Genomic characterization and development of therapies for pediatric sarcoma

点击翻译按钮获取中文摘要

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.