Transcriptome Sequence Analysis of Pediatric Acute Megakaryoblastic Leukemia Identifies An Inv(16)(p13.3;q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein As a Recurrent Lesion in 39% of Non-Infant Cases [2010]. Homo sapiens

Transcriptome Sequence Analysis of Pediatric Acute Megakaryoblastic Leukemia Identifies An Inv(16)(p13.3;q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein As a Recurrent Lesion in 39% of Non-Infant Cases: A Report From the St. Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project. Acute Megakaryoblastic Leukemia (AMKL) accounts for ~10% of childhood acute myeloid leukemia (AML). Although AMKL patients with down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS of less than 40%. With the exception of the t(1;22) translocation seen in infant non-DS-AMKL, little is known about the molecular genetic lesions that underlie this leukemia subtype. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases (discovery cohort) using the illumina platform. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of the CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. 6 cases in the discovery cohort fused exon 10 of CBFA2T3 to exon 3 of GLIS2, while 1 case carried a larger product that fused exon 11 of CBFA2T3 to exon 1 of GLIS2. Both products retain the 3 CBFA2T3 N-terminal nervy homology regions that mediate protein interactions, and the 5 GLIS2 C-terminal zinc finger domains that bind the Glis DNA consensus sequence, along with one of its N-terminal transcriptional regulatory domains. GLIS2 is a member of the GLI super family of transcription factors and has been demonstrated to play a role in regulating expression of GLI target genes as well as inhibiting WNT signaling through the binding of beta catenin. Although GLIS2 is not normally expressed in hematopoietic cells, the translocation results in high level expression of the CBFA2T3-GLIS2 fusion protein. In addition to CBFA2T3-GLIS2, chimeric transcripts were detected in 6/7 cases that lacked evidence of the inv(16)(p13.3;q24.3). Specifically, we detected GATA2-HOXA9, MN1-FLI1, NIPBL-HOXB9, NUP98-KDM5A, GRB10-SDK1 and C8orf76-HOXA11AS, each in an individual case. Importantly, several of the genes involved in these translocations either play a direct role in normal megakaryocytic differentiation (GATA2 and FLI1), or have been previously shown to be involved in leukemogenesis (HOXA9, MN1, HOXB9). Evaluation of a recurrency cohort of 42 samples including 14 additional pediatric cases and 28 adult cases by RT-PCR revealed 4 additional pediatric samples carrying CBFA2T3-GLIS2 for an overall frequency of 39% in pediatric AMKL. In addition to these somatic structural variations, we also identified mutations in genes previously shown to play a role in megakaryoblastic leukemia including activating mutations in JAK2 and MPL (36%). To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a mCherry expressing retroviral vector failed to form colonies after the second replating. By contrast, expression of either wild-type GLIS2 or the CBFA2T3-GLIS2 fusion resulted in a marked increase in the self-renewal capacity, with colony formation persisting through eight replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. Importantly, the CBFA2T3-GLIS2 cells remained growth factor dependent suggesting that cooperating mutations in growth factor signaling pathways are required for full leukemic transformation. Taken together these data identify a novel cryptic inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in approximately 40% of non-infant pediatric non-DS-AMKLs. Moreover, the majority of pediatric cases that lacked this lesion were shown by transcriptome sequence analysis to contain other chromosomal rearrangements that encoded fusion proteins that directly alter megakaryocytic differentiation and/or myeloid cell growth. The alteration of a key transcriptional regulator within the hedgehog signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway may have a therapeutic benefit in this aggressive form of AML. Overall design: Gene expression profiling was performed on 14 single diagnosis tumor samples

儿童急性巨核细胞白血病（Acute Megakaryoblastic Leukemia, AMKL）的转录组序列分析鉴定出inv(16)(p13.3;q24.3)编码的CBFA2T3-GLIS2融合蛋白在39%的非婴儿病例中为复发性病变：来自圣犹达儿童研究医院（St. Jude Children’s Research Hospital）-华盛顿大学（Washington University）儿童癌症基因组项目的报告。急性巨核细胞白血病（AMKL）约占儿童急性髓系白血病（acute myeloid leukemia, AML）的10%。尽管合并唐氏综合征（Down Syndrome, DS）的AMKL患者（DS-AMKL）具有极佳的5年无事件生存期（event-free survival, EFS），但非DS-AMKL患者的预后极差，3年无事件生存期不足40%。除了婴儿非DS-AMKL中可见的t(1;22)易位（translocation）外，人们对该白血病亚型背后的分子遗传病变知之甚少。为明确非DS-AMKL中发生的突变全景，我们采用Illumina平台对14例确诊病例的诊断性原始细胞（blasts）进行了转录组测序（发现队列，discovery cohort）。结果在12/14例病例中鉴定出可表达新型融合转录本（fusion transcript）的染色体重排（chromosomal rearrangement）。值得注意的是，7/14例病例中我们检测到16号染色体倒位[inv(16)(p13.3;q24.3)]，该倒位使ETO家族转录因子（ETO family of transcription factors）成员CBFA2T3与GLIS2相邻，形成CBFA2T3-GLIS2嵌合基因，编码框内融合蛋白（in frame fusion protein）。发现队列中6例病例的融合转录本由CBFA2T3的第10外显子（exon）与GLIS2的第3外显子拼接而成，另有1例病例的融合产物更长，由CBFA2T3的第11外显子与GLIS2的第1外显子拼接得到。两种融合产物均保留了CBFA2T3 N端的3个介导蛋白相互作用的神经同源结构域（nervy homology regions），以及GLIS2 C端的5个结合Glis DNA共识序列的锌指结构域（zinc finger domains），同时保留了其N端的一个转录调控结构域。GLIS2属于GLI超家族转录因子（GLI super family of transcription factors），已被证实可调控GLI靶基因的表达，并通过结合β连环蛋白（beta catenin）抑制WNT信号通路（WNT signaling）。尽管正常造血细胞（hematopoietic cells）中通常不表达GLIS2，但该染色体重排可导致CBFA2T3-GLIS2融合蛋白的高水平表达。除CBFA2T3-GLIS2外，在6/7例未检测到inv(16)(p13.3;q24.3)的病例中也检测到了嵌合转录本。具体而言，我们在各1例病例中分别检测到GATA2-HOXA9、MN1-FLI1、NIPBL-HOXB9、NUP98-KDM5A、GRB10-SDK1及C8orf76-HOXA11AS融合转录本。重要的是，这些易位所涉及的基因中，部分直接参与正常巨核细胞分化（megakaryocytic differentiation，如GATA2与FLI1），或此前已被证实与白血病发生（leukemogenesis）相关（如HOXA9、MN1、HOXB9）。我们通过逆转录聚合酶链反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）对包含14例额外儿童病例与28例成人病例的42例样本组成的复发性验证队列进行评估，额外发现4例儿童样本携带CBFA2T3-GLIS2融合，使儿童AMKL中该融合的总体发生率达到39%。除上述体细胞结构变异（somatic structural variations）外，我们还鉴定出此前被证实与巨核细胞白血病相关的基因突变，包括JAK2与MPL的激活突变（activating mutations，发生率36%）。为探究CBFA2T3-GLIS2促进白血病发生的机制，我们将该融合基因导入小鼠造血细胞，并以体外集落再接种试验（colony replating）作为自我更新（self-renewal）的替代检测手段评估其效应。仅表达mCherry的逆转录病毒载体（retroviral vector）转导的造血细胞在第二次集落再接种后无法形成集落。相比之下，表达野生型GLIS2或CBFA2T3-GLIS2融合蛋白的细胞的自我更新能力显著增强，集落形成能力可维持至第8次集落再接种。对表达CBFA2T3-GLIS2的集落进行免疫表型分析（immunophenotypic analysis），结果显示存在巨核细胞分化的特征。重要的是，CBFA2T3-GLIS2转导的细胞仍依赖生长因子，提示需要生长因子信号通路中的协同突变才能实现完全的白血病转化（leukemic transformation）。综上，本研究鉴定出一种新型隐匿性inv(16)编码的CBFA2T3-GLIS2融合蛋白，其在约40%的非婴儿儿童非DS-AMKL中为复发性驱动突变（driver mutation）。此外，转录组测序分析显示，多数未携带该融合病变的儿童病例存在其他可编码融合蛋白的染色体重排，这些融合蛋白可直接改变巨核细胞分化及髓系细胞生长。在近半数儿童AMKL病例中，刺猬信号通路（hedgehog signaling pathways）中的关键转录调控因子发生改变，这提示抑制该通路或可对这种侵袭性AML亚型产生治疗获益。总体实验设计：对14例单次确诊的肿瘤样本进行基因表达谱分析。