MOLECULAR TARGETS OF TRANSLOCATION T(4;14) IN MULTIPLE MYELOMA PATHOGENESIS

描述(申请人提供)：多发性骨髓瘤(MM)是美国第二大最常见的血液系统恶性肿瘤，每年约有14,500名美国人确诊，占所有癌症死亡的2%(seer.ancer.gov)。MM是一种无法治愈的恶性抗体分泌血浆B细胞，其病因尚不清楚。染色体易位t(4；14)(p16.3；q32.33)与总生存期缩短有关，在15%的多发性骨髓瘤患者中发现。候选癌基因WHSC1(MMSET)位于4p16.3，编码一种组蛋白甲基转移酶，是多发性骨髓瘤细胞t(4；14)过表达的结果。令人惊讶的是，WHSC1基因的过度表达在小鼠中表达时，未能转化细胞系或诱导任何表型。我们发现ACA11是一种编码在WHSC1中的孤儿盒H/ACA小核仁RNA(SnoRNA)，位于4p16.3，在含有t(4；14)的细胞系和MM患者样本中上调。ACA11调节氧化应激，促进MM细胞生长，并对化疗产生耐药性。ACA11是一种新的致癌非编码RNA，在其他类型的肿瘤中也有表达。骨髓瘤发病过程中与t(4；14)协同的突变尚不清楚，但13号染色体缺失(Del(13))与t(4；14)密切相关，在85-94%的t(4；14)患者中发现Del(13)，且预后不良。我们将多发性骨髓瘤患者样本中的最小缺失区域定位于13q14的视网膜母细胞瘤(RB1)基因。我们的模型是ACA11的表达与RB1的缺失协同导致预后不良的MM。我们建议描述ACA11在多发性骨髓瘤中的作用。具体目标1：ACA11和组蛋白甲基转移酶WHSC1之间的合作。我们假设ACA11与MMSET蛋白在骨髓瘤发病机制中的表达是协同的，并且WHSC1组蛋白甲基转移酶的活性是协同作用所必需的。我们将在细胞系和小鼠中单独或与ACA11共表达野生型WHSC1或HMT-Dead WHSC1。具体目标2：目标2：ACA11如何影响ROS水平和对化疗的敏感性？ACA11需要哪些域和合作伙伴才能实现这些效果？我们的数据表明，ACA11调节骨髓瘤中的活性氧物种和化疗耐药性。我们将确定这一功能需要哪些ACA11残基。我们推测，ACA11RNA结合残基对于ACA11‘S的氧化应激功能是必不可少的，但ACA11’S蛋白结合区将是关键。具体目标3：Rb1缺失背景下ACA11介导的恶性转化。50%的多发性骨髓瘤患者13号染色体长臂缺失，90%的t(4；14)患者缺失。我们假设在骨髓瘤的发病机制中，rb1基因缺失与t(4；14)基因协同作用。我们将在Rb1缺乏的情况下测试ACA11和WHSC1表达对B细胞发育、氧化应激和转化的影响。最后，我们将[通过对患者样本进行RNA测序来识别t(4；14)的目标基因。]深入了解t(4；14)基因在骨髓增生症中的作用对癌症生物学具有广泛的意义，并将有助于为预后不良的MM患者开发新的治疗方法。

DESCRIPTION (provided by applicant): Multiple Myeloma (MM) is the second most common hematologic malignancy in the United States, diagnosed in approximately 14,500 Americans each year, and is responsible for 2% of all cancer deaths (SEER.cancer.gov). MM is an incurable malignancy of antibody-secreting plasma B-cells whose etiology is poorly understood. Chromosomal translocation t(4;14)(p16.3;q32.33) is associated with shorter overall survival and is found in 15% of MM patients. The candidate oncogene WHSC1 (MMSET) at 4p16.3 encodes a histone methyltransferase over-expressed as a result of the t(4;14) in MM cells. Surprisingly, over-expression of WHSC1 cDNA's failed to transform cell lines or to induce any phenotype when expressed in mice. We identified ACA11, an orphan box H/ACA small nucleolar RNA (snoRNA) encoded hosted within WHSC1 at 4p16.3, to be up-regulated in cell lines and MM patient samples harboring t(4;14). ACA11 modulates oxidative stress, contributes to MM cell growth and confers resistance to chemotherapy. ACA11 is a novel oncogenic non-coding RNA and is also expressed in other cancer types. Mutations that cooperate with t(4;14) in myeloma initiation are unknown, but chromosome 13 deletions (Del(13)) are highly correlated with the t(4;14) in MM. Del(13) is found in 85-94% of patients with t(4;14) and associated with poor prognosis. We mapped a minimally deleted region in MM patient samples to the retinoblastoma (RB1) gene at 13q14. Our model is that ACA11 expression cooperates with RB1 loss to cause poor-prognosis MM. We propose to characterize the role of ACA11 in multiple myeloma. Specific Aim 1: Cooperation between ACA11 and the histone methyltransferase WHSC1. We hypothesize that ACA11 cooperates with MMSET protein expression in myeloma pathogenesis and that WHSC1 histone methyltransferase activity is required for cooperation. We will co-express wild-type WHSC1 or HMT-dead WHSC1 alone or with ACA11 in cell lines and mice. Specific Aim 2: Aim 2: How does ACA11 affect levels of ROS and sensitivity to chemotherapy? What domains and partners of ACA11 are required for these effects? Our data suggest that ACA11 modulates reactive oxygen species and resistance to chemotherapy in myeloma. We will determine which residues of ACA11 are required for this function. We hypothesize that ACA11 RNA binding residues will be dispensable for ACA11's oxidative stress function, but that ACA11's protein-binding domains will be critical. Specific Aim 3: ACA11-mediated malignant transformation in the context of Rb1 deletion. The long arm of chromosome 13 is deleted in 50% of all MM patients, and in 90% of patients with the t(4;14). We hypothesize that RB1 loss cooperates with the t(4;14) in myeloma pathogenesis. We will test the effects of ACA11 and WHSC1 expression on B-cell development, oxidative stress and transformation in the context of Rb1 deficiency. Lastly, we will [identify target genes of t(4;14) by RNA- sequencing patient samples.] A betting understanding of how the t(4;14) contributes to myelomagenesis has broad implications to cancer biology and will facilitate the development of novel therapeutic approaches to patients with poor-prognosis MM.