Tyrosine kinases are critical drug targets in oncology due to their role in tumorigenesis. Accordingly, the treatment of specific cancers has been revolutionized by the development of tyrosine kinase inhibitors (TKIs). However, the long-term effectiveness of TKIs is undermined by the emergence of drug resistance, often caused by mutations that prevent drug binding. Understanding whether a given mutation confers drug resistance can enable physicians to tailor treatment based on the patient’s tumor genotype. Current methods to identify resistance mutations are laborious and can only interrogate a small subset of possible mutations. To overcome this limitation, I have designed an assay to identify all possible single drug resistance mutations in the oncogenic kinase ALK in a single experiment. A key feature of this functional assay is that it can accurately discriminate between drug resistant and drug sensitive mutations. Moreover, this assay leverages the Ba/F3 cell line, a mammalian cell line that only proliferates when an active oncogenic tyrosine kinase variant is expressed. Thus, when treated with a TKI, cells expressing drug resistant kinases continue to proliferate while cells expressing sensitive kinases die. As a first step, I have genetically engineered the Ba/F3 cell line to enable single-copy integration of tens of thousands of ALK variants. Additionally, I have cloned ALK into a mammalian expression vector and have optimized transfection conditions for the Ba/F3 cell line. Next, I will transfect the cloned vectors into Ba/F3 cells and measure the growth rates of each transfected cell line. I anticipate that only cells harboring active ALK will grow, while those with inactive variants will not. This result would lay the foundation for further development of a system for the exhaustive identification of drug resistance mutations in oncogenic kinases.

The PTEN (phosphatase and tensin homolog) protein negatively regulates growth-promoting PI3K-Akt signaling in cells. Due to its function as a tumor suppressor, PTEN is often mutated in diverse cancers. Unfortunately, most PTEN variants have not been individually studied, making it difficult to ascertain their functionality within cells. Our lab recently demonstrated that thousands of PTEN missense variants exhibit decreased steady-state abundance when expressed in human cell lines and likely have reduced function. However, the mechanism behind their lower abundance is currently unknown. We hypothesized that many low-abundance PTEN variants are thermodynamically unstable and possess a reduced melting temperature. To test this, we fused EGFP, Enhanced Green Fluorescent Protein, to a panel of PTEN single amino acid variants, and these fusion proteins were expressed within human cell lines. Then, we cultured these cells at their standard growth temperature (37°C) and two lower temperatures (33°C and 30°C). We found that a subset of variants of intermediate abundance at 37°C exhibit WT-like abundance at decreased temperatures, while variants of extremely low-abundance remain unchanged. These results suggest that we can identify missense variants with reduced thermodynamic stability using this method. Next, we will repeat this experiment at high throughput to identify hundreds of temperature-dependent PTEN variants. We will determine biochemical properties shared by partially stable variants and compare them to computational predictors of protein folding. We will also identify variants of intermediate abundance that are not temperature-dependent, which may reveal other mechanisms by which variants lower a protein’s abundance. Our results demonstrate that we can characterize the thermodynamic stability of PTEN variants by measuring their abundances in cells grown at different temperatures. These results might be more physiologically relevant because the variants were studied in a cellular environment. Furthermore, our methods may be applied to other proteins that cannot be studied as purified protein.

Atherosclerosis is a disease in which cholesterol accumulates in the walls of arteries, limits blood flow, and causes heart attacks and strokes. Atherosclerosis could be prevented or reversed by expressing therapeutic genes in the artery wall. Gene therapy for atherosclerosis will require high-level therapeutic gene expression for two reasons: 1) to increase efficacy; and 2) to allow use of lower vector doses, thereby minimizing vector-related toxicity. Our goal is to increase expression of an apolipoprotein A-I (apoA-I) transgene, which both prevents and reverses atherosclerosis in rabbits. We used an in silico bioinformatics approach to identify endothelial cell-specific cis-regulatory modules (CRMs). CRMs are short enhancer regions of DNA that are expected – when introduced into endothelial cells – to increase transcriptional activity of a nearby gene. We identified 11 CRMs in the CDH5, EFEMP1, THBS1, and VWF genes. We hypothesize that insertion of these CRMs into a helper-dependent adenoviral vector encoding apoA-I will increase apoA-I expression in endothelial cells. We also hypothesize that the genomic regions surrounding these CRMs contain sequences that, along with the CRMs, could increase transgene expression above levels obtained with the CRMs alone. Our adenoviral vectors have a larger capacity for the insertion of DNA sequences than other gene-therapy vectors. This large capacity allows us to evaluate CRMs in their native genomic context (i.e., with several kilobases of neighboring DNA included). We cloned large CRM-containing regions of the 4 genes listed above and we are inserting the apoA-I gene into the translational start sites of these genes. We will test vectors containing individual CRMs and vectors containing the larger CRM-containing regions in cultured endothelial cells (in vitro) and in rabbit carotid arteries (in vivo) to identify CRM-containing constructs that express the highest apoA-I levels. This project may provide higher-expressing vectors for use in gene therapy for atherosclerosis.