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Characterization of select downstream effectors of oncogenic NOTCH1 in T-cell acute lymphoblastic leukemia Gusscott, Samuel D.

Abstract

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.

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Attribution-NonCommercial-NoDerivs 2.5 Canada

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