Precision Medicine in Pancreatic Cancer 517 views

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly human cancers due to a lack of early detection and its resistance to conventional chemo- and radiotherapy. Precision medicine using small molecule inhibitors is an alternative therapeutic strategy. The KRAS oncogene is mutated in >90% of pancreatic cancer tumors and presents an exploitable therapeutic opportunity. KRAS mutations render it constitutively active, signaling through effector molecules to stimulate growth and survival in cancer cells. One of the most predominant pathways through which KRAS signals is the RAF-MEK-ERK pathway. Many inhibitors of this pathway have gone to clinical trials, but ultimately failed in treating pancreatic cancer due to a lack of overall efficacy and an acquired resistance in tumors. Often, the studies noted that a method of selecting for patients who are predicted to respond to the therapy could improve therapeutic efficacy. Therefore, we set out to find markers that could be used to predict when MEK inhibition will be effective or should conversely be avoided. To discover such markers, we tested a panel of pancreatic cancer cell lines for sensitivity to the MEK inhibitor AZD6244. After grouping them as either sensitive or resistant, RTqPCR and cDNA microarray analyses identified genes that were differentially expressed between the two groups. Most notable were MERTK and MAPK8, which were both upregulated in resistant lines. Correlation studies with the protein products of these genes (MERTK and JNK1 respectively) showed both protein expression levels significantly correlated with resistance to the small molecule inhibitor. The MERTK protein in particular was found to be upregulated in both innate and acquired resistant cell lines. Knockdown of either MERTK or MAPK8 yielded a noticeable decrease in proliferation. Inhibitor targeting of JNK1 also resulted in combinatorial benefits with MEK inhibition, particularly in resistant cell lines. Our findings suggest that these two proteins can serve as markers of resistance and potential new therapeutic targets in pancreatic cancer. In addition to predictive and therapeutic targets, precision medicine needs effective techniques to select for moieties that can target these key proteins. One powerful approach for identifying such moieties is the use of phage display. The technique uses combinatorial peptide libraries on the surface of bacteriophages to offer a rapid, economical way to screen billions of peptides for specific binding properties. As a modification to this approach, we have created a system that enables specific insertion of selenocysteine (Sec) residues into the peptides displayed for screening. These Sec residues allow for site-specific tethering of small molecules to create a hybrid screening technique capable of much higher chemical diversity than current phage screens. As a proof of concept, we tethered a small molecule agonist of the adenosine A1 receptor to Sec phage and showed enhanced binding of this modified phage to the A1 receptor compared to unmodified phage. Further, we showed that the modified phage are capable of activating the receptor and its downstream signaling pathways because of the small molecule agonist tethered to the phage. This technique will provide new screening capabilities for small molecule-peptide hybrids and provide a new tool for advancing precision medicine.

PHD (Doctor of Philosophy)

English

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2015-04-24