Merkel cell carcinoma (MCC) is a rare, aggressive, neuroendocrine skin malignancy that is typically radiosensitive. Standard-of-care for loco-regional MCC consists of a wide local excision and sentinel node biopsy, followed by post-operative radiotherapy (PORT) to a total dose of 50 Gy in 25 fractions over 5 weeks. This approach has been shown to reduce the incidence of loco-regional recurrence. In cancers characterized by a rapid growth pattern, delay in starting PORT after surgery is associated with increased loco-regional failures. However, the impact of delayed PORT on recurrence patterns in MCC has not been characterized. We evaluated 152 MCC patients from a prospective registry with the following criteria: diagnosed with loco-regional disease, received definitive surgical resection followed by PORT, treated at the University of Washington Medical Center (UWMC) within 6 months of initial diagnosis, and had at least 6 months of follow up data from the date of surgery. Statistical analyses were conducted in STATA 14.0 using univariate tests to assess associations between variables and survival outcomes. The group was dichotomized according to UWMC recommendations to initiate adjuvant RT no later than 6 weeks after date of definitive excision. Sixty-three patients had short delay to PORT, defined as 6 weeks or less, and 89 patients had a long delay of > 6 weeks (range: 12-419 days). There were no differences between the two groups in regards to age at diagnosis, stage, sex, tumor site, tumor size, and immunosuppression status. Among the short delay and long delay groups, no significant survival differences were observed: loco-regional recurrence-free survival (p=0.69), and MCC-specific survival (p=0.48). These preliminary results suggest that delay for up to 6 weeks does not impact loco-regional control or cancer-specific survival, and may allow for improved wound healing. Further analysis is underway to determine the upper limit of safe PORT delay.

Cancer, the leading cause of death worldwide, results from a combination of mutagenesis and selection for malignant phenotypes. Mutations from DNA replication errors may initiate as many as two thirds of all human cancers. Accurate DNA replication requires proofreading domains found on the major DNA polymerases as well as mismatch repair (MMR) proteins that detect and repair replication errors after DNA synthesis. Cancer-causing defects in proofreading and/or MMR lead to “mutator phenotypes”, marked by elevated mutation rates and increased cellular mutation burden. Such mutator cells occur spontaneously and drive evolution by generating mutations that enhance population survival. However, mutator cells also accumulate detrimental mutations that compromise fitness. Combined defects in polymerase proofreading and MMR cause error-induced extinction (EEX), which imposes a strong selection for cells with “antimutator” mutations that suppress the mutator phenotype. Using yeast (Saccharomyces cerevisiae), we isolated mutants that survived EEX and mapped the underlying determinants to candidate mutations (APC1, MCM5, PMS1). We are engineering these mutations into yeast strains to test whether they confer an antimutator phenotype. Understanding how mutator cells suppress elevated mutation rates will give us insights into how cancer cells survive and thrive in the face of strong mutagenesis and may suggest novel therapeutic strategies to combat this disease.

Mutator phenotypes due to mutations in genes encoding DNA polymerases or mismatch repair proteins lead to increased error rates during DNA replication that accelerate the evolution of cancer cells and contribute to chemotherapy resistance. Work in the yeast Saccharomyces cerevisiae indicates that excessive DNA replication errors can lead to error-induced extinction (EEX), where every cell within the population dies due to a random lethal mutation. Thus, a possible direction for cancer therapy may be to target antimutators, which can modulate mutation rates of mutator cells and suppress mutation rates, in order to slow the rate of tumor evolution. In a screen isolating antimutators, we identified an EEX mutation in Chromosome Transmission Fidelity 18 (ctf18-K666fs). Ctf18 directly associates with the N-terminus of DNA Polε, including part of the proofreading domain, and thus may influence mutation rates by directly affecting Polε function — a novel finding that would expand our understanding of mutator polymerases. In the current study, I determined whether Ctf18 exerts its antimutator phenotype independent of the S phase checkpoint pathway, the pathway by which all other previously isolated antimutators, such as Dun1, are known to modulate mutation rates. To accomplish this, I compared the mutation rates of double mutant strains, dun1Δ ctf18-K666fs and dun1Δ ctf18Δ, to their respective single mutants in pol2-L439V cells, where lower mutation rates would indicate additive antimutator effects from independent pathways. I also observed the degree of activation of this pathway by fluorescently tagging Sml1, a protein downstream of Dun1 in the S phase checkpoint, expecting to see decreased expression of Sml1 in cells in which the S phase checkpoint was activated. Expanding our understanding of mechanisms by which antimutators can modulate mutation rates may contribute to novel approaches for cancer treatments by targeting the mutator phenotype.

We are using yeast to investigate how the DNA helicase mutation mcm4^Chaos3 interferes with the early events leading to DNA replication and how these replication defects may lead to its role in cancer development. Mcm4^Chaos3 encodes a mutation in a subunit of the MCM helicase, an essential complex required for unwinding double stranded DNA during replication. Mcm4^Chaos3 mice exhibit genomic instability and more than 80% of female mice homozygous for Mcm4^Chaos3 developed mammary tumors (Shima et. al 2007). Further work in yeast identified an origin-specific minichromosome loss phenotype, suggesting the mcm4^Chaos3 mutation may have particular sequence requirements at origins, where DNA replication initiates. To determine the basis of origin sequence specificity in mcm4^Chaos3 function, we had previously performed a plasmid maintenance competition assay using a mutARS317-seq library, containing 500+ plasmids with random single mutations within the ARS317 origin sequence. This assay identified 5 origin sequence variants that performed better in mcm4^Chaos3 yeast than wild type ARS317. To investigate mcm4^Chaos3 interaction with ARS317 variants, I first measured the loss rate for wild type ARS317 plasmids in mcm4^chaos3 vs wild type yeast. As predicted, wild type cells maintained the plasmids better than the mcm4^Chaos3 mutants (16.8% loss rate/generation compared to 5% in wild type). I am currently characterizing the 5 sequence variants, and based on the competition assay data, am predicting to see differences in plasmid loss rates across the ARS317 variants. Understanding the cause for this origin specificity could help us develop a greater understanding of the mechanics involved in DNA replication, genome stability, and cancer-causing mutations.

Breast Cancer type 1 gene (BRCA1) was first linked to a high risk for breast and ovarian cancer over two decades ago, yet much remains elusive about its functional mechanism and how inheritance of a variant of the ubiquitously-expressed gene can lead to markedly increased risk for tissue-specific cancers. A gene found in the nematode C. elegans was demonstrated to be homologous to human BRCA1 including at the protein product level. Its protein product was shown to have the same type of enzymatic activity as the human protein product of BRCA1, and to be involved in the same DNA repair pathway. Using purified protein products and both structural analysis via Nuclear Magnetic Resonance and in vitro biochemical enzyme activity assays, I have shown that the C. elegans and human BRCA1 share some structural similarities and that the enzymatic activity is conserved. These results suggest that C. elegans could be developed into a model system that would allow for experimentation to directly move between in vitro biochemical studies and in vivo genomic/genetic studies. My current work has focused on furthering the development of the C. elegans model system by using it to identify other protein-protein interactions, including the identification of new protein targets. Additionally, my work explores the effect that cancer associated variants have on the enzymatic activity of the C. elegans BRCA1. The development of C. elegans as a model system could lead to new insights into conserved functions of BRCA1 and a fuller understanding of its biological and developmental importance.

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.

Our project is on urothelial urinary bladder cancer, one of the more common types of cancers. Our lab focuses on protein synthesis control of ARID1A, a known tumor suppressor in the context of urothelial urinary bladder cancer. Our initial workup led us to believe that ARID1A loss is not important for tumor initiation but may be important for tumor progression. To prove this, we used CRE recombinase editing tool to knock out ARID1A after treatment with BBN, a known carcinogen that causes bladder cancer. The results confirmed our hypothesis. In our investigation of this mouse model, we discovered an unexpected relationship between ARID1A loss and lowered protein synthesis. There is a growing body of literature showing that high levels of protein synthesis are required for transformation. As such, we conducted an experiment with rpL24+/- mouse model to test this hypothesis. We found that rpL24+/- mice showed a significant delay in cancer initiation compared to wild type mice when treated with BBN, thus establishing the role of protein synthesis in cell transformation. Our focus now is to figure out the relationship between ARID1A loss and protein synthesis. Based on preliminary results that we have conducted, I hypothesize that ARID1A loss and decreased protein synthesis leads to a weakened cell state which makes them vulnerable to carcinogen induced DNA damage and cell death. This was shown by measuring DNA damage, apoptosis, and cell proliferation as cellular markers of cancer and then comparing them with protein synthesis levels in ARID1A knock out (KO) cells and WT cells upon short treatments with BBN. We used both a mouse model and in vitro model to address this hypothesis.

This research aims to examine the effects of nanoparticle-mediated inhibition of the phospholipid glutathione peroxidase (GPX4) pathway in mesenchymal state cells on radio-resistance in glioblastoma (GBM) therapy. GBM is a particularly deadly cancer with poor survival rates and relatively low treatment success, despite aggressive surgery and radiotherapy. Recent research has shown that biocompatible, tumor-targeted iron oxide nanoparticles (NPs) can serve to enhance radiotherapy through production of secondary electrons and subsequent reactive oxygen species (ROS) within the tumor volume. The presence of these NPs in the tumor during radiotherapy has shown to decrease damage to the healthy surrounding cells and prolong survival in mice with GBM tumors. This approach however, has not demonstrated the ability to eliminate the cancer completely, in part due to the presence of mesenchymal state cancer stem cells. The GPX4 pathway has shown to effectively kill mesenchymal state cells in sarcomas by inducing ferroptosis, an iron-dependent form of cell death. Here we plan to evaluate the efficacy of a range of GPX4 inhibitors on silencing of the GPX4 pathway and the ability to induce ferroptosis in primary human GBM cells. Inhibitors identified as effective silencers of the GPX4 pathway will be combined with a NP delivery vector to provide targeted delivery to GBM. The radioenhancement capabilities of iron oxide NPs in conjunction with targeted therapy against mesenchymal state cancer stem cells may provide a means to overcome radioresistance in GBM therapy.