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.

Hematopoiesis describes the formation and development of blood cells. All blood cells originate from a pluripotent hematopoietic stem cell (HSC) which has the capacity for long and short term self-renewal as well as differentiation into myeloid or lymphoid lineages. The balance between HSC differentiation and proliferation is tightly controlled by both extrinsic and intrinsic factors. During leukemogenesis, this regulation is disrupted as transformation events lead to changes in proliferation, differentiation, and survival. Two distinct experimental strategies were utilized to examine the role of the hematopoietic adaptor protein GADS (GRB2-related adaptor downstream of SHC) in both leukemogenesis and in normal hematopoiesis. Philadelphia chromosome positive (Ph+) leukemias, including chronic myeloid leukemia (CML) and B cell acute lymphoblastic leukemia (B-ALL), are mediated by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice when whole bone marrow is used as donor material. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads-deficient bone marrow expressing BCR-ABL. Gads-deficient mice were generated previously and display a decrease in mature T cells, indicating that GADS is critical to T cell differentiation. Through immunophenotyping analysis we observed that Gads-deficient mice have increased numbers of lymphoid progenitors and HSCs. The increased number of HSCs in Gads-deficient mice did not induce enhanced HSC function as Gads-deficient bone marrow cells have impaired repopulation potential. Our data suggests that the T cell defect previously observed in Gads-deficient mice may be due to an early block in differentiation and functional defect in the HSC. Taken together, our studies demonstrate that GADS is a critical mediator in the hematopoietic system for both normal and leukemic differentiation and proliferation.

Compared to radical nephrectomy (RN), partial nephrectomy (PN) leads to improved renal function preservation. However, PN may be infrequently utilized, particularly in patients susceptible to chronic kidney disease. We conducted a population-based, retrospective, observational study using the Canadian Institute for Health Information Discharge Abstract Database. All patients treated for a renal mass with either RN or PN from April 1, 1998 to March 31, 2008 were included in the analysis. Using descriptive statistics and multivariable regression modelling, we demonstrated low uptake of PN (17.5% overall); year, age, geographic region, Charlson score, hospital volume, and physician volume were independently associated with PN use, whereas DM, HTN and income quintile were not. In this contemporary analysis PN continues to be underutilized, and the rate of PN in DM, HTN and the elderly was less than expected given their known relationship to chronic renal failure.

Hematopoiesis describes the formation and development of blood cells. All blood cells originate from a pluripotent hematopoietic stem cell (HSC) which has the capacity for long and short term self-renewal as well as differentiation into myeloid or lymphoid lineages. The balance between HSC differentiation and proliferation is tightly controlled by both extrinsic and intrinsic factors. During leukemogenesis, this regulation is disrupted as transformation events lead to changes in proliferation, differentiation, and survival. Two distinct experimental strategies were utilized to examine the role of the hematopoietic adaptor protein GADS (GRB2-related adaptor downstream of SHC) in both leukemogenesis and in normal hematopoiesis. Philadelphia chromosome positive (Ph+) leukemias, including chronic myeloid leukemia (CML) and B cell acute lymphoblastic leukemia (B-ALL), are mediated by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice when whole bone marrow is used as donor material. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads-deficient bone marrow expressing BCR-ABL. Gads-deficient mice were generated previously and display a decrease in mature T cells, indicating that GADS is critical to T cell differentiation. Through immunophenotyping analysis we observed that Gads-deficient mice have increased numbers of lymphoid progenitors and HSCs. The increased number of HSCs in Gads-deficient mice did not induce enhanced HSC function as Gads-deficient bone marrow cells have impaired repopulation potential. Our data suggests that the T cell defect previously observed in Gads-deficient mice may be due to an early block in differentiation and functional defect in the HSC. Taken together, our studies demonstrate that GADS is a critical mediator in the hematopoietic system for both normal and leukemic differentiation and proliferation.

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.

Compared to radical nephrectomy (RN), partial nephrectomy (PN) leads to improved renal function preservation. However, PN may be infrequently utilized, particularly in patients susceptible to chronic kidney disease. We conducted a population-based, retrospective, observational study using the Canadian Institute for Health Information Discharge Abstract Database. All patients treated for a renal mass with either RN or PN from April 1, 1998 to March 31, 2008 were included in the analysis. Using descriptive statistics and multivariable regression modelling, we demonstrated low uptake of PN (17.5% overall); year, age, geographic region, Charlson score, hospital volume, and physician volume were independently associated with PN use, whereas DM, HTN and income quintile were not. In this contemporary analysis PN continues to be underutilized, and the rate of PN in DM, HTN and the elderly was less than expected given their known relationship to chronic renal failure.

FDA-approved drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. We compiled a library of such off-patent drugs to screen four leukemia cell lines and identified the anti-parasitic agent ivermectin that induced cell death at low micromolar concentrations. In cell death and clonogenic growth assays, low micromolar concentration of ivermectin significantly reduced viability of leukemia cell lines and patient samples compared to normal peripheral blood stem cells. In xenograft mouse models of leukemia, ivermectin decreased tumor volume and weight by up to 72% when compared to control without observable toxicity at pharmacologically achievable dosage. In this study, we further demonstrate that ivermectin activates chloride channels in leukemia cells leading to membrane hyperpolarization and increased reactive oxygen species generation. In addition, it demonstrated synergistic interaction when used in combination with Daunorubicin and Cytarabine. Therefore, this study highlights a potential new therapeutic strategy in repurposing ivermectin for the treatment of AML.

Each cancer is unique: it reflects the underlying genetic make-up of the patient and the stochastic mutational processes that occur within the tumour. This uniqueness suggests that each patient should receive a personalized type of therapy. Current predictions of a cancer patient’s outcome or prognosis are highly inaccurate. To aid in the prediction of patient prognosis based on highthroughput molecular datasets I have worked to optimize each step of the experimental pipeline: platform annotation, experimental design, consideration of tumour heterogeneity, data pre-processing and statistical analysis, and feature selection. First, a 12k CpG Island clone library was sequenced and annotated using a BLAT analysis. Second, microarrays built using this library were used in a fully-saturated study to evaluate the importance of ChIP-chip experimental design parameters. Third, intra-tumour heterogeneity was shown to influence specific pathways in a large fraction of genes. Fourth, a systematic empirical evaluation of 19,446 combinations of microarray analysis methods identified key steps of the analysis process and provided insight into their optimization. Finally, the combination of a two-stage experimental design and a novel semi-supervised algorithm yielded a six-gene, mRNA abundance-based classifier that could divide non-small cell lung cancer patients into two groups with significantly different outcomes in four independent validation cohorts. Further, a permutation study showed that millions of six-gene markers exist, but that ours ranked amongst the top 99.98% of all six-gene markers. The knowledge gained from these studies provides a key foundation for the development of personalized therapies for cancer patients.

Haematopoiesis requires strict regulation in order to maintain a balanced production of the various blood cell components. Escape from this regulation contributes to the development of cancers such as leukemia. SOCS2 is a member of the Suppressor of cytokine signalling (SOCS) family, and normally functions as a negative regulator of the JAK/STAT pathway. I examined gene expression and promoter methylation in acute myeloid leukemia (AML) cell lines and patient samples. SOCS2 expression was quite variable in AML patients, and very low in acute promyelocytic leukemia (APL) patients. Promoter hyper-methylation was found in these patients, particularly those with high white blood cell count and a FLT3-ITD. I speculate that SOCS2 interacts with an aspect of the signalling complex to inhibit cell growth in these patients, and silencing SOCS2 is necessary for leukemia progression. Treating these patients with a de-methylating agent, such as decitabine, may show promise in the clinic.