Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - April 2022)
Characterization of new pathways in Acute Myeloid Leukemia and T-cell Acute Lymphoblastic Leukemia which contribute to oncogenesis is necessary to relieve dependence on conventional chemotherapy for treatment of these diseases. In this dissertation, I characterized the role of signaling molecules (IGF1R) and transcription factors (RUNX1, RUNX3, NOTCH1) in regulating mechanisms of leukemia initiation and maintenance. I discovered that committed myeloid progenitor cells with genetically reduced levels of IGF1R were less susceptible to myelogenous leukemogenic transformation due, at least in part, to a cell-autonomous defect in clonogenic activity. Genetic deletion of IGF1R by inducible Cre recombinase however had no effect on growth/survival of established leukemia cells. I raise the possibility that IGF1R inhibitors in clinical development may be acting through alternate/related pathways. Second, in a retroviral insertional mutagenesis study, I cloned retroviral integration sites from hNOTCH1ΔE mouse leukemias to find genes which collaborate with Notch signaling in T-ALL initiation. Common integration sites include the previously identified Ikzf1, and a novel potentially Notch-collaborating gene, Runx3. Using a multicistronic lentiviral system, I show that RUNX1A, RUNX1B and RUNX3 were able to collaborate with the ΔEΔL allele of NOTCH1 to initiate leukemia. Finally, I sought to understand how RUNX1 and RUNX3 contribute to the biology of established T-cell leukemias. I found that both RUNX1 and RUNX3 contribute to T-ALL cell proliferation and survival. Although RUNX3 can induce cell proliferation, RUNX1 expression is finely tuned with overexpression and knockdown resulting in negative growth phenotypes. This may be in part to regulation of MYC, IL7R, IGF1R, and CDKN1B as well as affecting genome-wide H3K27Ac. I found that RUNX1 expression was targeted by the CDK7 inhibitor, THZ1. RUNX1 and RUNX3 are mediators of Notch-directed regulation of PKCθ, and as such are indirect regulators of LIC-activity. Finally, I showed that RUNX1 and Notch signaling provide complimentary, additive signals for growth of T-ALL cells. These experiments provide insight into the role of RUNX1 mutations in T-cell leukemia and point to a complementary role in supporting the Notch pathway.
Oncogenic NOTCH1 signalling is a major driver of T cell acute lymphoblastic leukemia (T-ALL) transformation and growth. Although some downstream effectors of this function are known, they cannot explain all observed pro-growth and leukemogenic phenotypes and there are undoubtedly other effectors yet to be described and investigated. This study identifies microRNAs (miRNAs) regulated by NOTCH1 in T-ALL and further characterizes the actions of insulin-like growth factor 1 receptor (IGF1R) and protein kinase C theta (PKCθ), two signalling molecules I was previously involved in identifying as being regulated by NOTCH1 in a T-ALL context. I found that NOTCH1 can negatively regulate miR-223 expression, contributing to its ability to enhance IGF1R expression. In turn, IGF1R signalling is important to maintain growth in a subset of T-ALL cell lines and is a major positive effector of the PI3K/AKT signalling pathway. IGF1R downstream signalling pathways may be negatively affected by PKCθ. As expression of PKCθ is negatively regulated by NOTCH1 in T-ALL, here, I have attempted to identify its direct phosphorylation targets in this context. I have done this through the combined use of an analog sensitive (AS) kinase screen and an ascorbate peroxidase (APEX) based chemical labelling proximity screen. Candidate direct PKCθ phosphorylation targets identified include potential IGF1R downstream signalling components such as IRS4, mTOR, RICTOR, RAF1 and ARAF. Some of these targets were also found to be proximal to PKCθ in a T-ALL cellular context. This suggests that, in addition to regulating IGF1R signalling at the transcript or protein (via miR-223 repression) level, NOTCH1 also has the potential to positively regulate this pathway through repressing PKCθ phosphorylation of downstream components. Further studies are required to validate this hypothesis. Other candidate direct PKCθ phosphorylation targets identified here may also be worth further investigation and may suggest the involvement of PKCθ in additional cellular processes in T-ALL. Further development of my novel combined approach for the identification of direct phosphorylation targets may prove to be useful for the investigation of other kinases in a broad range of cell types.
The Polycomb Group (PcG) is a highly conserved group of genes which serve to repress transcription via specific modifications of histones in chromatin. The PcG has well-established roles in development and is involved, by mutation or dysregulation, in many human diseases including cancer. This study identifies the gene PCGF5, which is a paralogue of the oncogene Bmi1, as a transcriptional target of Notch signalling in T cell acute lymphoblastic leukemia (T-ALL). Evidence suggests that this regulation is direct and that the Notch transactivation complex binds DNA at several regions near the PCGF5 gene. PCGF5 is found to be expressed at a higher level in T-ALL than other hematopoietic malignancies. PCGF5 is found to associate with the PcG proteins RING1A and RING1B and its overexpression results in increased ubiquitylation of histone H2A, suggesting it shares functional similarity to Bmi1. Despite their similarities, Bmi1 and PCGF5 have a different spectrum of binding partners and are targeted to different locations in the genome. Overexpression of PCGF5 does not significantly alter hematopoietic development in vivo; however, enforced expression of PCGF5 in bone marrow progenitors results in the generation of fewer colonies in a myeloid colony forming assay. This study suggests that PCGF5 may have as yet unappreciated roles in PcG biology and merits further study into its effects on development and hematopoietic neoplasia.
Master's Student Supervision (2010 - 2021)
T-cell acute lymphoblastic leukemia (T-ALL) is a blood malignancy that arises from T lymphoid progenitor cells. Even though the overall prognosis is favorable with the current treatment, patients who relapse have considerably worse outcomes. Moreover, pediatric patients suffer from significant long-term chemotherapy side effects. Therefore, we are in need of an improved therapy that targets key carcinogenic molecular mechanisms. There are several models that are commonly used to research T-ALL in vitro and in vivo, including cell lines and mouse models. However, all of them present major limitations that hinder the interpretation of the results in a natural human disease. To solve them, we have previously designed a synthetic T-ALL model developed from oncogene-transduced human cord blood cells. It effectively addresses constraints of other research systems and allows to reproducibly study early and late events in leukemia development. To support the notion that the synthetic model recapitulates bona fide T-ALL, I aimed to characterize it transcriptionally. As a result, I showed that NOTCH1-LMO2-TAL1-BMI1 (NLTB) transduced leukemia is similar to a wide range of patient-derived xenograft samples. Moreover, I discovered that HOXB and VEGF pathways (which are involved in various cancers) are also enriched in preleukemic cells compared to their normal counterparts. Interestingly, both of these pathways are clinically relevant in T-ALL patients, as their components negatively affect event-free survival. Using NLTB synthetic model, I further investigated functional roles of each of these pathways. Notably, I discovered that HOXB3 gene expression promotes preleukemic and leukemic cell growth in vitro. This suggests that it is involved in both initiation and maintenance of T-ALL. On the other hand, while VEGFR3 was overexpressed on the surface of preleukemic cells, it did not show a consistent effect neither on their growth nor on the growth of primary leukemia cells. This encourages further research into alternative mechanisms through which VEGFR3 influences cancer progression. It also suggests that cell context determines downstream effects of a particular pathway. Overall, this study provides a valuable insight into cellular mechanisms that are activated in leukemia initiation and maintenance and can possibly be used in targeted therapy for T-ALL patients.
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that affects both children and adults. Optimization of chemotherapy regimens has led to steady improvements in outcome for pediatric patients over the last 5 decades, with a long-term survival rate of 80%. However, the five-year survival rate for adults is still only 35-40% and there is a poor prognosis for relapse patients (Goldstone et al., 2008). Further improvements in outcome will undoubtedly require introduction of novel approaches and more specific targeted therapies. Research efforts in this area have been hampered by the lack of a reproducible model for in vitro growth of human T-ALL blasts. Most efforts to date have relied heavily upon established cell lines, which can be a useful tool to study malignancies but do not necessarily always represent bona fide disease biology, and in vivo studies involving patient samples expanded as xenografts in immunocompromised mice, which are costly, time-consuming and complicated by non-cell autonomous effects. Current methods for in vitro culture of patient T-ALL samples yield variable performance with high rates of apoptosis and less than robust proliferation. Development of an efficient and reproducible in vitro culture method for growth of primary human T-ALL blasts would greatly enhance the ability to test and validate novel therapies by allowing for direct assay for sensitivity/resistance of patient cells which have not been subject to extensive manipulation or selection. In this work we report an in vitro co-culture system using defined, serum-free media and a stromal feeder cell layer which supports robust growth and minimal apoptosis of patient T-ALL blasts. We have shown that the stromal feeder cell layer and supplemental IL-7 cytokine is critical for sustained patient T-ALL blast growth in this model. Finally, we have demonstrated the utility of this culture system as a platform that will facilitate ongoing efforts to identify growth factors/cytokines required for maintenance of leukemia cell self-renewal activity, aid in the study of signaling pathways important in T-ALL pathogenesis and maintenance, and allow for prospective testing of novel compounds for therapeutic efficacy on patients’ own tumor cells, thus enabling implementation of personalized medicine initiatives.