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RESEARCH POSTER PRESENTATION DESIGN © 2011 www.PosterPresentations.com Christina Murphy 1 , Berkley Gryder 2 , Javed Khan 2 , Jack Shern 3 Figure 5. Potential combination strategies for targeting the PAX3-FOXO1 fusion oncogene. In addition to the compounds isolated in the drug screen, Bromodomain inhibitors had previously been shown by our lab to specifically downregulate the transcriptional output of PAX3-FOXO1. We hypothesize that coupling Bromodomain inhibitors with our discovered compounds will have synergistic toxicity toward fusion- positive cell lines. Potential combination strategies include: Drug Screen RNA Isolation IncuCyte Experiments Discovery of candidate compounds A high-throughput drug screen identifies inhibitors of PAX3-FOXO in pediatric Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma afflicting children, with an incidence of 4.5 cases per 1 million adolescents yearly. 1 RMS arises from skeletal muscle precursor cells, though its anatomic sites of origin are highly varied and not restricted to striated muscle. Histological differences between embryonal and alveolar RMS subtypes were established as a basis for clinicopathological correlation. 2 Embryonal rhabdomyosarcoma (ERMS) has earlier age of onset and better prognosis, 3 while alveolar rhabdomyosarcoma (ARMS) is associated with different primary sites, is more aggressive, usually presents with metastasis and shows severely lagging 5-year survival rates when compared to ERMS. 1 The dawn of next-generation sequencing revealed 2 distinct RMS genotypes, characterized by presence or absence of a gene fusion resulting from PAX translocation with FOXO1 6 . Fusion- positive tumors carry significantly fewer genetic mutations when compared to fusion-negative tumors 7 . Molecular profiling of RMS has become invaluable in initial diagnosis, as ARMS is typically identified by fusion-positive status 8 . The fusion event yields a chimeric transcript consisting of the N- terminus of PAX, which is a DNA-binding domain, and the C- terminus of FOXO1, an activating domain 9,10 .The oncogenic consequences of this fusion protein include heightened activation of normal downstream PAX3 targets as well as altered gene expression patterns 11 . Presence of PAX-FOXO rather than histology seems to be the primary cause of poor outcomes associated with the specific subtype. BACKGROUND PURPOSE RESULTS Figure 4. Development of a secondary screen to evaluate candidate compounds. To evaluate the transcriptional effects of the compounds discovered in the primary screen, we developed an assay to rapidly elevated expression changes of the genes downstream of PAX3-FOXO1. Cells were treated with all candidate compounds at 1 uM for 6 hours and RNA was isolated. Gene selection. RNAsequencing data previously generated from fusion-positive Rhabdomyosarcoma tumors was mined to discover genes distinctly upregulated in P3F tumors. RESULTS RESULTS Figure 7. Potential for BET-inhibitor, topoisomerase-inhibitor synergy. CONCLUSION ACKNOWLEDGEMENTS Thanks to Dr. Jack Shern and the rest of the Shern lab, including Dr. Fountaine and Dr. Sayers. Thanks also to the National Institutes of Health for their support through the Summer Internship Program. 1 University of Notre Dame, Notre Dame, IN 60040, USA 2 Genetics Branch, NCI, NIH, Bethesda, MD, USA 3 Pediatric Oncology Branch, Oncogenomics Section, Center for Cancer Research, National Institutes of Health, Gaithersburg, MD, 20877, USA Mechanism Analysis Pre-Clinical Validation Clinical Trial FUTURE DIRECTIONS • RNA sequencing to determine genetic profile of drug-treated RH4 cells. • Mechanistic investigation of main classes of drugs pulled down from drug screen. • Optimization of a low dose BET-inhibitor, topoisomerase- inhibitor combination treatment for clinical trial. Figure 1. RH4 cell line and pGreenFire Reporter used for the primary screen. The chemotherapeutic backbone for treating RMS has remained relatively unchanged since 1975, and intensified regimen yields only marginal results for the most aggressive of RMS cases. Significant toxicity and long-term morbidity are especially concerning in these young patients. An enhanced understanding of the tumor biology and discovery of new drugs for precision targeting will improve outcomes for these children. Being that fusion-positive RMS is associated with the cases having some of the most grim outcomes, the PAX3-FOXO fusion oncogene warrants further investigation as a therapeutic target. EXPERIMENTAL DESIGN Figure 2. P3F-dependent enhancer sequence used in the reporter construct. RNAseq espression of selected genes in primary Rhabdomyosarcoma tumors and normal tissues. Log2FPKM value of the genes selected for inclusion in Nanostring assay design were obtained from RNAseq data on a panel of tumors (left) and normal tissue samples (right). Secondary screen of candidate compounds: Nanostring Expression Assay Figure 3. Primary PAX3-FOXO1 inhibitor compound screen. Figure 6. Real-time quantitative live-cell analysis by IncuCyte of RH4 cell line 48h following drug treatment. This high-throughput drug screen provided many potential leads toward projects investigating PAX3-FOXO target for development of future therapies. The Nanostring assay will elucidate the different mechanisms of potentially specific, less toxic compounds to be included in a new generation of fusion- positive RMS chemotherapeutics. BET inhibitors in past studies in our research group have shown to inhibit PAX3-FOXO1- dependent transcription. Topoisomerase inhibitors were a recurring class of drugs pulled down in the compound screen. Our understanding of the PAX3-FOXO super enhancer leads us to believe that topoisomerases are especially crucial to PAX3- FOXO-driven transcription due to the torsional strain accrued from extreme chromatin remodeling. Topoisomerases were effective in treating RH4 cells, even at low nanomolar doses. When combined with BET-inhibitors, a potentially synergistic effect was observed that warrants further investigation. A combination treatment of these two inhibitory drugs could potentially provide a new low-dose option for chemotherapeutic treatment. Both teniposide and PLX2 are commercially available and could be rolled into clinical trial after further experimentation and pre-clinical validation. Topoisomerase Inhibitors 20 2 0.2 0.02 0.002 16 32 64 128 256 Concentration Normalized Luciferase CMV ALK XTT Teniposide 20 2 0.2 0.02 0.002 8 16 32 64 128 256 512 1024 Concentration Normalized Luciferase CMV ALK XTT Amonafide Amsacrine 20 2 0.2 0.02 0.002 8 16 32 64 128 Concentration Normalized Luciferase CMV ALK XTT Other HDAC Inhibitors 1-alaninechlamydocin 20 2 0.2 0.02 0.002 2 4 8 16 32 64 128 256 512 Concentration Normalized Luciferase CMV ALK XTT 20 2 0.2 0.02 0.002 16 32 64 128 256 Concentration Normalized Luciferase CMV ALK XTT Camptothecin derivative Menogaril 20 2 0.2 0.02 0.002 4 8 16 32 64 128 256 Concentration Normalized Luciferase CMV ALK XTT Psammaplin A Tetrocarcin A 20 2 0.2 0.02 0.002 0.125 0.25 0.5 1 2 4 8 16 32 64 128 Concentration Normalized Luciferase CMV ALK XTT 20 2 0.2 0.02 0.002 0.03125 1 32 1024 Concentration Normalized Luciferase CMV ALK XTT Midostaurin 20 2 0.2 0.02 0.002 16 32 64 128 256 512 Concentration Normalized Luciferase CMV ALK XTT PD-407824 20 2 0.2 0.02 0.002 16 32 64 128 256 512 Concentration Normalized Luciferase CMV ALK XTT 0 50 100 0 50 100 Hours % Confluence RH4 PLX2 dose response 10uM PLX2 +Teniposide 5uM PLX2 + 100nM Teniposide 2.5uM PLX2 +Teniposide 1.25uM PLX2 + Teniposide 0.625uM PLX2 +Teniposide 0.312uM PLX2 +Teniposide 0.156uM PLX2 + Teniposide 0.078uM PLX2 + Teniposide 0.039uM PLX2 + Teniposide DMSO + 100nM Teniposide 10uM PLX2 5uM PLX2 2.5uM PLX2 1.25uM PLX2 0.625uM PLX2 0.312uM PLX2 0.156uM PLX2 0.078uM PLX2 0.039uM PLX2 DMSO alone Dose Response curves of normalized luciferase values of the ALK enhancer construct, a CMV only promotor construct and viability (DTT assay). Bromodomain inhibitor + TOPO1 inhibitor Bromodomain inhibitor + HDAC inhibitor Bromodomain inhibitor + TOPO2 inhibitor Bromodomain inhibitor + Kinase inhibitor (Midostaurin) Fusion Positive Tumors Normal Tissue Samples
