Functional Genomics of Sarcoma

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Our laboratory has had a long-standing interesting sarcoma genome biology. Our recent efforts are focused on the investigation of important emerging aspects of pediatric and adult sarcoma multi-omics analyses. We have investigated the molecular consequences of specific mutations which occur in sarcoma, particularly the common chromosome translocations which produce the fusion gene transcription factors characteristic of several pediatric sarcomas. Using chromatin immunoprecipitation and DNA sequencing technology (ChIP-Seq), we have identified the binding sites of oncogenic transcription factors and integrating this information with the known expression profiles of these diseases. In Ewing sarcoma, we have also used RNA interference technology to target the oncogenic transcription factor EWS-FLI1 to study the genes regulated by this protein. In alveolar rhabdomyosarcoma we have used ChIP-Seq to identify the genes which are targeted by the oncogenic fusion protein PAX3-FKHR. This has provided the framework for building the network of dysregulated genes downstream of these critical oncogenic events. Currently our efforts are focused primarily on pursuing leads arising from studies of osteosarcoma. Because of the highly chaotic nature of the osteosarcoma genome, prioritizing candidate genes for investigation is challenging. We now understand that the near universal presence of defects in cell cycle regulators underpins genome complexity. Osteosarcoma cell line models capture many aspects of this process providing a platform for functional investigations. These models also can be useful for study aspects of chromosome biology and genome instability. For example, we recently determined that osteosarcoma cells often maintain their telomeres through the Alternative Lengthening of Telomeres Pathway (ALT). Data from TCGA suggests that ALT tumors have more mis-repaired double strand breaks than TERT expressing tumors. We are interested to uncover the potential role of ALT in osteosarcoma genome structural instability as well as to identify possible dependencies of cells in the ALT state. The G292 osteosarcoma cell line has a defect in the DAXX gene as the molecular basis of its ALT phenotype. We modified G292 cells with an inducible DAXX construct has allowed us to investigate changes during the inducible suppression and re-expression of ALT. Using our published G292 iDAXX model, we observed that gene expression changes are minimal, suggesting that post-transciptional events are most important to transitions to and from the ALT state. In iDAXX we have observed that the APBs (ALT PML Bodies) ALT specific nuclear structures observed in the ALT state disappear upon ALT suppression. We are investigating this and related changes with imaging, genomics and biochemical techniques. Currently, we are using Split-Pool Recognition of Interactions by Tag Extension (SPRITE) to identify changes in large scale chromatin associations in iDAXX in the ALT vs. nonALT states. Also in osteosarcoma cell line models, we are investigating the functional properties of a novel target for immunotherapy target, LRCC15. This protein is a membrane anchored protein found on the surface of osteosarcoma cells as well as some stromal cells. It appears to bind important components of the bone microenvironment, notaby Type I collagen. We have recently reported the activity of an LRRC15 ADC in osteosarcoma cell line xenografts. Concurrently, we established that high LRRC15 expression can be induced even in cell lines with low baseline expression. These observations have led us to investigate the biology of LRRC15. Using a panel of osteosarcoma cell lines modified for inducible suppression of LRRC15 expression, we are currently identifying the biological consequences of depleting this molecule using inducible knockdowns of LRRC15 in conjunction with biochemical, molecular, imaging, and cell biology assays.