APLF (Aprataxin and Polynucleotide kinase/phosphatase-Like Factor) is an FHA (forkhead-associated)-domain-containing nuclear protein that facilitates the repair of single-strand and double-strand breaks (SSBs and DSBs). Specifically, the APLF-FHA domain mediates interactions with XRCC1 and XRCC4, factors involved in SSB and DSB repair, respectively. A novel substitution was identified in pancreatic cancer patients, where a conserved histidine was substituted to leucine (H42L) in the APLF-FHA domain. The functional and biological characterization of this “variant of unknown significance” was investigated. This thesis shows that the H42L substitution affects APLF phosphorylation, impairs APLF retention at laser-induced DNA breaks, disrupts protein-binding to XRCC1 but not to XRCC4, and reduces cell survival following irradiation and etoposide exposure. Collectively, these data suggest that the H42L substitution impacts APLF participation in global DNA damage repair. The findings from this work could provide insight into the role of APLF in genomic integrity and, moreover, in cancer predisposition.

We demonstrate here that ultrasound-stimulated microbubbles can lead to enhanced cell death within tumors when combined with radiation. The aim of this study was to investigate different ultrasound parameters in conjunction with different concentrations of microbubbles with regards to this effect. Prostate xenograft tumors in Severe Combined Immunodeficient mice were subjected to ultrasound treatment that involved various peak negative pressures (250 kPa, 570 kPa, and 750 kPa), microbubble concentrations (8 µL/kg, 80 µL/kg, and 1000 µL/kg), and different radiation doses (0 Gy, 2 Gy, and 8 Gy). Twenty-four hours after treatment, tumors were excised and assessed for cell death. Histological analyses demonstrated that increases in radiation dose, microbubble concentration, and ultrasound pressure promoted apoptotic cell death and cellular disruption within tumors. Comparable increases in ceramide, a cell death mediator, were identified using immunohistochemistry. We also demonstrate that clinically-utilized microbubble concentrations combined with ultrasound can induce an enhancement in cell death.

Effective therapeutics are urgently needed to improve the treatment and survival of cancer patients and we believe that targeting the MYC oncogene would fill this gap. Our strategy to achieve this goal involves the design of minimalist hybrid protein inhibitors (MHP) - 25-75 amino acid proteins composed of subdomains of known transcription factor families to generate hybrids that act as structural competitive inhibitors of the Myc/Max:DNA E-box interaction. We have established cell systems, reagents and assays using our prototype MHP, ME47 to evaluate the biological effectiveness of this putative inhibitor as well as subsequently designed MHPs using cell-based proliferation and transformation assays. Omomyc was included as a proof-of-concept control to optimize our systems and gauge the performance of ME47. This research demonstrates for the first time that ME47 exerts desirable biological effects in human cells lines and provides support for the validity of our MHP strategy thus warranting further investigation.

The goal of this research is to improve the breast cancer screening process based on magnetic resonance imaging (MRI). In a typical MRI breast examination, a radiologist is responsible for visually examining the MR images acquired during the examination and identifying suspect tissues for biopsy. It is known that if multiple radiologists independently analyze the same examinations and we biopsy any lesion that any of our radiologists flagged as suspicious then the overall screening process becomes more sensitive but less specific. Unfortunately cost factors prohibit the use of multiple radiologists for the screening of every breast MR examination. It is thought that instead of having a second expert human radiologist to examine each set of images, that the act of second reading of the examination can be performed by a computer-aided detection and diagnosis system. The research presented in this thesis is focused on the development of a computer-aided detection and diagnosis system for breast cancer screening from dynamic contrast-enhanced magnetic resonance imaging examinations. This thesis presents new computational techniques in supervised learning, unsupervised learning and classifier visualization. The techniques have been applied to breast MR lesion data and have been shown to outperform existing methods yielding a computer aided detection and diagnosis system with a sensitivity of 89% and a specificity of 70%.

CK2 is a ubiquitous serine/threonine protein kinase implicated in numerous cellular processes as well as in tumorigenesis. CK2 is composed of two catalytic (αα, αα’, α’α’) subunits and two regulatory (ββ) subunits that assemble to form the active CK2 holoenzyme. CK2 has been shown to phosphorylate, interact with, and regulate other proteins, including other protein kinases. CK2 substrates can be initially bound by the CK2β regulatory subunit, which acts as a docking site to facilitate phosphorylation and mediate CK2 substrate specificity. In a screen to identify novel CK2β interacting proteins, I identified three novel CK2β interactors, including the mitogen- and stress-activated kinase 2 (MSK2), which I pursued for further characterization. MSK2, and the closely related isoform MSK1, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38-MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. In my thesis, I demonstrate that CK2, which contributes to NF-κB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue serine-324 and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-κB p65 at serine-276 in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV-induced p65 transactivation capacity was dependent on MSK2, MSK2 residue S324, and p65-S276. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at S276 that positively regulates NF-κB activity in MDA-MB-231 cells.

Anti-cancer drugs gain access to solid tumors via the blood, and must penetrate tissue to reach all viable cancer cells. This thesis aims to compare the distribution of anticancer drugs in normal tissues and tumours, to examine whether drug distribution is modifiable and quantifiable in solid tumours, and to determine whether extracellular drug distribution can be improved by modifying intracellular drug distribution. The time-dependent spatial distribution of three anticancer drugs, doxorubicin, mitoxantrone and topotecan, were studied in normal tissues and tumours. Ten minutes after drug administration, there was fairly uniform distribution in the heart, kidney and liver whereas drug distribution within tumours was limited to perivascular regions. Doxorubicin distribution in P-glycoprotein (PgP) over-expressing tumours was compared to that in wild-type tumours and changes in distribution were evaluated with the use of PgP inhibitors. There was better doxorubicin distribution in PgP-over-expressing tumours compared to wild-type tumours, and pretreatment of PgP-over-expressing tumours with PgP inhibitors decreased doxorubicin distribution. These data suggest that reduced uptake of drug into cells may enhance extracellular drug distribution, and the dual effects of PgP inhibitors (increased drug uptake in proximal cells, but poorer drug distribution) may explain, in part, why these agents have not provided clinical benefit. The effect of the proton pump inhibitor pantoprozole on intracellular and extracellular drug distribution was determined. Pantoprazole increased endosomal pH in cells, leading to less sequestration of doxorubicin within them, and increased the toxicity of doxorubicin for cultured cells. In wild-type MCF7 tumours, pretreatment with pantoprazole enhanced doxorubicin distribution and tumour growth delay without apparent increase in toxicity. These studies have led to initiation of a phase I clinical trial of pantoprazole and doxorubicin for patients with solid tumours. The work completed in this thesis demonstrates that drug distribution can be modified and that these changes can be quantified, and may correlate with improved anti-tumour effects. Improving drug distribution through the use of proton pump inhibitors may be an effective strategy to improve chemotherapeutic efficacy.

For the treatment of cancer, immunotherapy has some inherent advantages over other treatment modalities: disseminated disease can be eradicated due to the systemic nature of immunity, the immune system is effective against a wide range of targets, long-term memory can offer added protection against disease relapse, immunotherapy should be relatively non-toxic, and it can be synergistically combined with other treatment platforms such as radiation and chemotherapy. Type 1 immune responses are thought to be superior for the treatment of cancer and, as the quintessential Th1 polarizing cytokine, interleukin-12 (IL-12) holds much promise; however, optimal therapeutic protocols have yet to be developed and clinical results have fallen short of this promise. The in vivo IL-12 experiments described here highlight a characteristic of cellular therapy that has not previously been appreciated. That is, the effect of cell-mediated cytokine delivery on the immediate microenvironment and how that affects the immune response initiated. This observation has implications for the clinical application of IL-12 therapy but may also prove to be an important consideration when studying other immunostimulants. I have herein developed a novel in vitro assay system that I have used to dissect the cellular responses to IL-12 and to identify the signals that are required for activation of a cluster of differentiation 4 (CD4)+ effector population that affects leukaemia cell clearance both in vitro and in vivo. This work, and the future studies proposed, will expand our understanding of the potential of IL-12 immunotherapy and enhance our ability to manipulate therapeutic conditions to favour the desired response. Moreover, the in vitro assay system offers a method for further characterization of CD4+ effector cells and the development of protocols to initiate their potent anticancer activity.

Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS-mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors.

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifiers. Like ubiquitin, most Ubls are covalently attached to a lysine residue on target proteins. The small ubiquitin-related modifiers (SUMO) play important roles in a number of critical biological processes, such as proliferation and regulation of the cell cycle, yet their specific cellular functions have remained poorly understood. Like ubiquitin, SUMO proteins can also form oligomeric “chains”, but the functions of these structures were even less well understood. To this end, I created the first spectral library for the identification of Ub/Ubl proteins and Ub/Ubl chain linkages in mass spectrometry experiments. This tool has dramatically improved our ability to use MS to analyze the contents of biological samples for Ub and Ubls, and to identify specific types of Ub and Ubl chains in model organisms. I also used MS to conduct the first comprehensive SUMO system protein-protein interactome in any organism. In total, 452 high confidence protein-protein interactions were detected for S. cerevisiae SUMO system proteins, encompassing a total of 321 interacting partners. Yeast SUMO system components were found to interact with proteins involved in a number of different biological processes, and my mapping effort increased the number of known SUMO system interacting partners >50-fold. This study revealed that a number of transcriptional co-repressors and chromatin remodelling proteins interact physically with specific SUMO system components, with a clear division of labour between SUMO system enzymes. Finally, I conducted the first global analysis of SUMO chain function, using a combination of genetic, high-content microscopy, and high-density transcriptomics screens. Consistent with my interactomics work, this study demonstrated that inhibition of SUMO chain synthesis leads to severe chromatin condensation defects, which in-turn leads to chromosome missegregation, unscheduled transcription of stress-and nutrient-regulated genes, and aberrant intragenic transcription. Together, my work thus revealed a major role for the SUMO system in the maintenance of higher order chromatin structure and transcriptional repression.

This work is a part of a large project in our laboratory that is aimed toward characterization of prokaryotic transcription regulators from different families and their interactions with small-molecule effectors. My study was focused of IclR family of transcriprion regulators, specifically on its founding member Isocytrate Lyase Regulator (IclR) from E.coli and AllR regulator from E.coli, which share 42% sequence identity with IclR. I used a combination of biophysical, biochemical and structural biology techniques to explore the mechanisms by which IclR and AllR interact with their effectors. I performed site-directed mutagenesis experiments in order to research the role of individual amino acids in interaction of AllR regulator with its previously identified effector glyoxylate and to test whether oligomerization plays a role in effector-induced signal transduction by AllR. Using differential light scattering, which allows high-throughput screening of small molecules for thermostabilization of proteins, I identified potential effctors for the IclR regulator. The physiological relevance of these candidate molecules was tested in-vitro and in-vivo and their interaction with IclR was characterized by Isothermal Titration Calorimetry and X-ray Crystallography.