In Vivo Functional Analysis of Chromosome 7q22 Deletions in Myeloid Malignancies

摘要 大片染色体区域的反复丢失是儿童和成人癌症基因组的一个标志 为揭示这些缺失如何导致恶性生长带来了特殊的挑战。单体7 (-7)和del(7q)(-7/del(7q))是新发髓系恶性肿瘤中反复出现的细胞遗传学异常 与骨髓增生异常综合征(MDS)和急性髓系白血病(AML)的发生密切相关 在儿童和青少年/青壮年(Aya)患者中，遗传性癌症易感性患者和那些 原发癌症治疗后发展为髓系恶性肿瘤。治疗诱发的MDS和AML(t-MDS 和t-AML)由于现代强化治疗，与儿科和Aya人群特别相关 治疗许多实体癌症的方案，这些癌症通常是治愈的。因此，有一个巨大的并在不断增长的 “高危”儿科和AYA癌症幸存者群体。不幸的是，t-MDS/t-AML和其他髓系 7号染色体缺失的恶性肿瘤对目前的治疗方法非常难治。广泛的细胞遗传学 全基因组分析研究表明染色体带7q22的缺失与白血病的发生有关；然而， 测序研究和转录组分析没有发现任何频繁的纯合失活 髓系恶性肿瘤候选7q抑癌基因。这些数据意味着单倍体不足。 或更多的7q基因在白血病发生中，这对阐明潜在的 分子机制。为了解决这个根本问题，我们部署了染色体工程来 建立5A3+/del和5G2+/del小鼠，它们分别存在小鼠染色体5A3和5G2+/del的缺失 5G2。这些缺失跨越了约4MB的基因组DNA，这与最常见的7q22缺失是一致的 在人类患者中被发现。5a3+/del造血干/祖细胞(HSPC)表现为“白血病前期” 异常，但这些小鼠不会自发地发展成MDS或AML。5G2+/del小鼠的初步研究 还发现了HSPC的异常，并将该菌株暴露于N-乙基-N-亚硝脲(ENU)中加速了 血液病的发展。我们将利用这些7q22缺失的新模式来追求 目的：(1)功能检测5G2+/del和5A3+/del/5G2+/del小鼠的造血功能； 观察小鼠发生髓系恶性肿瘤的队列情况；(2)观察DNA对小鼠的影响 5A3+/del/5G2+/del HSPC的损伤剂；以及，(3)建立髓系恶性肿瘤的复杂遗传学模型 通过在5A3+/del/5G2+/del HSPC中引入协同突变并对其表型进行鉴定，得到-7/del(7q) 以及体内的功能后果。精确建立复发模型的基因工程小鼠 在人类髓系恶性肿瘤中发现的染色体带7q22缺失是一种执行 功能研究和测试新的治疗策略。

ABSTRACT Recurring losses of large chromosomal regions are a hallmark of pediatric and adult cancer genomes that pose exceptional challenges for uncovering how these deletions contribute to malignant growth. Monosomy 7 (-7) and del(7q) (-7/del(7q)) are recurring cytogenetic abnormalities in de novo myeloid malignancies that are strongly associated with cases of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) arising in children and in adolescent/young adult (AYA) patients with inherited cancer predispositions and in those who develop myeloid malignancies after treatment for a primary cancer. Therapy-induced MDS and AML (t-MDS and t-AML) are particularly relevant to the pediatric and AYA population due to modern intensive treatment protocols for many solid cancers, which are frequently curative. As a result, there is a large and growing population of “at risk” pediatric and AYA cancer survivors. Unfortunately, t-MDS/t-AML and other myeloid malignancies with chromosome 7 deletions are highly refractory to current therapies. Extensive cytogenetic and genome wide analysis studies implicate deletions of chromosome band 7q22 in leukemogenesis; however, sequencing studies and transcriptome analysis did not reveal frequent homozygous inactivation of any candidate 7q tumor suppressor gene in myeloid malignancies. These data implicate haploinsufficiency for one or more 7q genes in leukemogenesis, which pose formidable challenges for elucidating the underlying molecular mechanisms. To address this fundamental problem, we deployed chromosome engineering to create 5A3+/del and 5G2+/del mice, which respectively harbor deletions in mouse chromosome bands 5A3 and 5G2. These deletions span ~4 MB of genomic DNA that is syntenic to the most common 7q22 deletions identified in human patients. 5A3+/del hematopoietic stem and progenitor cells (HSPC) exhibit “preleukemic” abnormalities, but these mice do not spontaneously develop MDS or AML. Preliminary studies of 5G2+/del mice also revealed HSPC abnormalities and exposing this strain to N-ethyl-N-nitrosourea (ENU) accelerated the development of hematologic cancer. We will utilize these novel models of 7q22 deletions to pursue the following specific aims: (1) to functionally interrogate hematopoiesis in 5G2+/del and 5A3+/del/5G2+/del mice and to observe cohorts of mice for the development of myeloid malignancies; (2) to investigate the effects of DNA damaging agents on 5A3+/del/5G2+/del HSPC; and, (3) to model the complex genetics of myeloid malignancies with -7/del(7q) by introducing cooperating mutations into 5A3+/del/5G2+/del HSPC and assessing the phenotypic and functional consequences in vivo. Genetically engineered mice that accurately model recurrent chromosome band 7q22 deletions found in human myeloid malignancies are a versatile system for performing functional studies and testing new therapeutic strategies.