经鉴定出患者来源的肝癌细胞系在KMT2B中携带乙肝病毒整合。使用这些电池 KMT2B中整合了乙肝病毒的肝癌细胞株和RNA-seq数据，我们发现这些整合导致 将N-末端截短的KMT2B表达为697-906个氨基酸残基。淘汰赛 携带整合体的细胞系中的KMT2B截断在体外抑制了细胞的增殖。的过度表达 KMT2B截短基因在体外转化人正常肝细胞及体内诱导肿瘤生长 基于流体力学注射的小鼠模型。从力学上讲，我们发现KMT2B截断隔离了 来自KMT2A/B组蛋白甲基转移酶复合体和染色体的肿瘤抑制因子Menin。A KMT2B 携带阻断Menin结合的突变的截断未能促进体内肿瘤的形成。基于 这些初步数据，我们的中心假设是，乙肝病毒在KMT2B中的整合产生截断，这 从KMT2B复合体中隔离Menin并促进肝细胞癌。为了检验这一假设， 我们将使用不同的体外和体内模型来确定KMT2B截断的致癌功能(AIM 1)，并确定了KMT2B截断触发的肿瘤发生机制(AIM2)。具体地说，新的 基因工程小鼠肝细胞癌模型的建立及针对携带乙肝病毒的肝细胞癌的潜在靶向治疗 将测试KMT2B中的集成。建议的研究旨在建立致癌驱动功能和 肝细胞癌中HBVKMT2B整合的机制，每年导致3.5万例肝细胞癌。它是世界上 最常见的致癌基因改变，但对其研究严重不足。该计划的完成 拟议的研究将发现一种新的肿瘤病毒驱动的、肝癌发生和发展的表观遗传学机制 为制定预防方法、早期检测分析和靶向治疗提供信息 受影响的个体。

ABSTRACT Chronic infection by hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC). HBV alone is responsible for more than 3% of worldwide cancer deaths of all cancer sites combined. How HBV promotes carcinogenesis is not fully understood, leading to limited therapeutic options for advanced diseases. We previously revealed that 10% of HBV-associated HCC tumors carry clonal integrations of HBV in the KMT2B gene locus in cancer cell genomes. All these integrations are located between exons 3 and 6 of the KMT2B gene. KMT2B is the only close homolog of KMT2A (also known as MLL1). It has been well established that the chromosomal translocations affecting KMT2A are the primary drivers of infant mixed lineage leukemia. HBV integrations in KMT2B in HCC may mirror KMT2A rearrangements in leukemia, but its function has not been studied. We have identified patient-derived HCC cell lines carrying HBV integrations in KMT2B. Using these cell lines and RNA-seq data of HCC tumors with HBV integrations in KMT2B, we found that these integrations lead to expression of truncated KMT2B from the N-terminus to 697 to 906 amino acid residues. Knockout of the KMT2B truncation in an integration-carrying cell line suppressed cell proliferation in vitro. Overexpression of a KMT2B truncation transformed normal human hepatocytes in vitro and induced tumor growth in vivo in hydrodynamic injection-based mouse models. Mechanistically, we found the KMT2B truncation sequestered the tumor suppressor MENIN from the KMT2A/B histone methyltransferase complex and chromosome. A KMT2B truncation carrying a mutation that blocks MENIN binding failed to promote tumor formation in vivo. Based on these preliminary data, our central hypothesis is that HBV integrations in KMT2B produce truncations, which sequester MENIN from KMT2B complex and promote hepatocellular carcinoma. To test this hypothesis, we will determine the oncogenic function of KMT2B truncations, using various in vitro and in vivo models (Aim 1), and determine the mechanism of KMT2B truncation-triggered tumorigenesis (Aim2). Specifically, a new genetic engineered mouse model of HCC will be generated and a potential targeted agent for HCC with HBV integrations in KMT2B will be tested. The proposed studies aim to establish the oncogenic driving function and mechanism of HBV integrations in KMT2B in HCC, which cause 35,000 HCC cases every year. It is among the most common cancer-causing genetic alterations but it is significantly understudied. The completion of the proposed studies will discover a novel oncovirus-driving, epigenetic mechanism of HCC development and provide information for developing prevention approaches, early detection assays, and targeted therapeutics for affected individuals.