Genomic characterization and development of therapies for pediatric sarcoma

1. 横纹肌肉瘤（Rhabdomyosarcoma， RMS）是一种肌源性癌症，是儿童时期最常见的软组织肉瘤。随着多模式化疗方案的发展，局部疾病患者的无复发生存率已提高到70-80%，尽管有明显的毒性。不幸的是，尽管有积极的治疗，转移性疾病患者的5年生存率仍然只有30%。目前的治疗分配是基于临床病理特征，并使用这些标准，可以确定三个不同的亚组患者（低、中、高风险）。然而，许多患者属于中等风险类别（约占所有患者的50%），并且具有异质性的临床结果。这表明，这些儿童中的一些可以用不那么积极的治疗，或者应该被认为患有更严重的疾病。为了进一步表征这种肿瘤类型的遗传事件，我们的团队与儿童肿瘤组（COG）合作，使用全基因组、全外显子组和全转录组测序以及高分辨率SNP阵列进行了大量测序工作，以表征147对肿瘤/正常的体细胞改变。我们的研究结果描述了一组异质性的遗传事件似乎驱动RMS，最明显的是肺泡亚型中的PAX 3/7-FOXO1融合和融合阴性肿瘤中多个RAS通路基因的突变，包括10种癌症共识基因（NRAS， KRAS， HRAS, PIK3CA, bor, TP53, NF1, FGFR4, FBXW7, CTNNB1）的复发性遗传病变。虽然这些突变中的大多数似乎是相互排斥的，但有一部分肿瘤似乎在同一肿瘤内存在共存的病变；也许表明了这些肿瘤的生物学相关进展。目前的工作重点是利用这些结果来启发前瞻性临床试验。这包括实施CLIA认证的检测和信息学管道，以集中分析所有参加儿童肿瘤组试验的横纹肌肉瘤患者。这项工作正在与儿童癌症数据倡议（CCDI）和COG项目：每个儿童方案相关的分子分析方案进行协调。该实验室正积极致力于通过临床和基因组数据的联系来丰富这些可公开访问的数据。为了配合COG和新的低风险研究（ARST2032），需要对TP53和MYOD1突变进行中心测序。除了这些努力之外，今年我的小组继续利用在中高风险临床试验中收集的回顾性样本，生成与患者肿瘤样本相关的分子数据。我们目前的工作重点是了解发生在青少年和年轻成人人群中的横纹肌肉瘤与年轻患者之间的基因组差异。这项工作已经扩展到包括多形性横纹肌肉瘤，这是一种几乎只在成人中发现的肿瘤类型，预后较差。这些肿瘤背后的基因组学是相对未知的。在这项正在进行的工作中，我们试图了解这些组之间是否存在分子差异，这将解释老年和年轻患者之间观察到的生存差异。此外，我们正在与儿童肿瘤组和Fred Barr博士（NCI CCR病理学实验室）合作，以了解肿瘤细胞中发生的转移性和难治性疾病的基因组变化。我们已经与COG （ARST19B1）进行了批准的生物学试验，并已开始使用全外显子组，RNAseq和甲基化阵列对这些样品进行分子分析。我们预计这项工作将在临床特征组患者标本中提供治疗耐药的遗传机制。最后，我们发起了一个合作工作组来检查和靶向TP53突变型横纹肌肉瘤。今年，我们组建了一个国际专家团队，拥有相关的患者样本、临床前模型和新的靶向策略。在过去的一年里，该实验室对RMS中TP53改变的基因型-表型相关性进行了更深入的分析，揭示了导致治疗失败的TP53突变背景的更多见解。2. 在恶性周围神经鞘肿瘤（MPNST）方面的努力本工作的目的是进一步了解丛状神经纤维瘤向恶性周围神经鞘肿瘤（MPNST）转化的分子癌发生的遗传、表观遗传和转录机制。MPNSTs是一种破坏性肉瘤，常发生在1型神经纤维瘤病（NF1）患者中。利用儿科肿瘤科的专业知识和患者群体，在这个项目中，我们使用临床前模型系统来分析NF1肿瘤从良性前体向侵袭性MPNST转变过程中发生的遗传和表观遗传变化。具体来说，我们正在研究多梳复合体（PRC2）成员之一SUZ12或EED突变或缺失的影响，作为向MPNST转化的最后一步。PRC2复合体是细胞内主要的转录抑制因子，在患者样本的基因组测序研究中发现了该复合体成员的复发性改变。本研究使用体外诱导模型系统，结合ChIPseq、RNAseq和ATACseq来指定在这些基因缺失后表达改变的基因，主要目的是鉴定PRC2缺失下游的潜在治疗靶点基因。毫不奇怪，有数百个基因的表达在PRC2复合体重组后发生改变，其中包括表达减少和表达增加的基因群。对这组基因的通路分析表明，与PI3K信号通路、WNT信号通路和细胞周期相关的基因显著富集。有趣的是，由诱导胜任PRC2驱动的转录变化与H3K27me3标记的获得不完全重叠，因此只有一个独特的基因子集似乎在RNA水平上由抑制标记的获得直接调节。对145个基因的分析发现，PRC2的主要下游靶点是显著富集的二价基因，它们的表达在重新引入SUZ12后发生变化。令人感兴趣的是几个谱系特异性转录因子，它们似乎是MPNST细胞中独特的脆弱性。在过去的一年里，我们利用生化、分子生物学和CRISPR技术进一步研究了这些基因与MPNST的功能相关性。这项工作已经鉴定出FOXC1、HOXB8和SNAI2是与原始间充质细胞相关的谱系特异性转录因子。这些转录因子是相对高表达的，当MPNST丢失时，它们是有效的杀手，使它们成为潜在的治疗靶点。用小分子或其他策略靶向这些转录因子的积极努力正在进行中。该实验室通过评估可能成为肿瘤特异性免疫治疗有用靶点的细胞表面分子，扩大了我们对MPNST潜在靶点的研究。迄今为止，我们已经从大量RNA和单细胞RNA测序数据中生成了预测靶点的综合分析。候选靶点正在通过免疫组织化学和流式细胞术检测进行评估。我们目前正在扩大这些努力，以确定患者来源的异种移植模型，该模型将用于测试鉴定的细胞表面抗原的治疗靶向性。

1. Efforts in Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is a myogenic cancer that is the most common soft tissue sarcoma of childhood. With the development of multimodal chemotherapy regimens, relapse-free survival rates have improved to 70-80% in patients with localized disease, albeit with significant toxicity. Unfortunately, despite aggressive therapy, the 5-year survival rate for patients with metastatic disease remains only 30%. Therapy assignment is currently based on clinicopathologic features and using these criteria, three distinct subgroups of patients can be identified (low, intermediate, and high risk). However, many patients fall into the intermediate risk category (which accounts for about 50% of all patients) and have a heterogeneous clinical outcome. This suggests that some of these children could be treated with less aggressive therapy or alternatively should be considered to have more aggressive disease. In an effort to further characterize the genetic events underlying this tumor type, our group in collaboration with the Children's Oncology Group (COG) performed a large sequencing effort using a combination of whole-genome, whole-exome and whole-transcriptome sequencing along with high resolution SNP arrays to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Our findings describe a heterogenous group of genetic events appears to drive RMS most notably the PAX 3/7-FOXO1 fusion in the alveolar subtype and mutation of multiple RAS pathway genes in fusion negative tumors including recurrent genetic lesions in 10 cancer consensus genes (NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1). While the majority of these mutations appear to be mutually exclusive, a subset of tumors appears to have coexisting lesions within the same tumor; perhaps indicating a biologically relevant progression in these tumors. Current efforts are focused on using these results to enlighten prospective clinical trials. This includes implementation of a CLIA certified assay and informatics pipeline to centrally profile all rhabdomyosarcoma patients enrolled on Children's Oncology Group trials. This effort is being coordinated with the molecular profiling protocol associated with the Childhood Cancer Data Initiative (CCDI) and the COG Project:EveryChild protocol. The laboratory is actively working to enrich this publicly accessible data through linkages of the clinical and genomic data. In coordination with the COG and new low risk study has been developed (ARST2032) that requires central sequencing for TP53 and MYOD1 mutations. In addition to these efforts, this year my group continued to generate molecular data associated with patient tumor samples using retrospective samples collected on intermediate and high-risk clinical trials. A current focus of our efforts is to understand the genomic differences between rhabdomyosarcoma that occurs in the adolescent and young adult populations when compared to younger patients. This work has expanded to include Pleomorphic rhabdomyosarcoma, a tumor type that is nearly exclusively found in adults and associated with a poor prognosis. The genomics underlying these tumors is relatively unknown. In this ongoing work, we are attempting to understand if there are molecular differences between these groups that would explain the observed survival differences between older and younger patients. In addition, we are working in collaboration with the Childrens Oncology Group and Dr. Fred Barr (NCI CCR Laboratory of Pathology) to understand the genomic changes that occur in the tumor cell that underlie metastatic and refractory disease. We have an approved biology trial with the COG (ARST19B1) and have begun performing molecular profiling of these samples using whole exome, RNAseq and methylation arrays. We anticipate that this work will deliver genetic mechanisms of therapeutic resistance in a clinically characterized group of patient specimens. Finally, we have initiated a collaborative working group to examine and target TP53 mutant rhabdomyosarcoma. This year we have assembled an international team of experts with relevant patient samples, preclinical models, and novel targeting strategies. Over the past year, the laboratory has added a deeper analysis of the genotype-phenotype correlation of TP53 alterations in RMS, uncovering additional insights into the TP53 mutational background that leads to therapeutic failure. 2. Efforts in Malignant Peripheral Nerve Sheath Tumor (MPNST) The goal of this work is to provide further understanding of the genetic, epigenetic and transcriptomic mechanisms of the molecular oncogenesis underlying the transformation of a Plexiform Neurofibroma to a Malignant Peripheral Nerve Sheath Tumor (MPNST). MPNSTs are a devastating sarcoma that frequently occurs in patients with Neurofibromatosis Type 1 (NF1). Leveraging the expertise and patient population within the Pediatric Oncology Branch, in this project we are using preclinical model systems to dissect the genetic and epigenetic changes that occur as NF1 tumors transition from benign precursors to the aggressive MPNST. Specifically, we are studying the effects of mutation or deletion in one of the polycomb complex (PRC2) members SUZ12 or EED as the final step in the transformation to MPNST. The PRC2 complex is a major transcriptional repressor within the cell and recurrent alterations of members of this complex have been discovered in genomic sequencing studies from patient samples. This work uses inducible in vitro model systems coupled with ChIPseq, RNAseq and ATACseq to nominate genes whose expression is altered upon loss of these genes with the primary objective to identify genes downstream of the PRC2 loss that are potential therapeutic targets. Not surprisingly, there are hundreds of genes whose expression is altered upon reassembly of the PRC2 complex and includes groups of genes whose expression decreases and those whose expression increases. Pathway analysis of this group of genes demonstrated a marked enrichment for genes involved in PI3K signaling, WNT signaling and cell cycle. Interestingly, the transcriptional changes driven by induction of competent PRC2 imperfectly overlapped with gain of the H3K27me3 mark, whereby only a distinct subset of genes appeared to be directly regulated at the RNA level, by the gain of the repressive mark. Analysis of this interesting group of 145 genes discovered a remarkable enrichment of bivalent genes as the primary downstream targets of PRC2 whose expression changes in response to reintroduction of the competent SUZ12. Of interest are several lineage specific transcription factors that appear to be unique vulnerabilities in MPNST cells. In the past year we have further interrogated these genes for functional relevance to MPNST using biochemical, molecular biology and CRISPR technologies. This work has led to identification FOXC1, HOXB8 and SNAI2 that are lineage specific transcription factors associated with primitive mesenchymal cells. These transcription factors are relatively highly expressed and potent killers of MPNST when lost making them potential therapeutic targets. Active efforts to target these transcription factors with small molecules or other strategies is underway. The laboratory has expanded our search for potential targets in MPNST by evaluating cell surface molecules that might be useful targets for the development of tumor specific immune therapy. To date, we have generated a comprehensive analysis of predicted targets from bulk RNA and single cell RNA sequencing data. Candidate targets are being evaluated by immune-histochemistry and flow cytometry assays. We are currently scaling these efforts to identify patient derived xenograft models that will be used to test therapeutic targeting of the identified cell surface antigens.