Ewing sarcoma (ES) is a bone and soft tissue tumor that occurs in children and young adults. The tumor is driven by an oncogenic fusion gene that fuses the EWSR1 gene to FLI1, an ETS family transcription factor. The EWS-FLI1 fusion promotes tumorigenesis through transcriptional and epigenetic dysregulation. Despite maximally intensive chemotherapy, the outcomes for metastatic ES patients remains poor, thus the need to identify new therapeutic strategies. Given ES’s epigenetic dependencies, there is strong rationale to investigate epigenetic modifying drugs. Bromodomain and extra terminal domain (BET) proteins function as epigenetic readers that facilitate transcription. I have shown that BET inhibitors (BETi) slow the growth of ES cells in vitro but will not be successful as a single agent. I hypothesize that the combination of BETi with other biologically targeted agents will be synergistic. Based on our preliminary results from RNA-seq data and an in silico drug screen on BETi-treated ES cells, we prioritized testing of top predicted small molecule inhibitors. Using standard in vitro cytotoxic assays and calculating synergy using the Chou-Talalay method, my preliminary results showed strong synergy between Copanlisib and BMS-986158. Ongoing studies are testing the selected kinase inhibitors, and promising combinations will be tested in in vivo xenograft models. It is our goal to identify drug combinations that will enhance the cytotoxic effects of BETi in ES.

The Raf family of kinases are highly conserved and important for animal development and cell proliferation. In normal cells, Raf activity level is tightly regulated and Raf dysregulation is implicated in many diseases and cancers. About 70% of melanomas carry gain-of-function mutations in a human BRAF gene. On the other hand, some Noonan and LEOPARD syndrome patients carry mutations in another Raf gene, RAF1, that alter the binding site for a binding partner called 14-3-3. Currently, we do not know all the mechanisms by which mutations found in the RAF1 gene change signaling activity. We hypothesize that mutation of the 14-3-3 binding site alters Raf protein stability. Caenorhabditis elegans are microscopic, translucent nematodes that have one Raf ortholog, termed LIN-45, which is important for development. We will use C. elegans to investigate the effects of protein interactions between Raf and 14-3-3 proteins, with the goal of generating new mutants that prevent LIN-45 binding to 14-3-3 proteins. We will use CRISPR-Cas9 methods, which allow us to edit, add, or remove specific sites of DNA. To test our hypothesis about Raf protein stability, we will analyze how mutations of the 14-3-3 binding sites change LIN-45 protein levels in living animals. Understanding the impact of these mutations may provide insight into the normal regulation of Raf protein levels as well as mechanisms underlying Noonan and LEOPARD syndromes.  

Research studies have found that the origin of 80% of ovarian cancers (OvCa) is epithelial. Genetically and historically, there are four subtypes of ovarian cancer: serous, endometrioid, clear cells, and mucinous. Among the four subtypes of ovarian cancer, high-grade serous OvCa is associated with a high mortality rate (5-year survival rate of 30%). High-grade serous OvCa is usually diagnosed at an advanced stage where cancer has metastasized. Non-specific symptoms of advanced-stage ovarian cancer are due to the lack of precise biomarkers, screening tools, and limited imaging modalities. We have developed an in vitro 3-dimensional (3D) microtumor model to examine high-grade serous OvCa. This study aims to determine if 3D microtumor culture replicates OvCa pathology. This research study explores the hypothesis that 3D ovarian microtumors secrete varying amounts of the extracellular matrix of the basement membrane (collagen type IV and laminin), inflammatory markers (C-reactive protein, and Interleukin-6), and invasion/migration markers (fibronectin) when compared to ovarian cancer cells cultured in 2 dimensions (2D). This research compares two ovarian cancer cell types, PEO4, and SKOV3 cells, in 2D and 3D cell cultures. We hypothesize that high-grade serous cancer cells (PEO4) have a greater change to 3D culture conditions than low-grade serous ovarian cancer cells (SKOV3). A novel RNA isolation technique was first identified to collect high-quality mRNA from 3D hydrogel scaffolds. Quantitative real-time polymerase chain reaction was used to examine gene expression in 2D and 3D hydrogel scaffold cell cultures.