Exosomes have been known to secrete an increased amount of miRNA and noncoding genes that are abnormally expressed in various cancer subtypes. Thus, they may be an early marker for pediatric cancer types that are more difficult to diagnosis without invasive techniques, and may also help identify progression of the disease. In the project, six types of pediatric cancer cell lines, along with their extracted exosomes, were analyzed and tested for different monoclonal antibodies through western blot analysis. The genes EWS-FLI1 and FGFR4 were also identified in some cancer cell lines through Reverse-Transcriptase Polymerase Chain Reaction analysis (RT-PCR). The results were indicative of similar protein markers being found in both the originating cells and their corresponding exosomes.

Difficult to treat cancer patients, specifically those tumors that are metastatic and drug-resistant, prove to have the lowest survival rates when compared to more localized types. The commonplace combination therapies, surgery, chemotherapy, and radiation, do not usually result in remission and sometimes cannot be done with these specific patients. RNA interference therapeutics, especially those that use short-interfering RNA (siRNA), have given rise to a novel field that employs the mechanisms in the body to silence the gene expression post-transcriptionally. The main cell types used in this research were Ewing Sarcoma, Acute Myelogenous Leukemia, and Rhabdomyosarcoma cells. Initial assays involved the testing of the cells' responsiveness to a panel of siRNA compounds, to better understand the most effective ones. The siRNA UBBs1 proved to be the most cytotoxic to all cell lines tested, allowing for further investigation through transfection procedures for cellular assays and RNA purification for expression analysis. The data showed decreased cell viability for the UBBs1 treated group for both RD and RH-30 Rhabdomyosarcoma cell lines, especially at a much lower concentration than traditional chemotherapy drug dose response assays. The RNA purification and quantification of the transfected cells over time showed the biggest decrease in gene expression when treated with UBBs1. The use of siRNA in future therapeutics could be a highly-specific method to induce cytotoxicity of cancer cells, but more successful clinical testing and better manufacturing processes need to be established first.

Acute Myeloid Leukemia (AML) is a disease that occurs when genomic changes alter expression of key genes in myeloid blood cells. These changes cause them to resume an undifferentiated state, proliferate, and maintain growth throughout the body. AML is commonly treated with chemotherapy, but recent efforts to reduce therapy toxicity have focused on drugs that specifically target and inhibit protein products of the cancer’s aberrantly expressed genes. This method has proved difficult for some proteins because of structural challenges or mutations that confer resistance to therapy. One potential method of targeted therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes. G-quadruplexes are present in the promoter region of many potential oncogenes and have regulatory roles in their transcription. This study analyzes the therapeutic potential of the compound GQC-05 in AML. This compound was shown in vitro to bind and stabilize the regulatory G-quadruplex in the MYC oncogene, which is commonly misregulated in AML. Through qPCR and western blot analysis, a GQC-05 mediated downregulation of MYC mRNA and protein was observed in AML cell lines with high MYC expression. In addition, GQC-05 is able to reduce cell viability through induction of apoptosis in sensitive AML cell lines. Concurrent treatment of AML cell lines with GQC-05 and the MYC inhibitor (+)JQ1 showed an antagonistic effect, indicating potential competition in the silencing of MYC. However, GQC-05 is not able to reduce MYC expression significantly enough to induce apoptosis in less sensitive AML cell lines. This resistance may be due to the cells’ lack of dependence on other potential GQC-05 targets that may help upregulate MYC or stabilize its protein product. Three such genes identified by RNA-seq analysis of GQC-05 treated cells are NOTCH1, PIM1, and RHOU. These results indicate that the use of small molecules to target the MYC promoter G-quadruplex is a viable potential therapy for AML. They also support a novel mechanism for targeting other potentially key genetic drivers in AML and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes.