Survival rates for pediatric B-cell acute lymphoblastic leukemia (B-ALL) have improved dramatically, but outcomes for the 15% who relapse and for adults with B-ALL remain poor. Up to 40% of pediatric B-ALL patients require central nervous system (CNS) prophylaxis that causes significant treatment-related morbidities. p53-/- Rag-2-/- Prkdcscid;scid triple mutant (TM) mice spontaneously develop B-ALL that disseminates to the CNS. We used this model to investigate molecular mechanisms that drive CNS dissemination of leukemic B-cells. We show that CNS-disseminating B-ALLs had recurrent genomic rearrangements that replaced N-terminal Fms-like tyrosine kinase 3 (Flt3) exons with endogenous retrovirus (ERV) transcriptional control elements. ERV-Flt3 fusion genes encoded truncated FLT3 (trFLT3) proteins that induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, trFLT3 promoted de novo development of CNS-disseminating B-ALL from hematopoietic progenitors. Thus, a novel mutational mechanism involving ERV-mediated FLT3 activation can drive the development of B-ALL characterized by high degree of CNS-invasion. Ectopic expression of trFlt3 suggested that TM B-ALLs initiate prior to B-cell commitment, since Flt3 is normally repressed by PAX5 upon B-cell commitment, co-incident with Cd19 expression. In support of this idea, we report evidence of Flt3 amplification in a rare subset of CD19- progenitors, and we show that CD19- FLT3+ cells from leukemic TM mice contain leukemia-initiating cells. Finally, we compared gene expression profiles of trFl3+ and trFl3- B-ALLs to identify potential Flt3 effectors important for CNS dissemination. TM B-ALLs uniquely expressed RANKL, a key regulator of osteoclast differentiation and normal B-cell development. FLT3 inhibition decreased RANKL expression, suggesting at least partial dependence on trFLT3 signaling. RANKL-expressing TM B-ALLs decreased trabecular bone density after adoptive transfer to normal mice, demonstrating a role for RANKL in leukemia-associated bone pathology. Importantly, a RANKL biologic antagonist inhibited CNS dissemination of TM B-ALLs in adoptive transfer experiments. Thus, my studies identified novel ligand-independent Flt3 mutations that arise prior to B-cell commitment and promote development of CNS-disseminating B-ALLs. Furthermore, I identified RANKL as a potential therapeutic target that may limit leukemia CNS dissemination and leukemia-associated bone pathology.

MicroRNA is a single-stranded RNA molecule of about 22 nucleotides in length and is expressed endogenously. It functions as a gene regulator by pairing imperfectly with 3’ untranslated region (3’UTR) of target mRNAs, leading to translational inhibition. MicroRNA is implicated in many regulatory pathways and hence affects various cellular activities. In the development of cancer, genetic alterations occurred at miRNA locus and its expression level is dysregulated in various cancers versus normal tissue counterparts. It is thus important to find the targets of dysregulated microRNAs contributing to progression of cancer. To facilitate long term functional studies, a microRNA expression construct with unique futures was generated. Stable expression of miR-378 enhanced cell survival, reduced caspase-3 activity, and promoted tumor growth and angiogenesis. By algorithmic predictions and proteomic analysis, two tumor suppressors, SuFu and Fus-1, were found to be translationally regulated by miR-378. Target validation was confirmed by co-transfection experiments and luciferase activity assays, reassuring its oncogenic role by regulating two tumor suppressor genes simultaneously. Conversely, microRNA can also function as a tumor suppressor by modulating expression of Versican, an extracellular matrix protein known to facilitate tumorigenesis and angiogenesis. By a novel PCR method, more than one microRNA were found to bind to Versican 3’UTR. iii Among these microRNAs, targeting of Versican and Fibronectin by miR199a-3p was validated. Expression of a fragment of Versican 3’UTR was expected to antagonize the function of miR-199a-3p. Stable expression of Versican 3’UTR resulted change in cell morphology and increased cell-cell adhesion. Analysis of primary tissues from transgenic mice expressing versican 3’UTR showed an increase expression of Versican and Fibronectin, and organ adhesion was found between liver and its surrounding tissues. In addition, 3’UTR also modulated the level of miR-199a-3p and miR-136, alleviating translation of negative cell cycle regulators, PTEN and Rb1. This resulted in reduced cell proliferation and hence diminished tumor growth. These findings suggest a role of microRNA in tumor growth, providing a valuable target for therapeutic intervention.

Chemotherapy is often administered once every three weeks to allow repopulation of essential normal tissues such as the bone marrow. Repopulation of surviving tumour cells can also occur between courses of chemotherapy and can decrease the efficacy of anticancer treatment. This thesis aims to characterize repopulation, to study the effect of targeted cytostatic agents to inhibit repopulation, and to determine the optimal scheduling of chemotherapy and molecular targeted treatment. The distribution of proliferating and apoptotic cells in human squamous cell carcinoma (A431) xenografts was studied following chemotherapy using fluorescence immunohistochemistry. There was an initial decrease in cell proliferation and in the total functional blood vessels, and an increase in apoptosis observed following treatment with paclitaxel chemotherapy. A rebound in cell proliferation occurred approximately 12 days following treatment, which corresponded with a rebound in vascular perfusion. The effect of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, to inhibit repopulation between courses of chemotherapy was determined using EGFR-overexpressing A431 cells and xenografts. Furthermore, concurrent and sequential schedules of combined chemotherapy and molecular targeted treatment were compared. Gefitinib inhibited the repopulation of A431 cells in culture when administered sequentially between chemotherapy; sequential treatment was more efficacious than concurrent treatment probably because concomitant scheduling rendered quiescent cells less responsive to chemotherapy. However, in vivo studies using chemotherapy in combination with gefitinib or temsirolimus, a mammalian target of rapamycin (mTOR) inhibitor, showed that concurrent scheduling of combined treatment was more effective at delaying regrowth of xenografts than sequential treatment; this was likely due to dominant effects on the tumour microenvironment. The work completed in this thesis has shown that repopulation occurs in A431 xenografts following paclitaxel treatment, and these changes are associated with changes in the tumour vasculature. Repopulation of A431 cells was inhibited by gefitinib administered sequentially with paclitaxel. However, studies in mice showed better inhibitory effects when chemotherapy was given concomitantly with cytostatic agents such as gefitinib or temsirolimus. Our in vivo data highlight the importance of characterizing changes in the tumour microenvironment when determining optimal scheduling of chemotherapy and molecular targeted treatment.

In Canada, ovarian cancer kills about 67% of diagnosed patients, largely due to difficulties in early diagnosis. Current treatment consists of debulking surgery and intermittent chemotherapy every three weeks. This approach leads to insufficient drug concentrations at disease sites, and long treatment-free intervals cause accelerated tumor proliferation and drug resistance, resulting in a 5-year survival rate of only 25-35%. Drug resistance development is the ultimate cause of the majority of patient deaths. Improvements yielding more effective treatment are fundamental for successful management of this disease. This thesis investigated a continuous chemotherapy strategy devoid of treatment-free intervals for ovarian cancer treatment. A biocompatible, biodegradable polymer-lipid injectable formulation PoLigel, was used for continuous DTX delivery. The formulation was well tolerated; no alterations in body weight, behaviour, histology of peritoneal tissues, or interleukin-6 levels were seen in CD-1 mice treated with the PoLigel. Continuous DTX therapy via the PoLigel was considerably more efficacious than intermittent therapy, resulting in significantly less tumor burden and ascites fluid in models of human and murine ovarian cancer. Continuous therapy resulted in less tumor cell proliferation and angiogenesis, and more tumor cell death than intermittent DTX. The presence and length of treatment-free intervals was shown to contribute to the development of drug resistance. Eliminating these intervals by continuous dosing resulted in superior antitumor efficacy in both chemosensitive and chemoresistant xenograft models of human ovarian cancer, and prevented drug resistance increase after a 21-day treatment period. Survival studies revealed that intermittent dosing led to a mild survival prolongation of 36% and 10% in chemosensitive and chemoresistant models, respectively, whereas continuous DTX prolonged survival by a striking 114% and 95%. Although long-term continuous chemotherapy substantially improved survival, increased drug resistance mechanisms were found at the endpoint. Overall, results presented here encourage the clinical implementation of continuous chemotherapy due to greater achievable therapeutic advantages.

The Hedgehog (Hh) signaling pathway plays a critical role in modulating various developmental processes that requires fine tuning of the Hh signal, such that dysregulation can lead to cellular events involved in cancer. To elucidate the factors responsible for aberrant Hh activation and subsequent tumorigenesis, I investigated three distinct modulators of Hh signaling: (1) p53 tumour suppressor (2) primary cilia (3) PTHLH. During chondrocyte development, abnormal Hh signalling can result in benign cartilage tumours, called Enchondroma. As precursor lesions, enchondroma may progress to malignant neoplasia, collectively known as chondrosarcoma. Although the molecular events involved in this progression are poorly understood, inactivation of the p53 tumour suppressor has been identified in approximately one-third of chondrosarcoma. Using an enchondroma mouse model, I showed that p53 deficiency can cause chondrosarcoma to develop. The combined inhibitory effects of Hh and p53 pathways on the pro-apoptotic factor, IGFBP-3, suppressed apoptosis and was demonstrated to play a critical role in the progression to chondrosarcoma. The primary cilium is an organelle that serves as a signaling centre for the Hh pathway to allow for greater control of the signal output. Loss of primary cilia results in abnormal Hh signaling that is associated with cancer and various developmental defects. I observed a depletion of primary cilia in both human Chondrosarcoma and Enchondroma tumours compared to normal cartilage. Analysis of cilia-deficient mice revealed that defective ciliogenesis alone could lead to the formation of benign cartilage tumours. Furthermore, loss of primary cilia potentiated the effect of Hh signaling activation, revealing a novel role in cartilage tumorigenesis. Parathyroid-like hormone (PTHLH) is an essential inhibitor of the Hh pathway during chondrocyte development, however its function as a regulator of Hh in other tissue types are largely unknown. Through activation of PKA, PTHLH suppresses the activation of Gli transcription factors; downstream effectors of the Hh pathway. Using irradiated Ptch+/- mice that exhibit a high incidence of skin and brain tumours, I demonstrated that treatment with PTHLH agonist, PTH (1-34), results in inhibition of the Hh pathway, increased survival and a reduction in tumour incidence and size. Thus, PTH (1-34) may have therapeutic potential for Hhrelated cancers, especially given its known clinical safety in treating osteoporosis.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by an excess production of a specific, fully functional blood cell type. Many cases involve deregulation of a protein tyrosine kinase. JAK2 is one such kinase, involved in a subset of MPNs. JAK2-selective inhibitors are currently being evaluated in clinical trials. In order to identify inhibitor-resistant JAK2 mutations before they appear in the clinic, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signalling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles. Chronic myeloid leukemia is an MPN characterized by the presence of the BCR-ABL fusion gene. We determined that a specific cohort bearing deletions near the ABL gene, which is associated with poor prognosis, do not suffer from genomic instability. We also examined the role of a putative tumour suppressor gene EXOSC2 as an explanation for the reduced survival time, and suggest it may have a role in disease progression.

Introduction: Objectives were to describe time intervals between presentation to a tertiary care center, diagnosis and treatment in pediatric acute lymphoblastic leukemia (ALL) and Hodgkin disease (HD), and measure their impact on overall survival (OS) and event-free survival (EFS). Methods: Children in POGONIS (Pediatric Oncology group of Ontario Networked Information System) with ALL or HD from 1997-2007 were eligible. Time intervals were dichotomized at clinically defined cut-points. OS and EFS were examined with univariate and multivariable Cox proportional hazards (CPH) models. Results: In ALL, in multivariable analysis, those with treatment > 3 days after diagnosis had inferior OS (adjHR=2.49; 95%CI 1.4-4.43;p=0.002), and inferior EFS (adjHR=1.73; 95%CI 1.01-2.96;p=0.047). In HD, in multivariable analysis, those with treatment > 7 days after diagnosis had superior EFS (adjHR=0.37; 95%CI 0.18-0.77;p=0.008). Conclusions: Time to treatment is associated with survival in ALL and HD. Future research will further delineate the relationship between time to treatment and outcome.

BACKGROUND: The incidence of liver metastases from colorectal cancer (CLM) is on the rise. Older cancer patients are frequently subject to under-treatment. METHODS: A Markov decision model was built to examine the effect on life expectancy (LE) and quality-adjusted life expectancy (QALE) of four strategies – best supportive care (BSC), systemic chemotherapy (SC), radiofrequency ablation (RFA), and hepatic resection (HR). The model was designed to account for both age and comorbidities. RESULTS: In the base case analysis, BSC, SC, RFA, and HR yielded LEs of 11.9, 23.1, 34.8, and 37.0 months, respectively, and QALEs of 7.8, 13.2, 22.0, and 25.0 months, respectively. Model results were sensitive to several variables including age, comorbidity status, and length of model simulation. CONCLUSION: Hepatic resection may be the optimal treatment strategy for healthy older patients with CLM. Treatment decisions in older cancer patients should be individualized and account for patient age, comorbidities, and values.

The pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) is highly aggressive with a poor prognosis for diagnosed patients. Here, we demonstrate that targeting the unique oncogene integrin-linked kinase (ILK) in ARMS cells in conjunction with the common chemotherapy agent vincristine, a synergistic effect is found in the reduction of cell viability in vitro. This result was achieved by both RNAi-mediated depletion of ILK and using a small molecule kinase inhibitor specific for ILK. Both techniques were found to disrupt important protein interactions at the site of the centrosome. Combination ILK disruption and vincristine treatment of cells induced the expression of apoptotic markers and arrested cells in the G2/M stage of the cell cycle. Interestingly, protein levels of JNK and its target c-Jun were regulated with combined treatment. Altogether, these findings indicate that the use of molecular targets like ILK may further improve the clinical treatment of ARMS.

Polo-like kinase 4 (Plk4) has been identified as a molecular marker of resistance to therapy in cancer. Our laboratory has recently shown a motility defect in Plk4+/- murine embryonic fibroblasts (MEFs) compared to Plk4+/+. I hypothesized that Plk4 augments cancer cell motility. Plk4 depletion with siRNA in Plk4+/+ MEFs and HeLa cells suppressed invasion compared to control. Transient over-expression of Flag-Plk4 in cancer cell lines did not consistently increase invasion. However, modest Plk4 over-expression using stable clones showed higher invasion rates than non-induced. The RhoGEF Ect2, an important Plk4 substrate, transiently localized to protrusions in MEFs, suggesting a RhoA-based signalling cascade in motility. The effect of Plk4 heterozygosity on metastasis was tested in a transgenic mouse model but there was no significant difference in developing metastasis compared to wild type. Further studies are required to characterize the effect of Plk4 on motility, and its potential as a therapeutic cancer target.