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Delineating the biology of Anaplastic Lymphoma Kinase (ALK) and its resistance to crizotinib

  • Author / Creator
    Alshareef, Abdulraheem
  • Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase receptor that was initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of NPM-ALK fusion protein. Various forms of oncogenic ALK proteins have been identified, subsequently, in various types of human cancers. The focus of this thesis was to get more insight into the ALK biology and how this could impact cancer phenotypes. 1) Crizotinib is an ALK inhibitor that has been found to be therapeutically useful against a subset of ALK-positive tumors. However, clinical resistance to this drug has been well known and the mechanism of this resistance is incompletely understood. In this part of my thesis, I hypothesized that the crizotinib—ALK binding is a determining factor for the sensitivity to crizotinib in ALK-positive cell lines derived from various types of cancers. Using the cellular thermal shift assay (CETSA), I measured the crizotinib—ALK binding in a panel of ALK-positive cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. I found that the biological response to crizotinib treatment significantly correlated with crizotinib—ALK binding. Additionally, I found that the crizotinib-resistant cell lines expressed higher protein levels of β-catenin. Furthermore, the siRNA knockdown of β-catenin in these cells restored crizotinib—ALK binding and was associated with a significant lowering of IC50. Taken together, this part of the thesis showed that the crizotinib—ALK binding measurable by CETSA is useful in predicting crizotinib sensitivity in ALK-positive cancer cells and crizotinib—ALK binding is dictated by the structure of ALK and some of its binding partners. 2) Since I have highlighted the importance of the physical binding between crizotinib and ALK in determining the crizotinib sensitivity, here I asked if these observations hold true for the stem-like cells in neuroblastoma (NB) cells, which were purified based on their responsiveness to a Sox2 reporter. NB is the most common pediatric extra-cranial solid tumor and the survival for metastatic NB remains <50% despite intensive multi-modality therapies. Notably, the sensitivity of NB cells to crizotinib is highly variable. In this study, I found that compared to bulk, reporter unresponsive (RU) cells, reporter responsive (RR) cells had significantly higher neurosphere formation ability, expression of CD133/nestin, and chemo-resistance. Using CETSA, I found that RR cells exhibited higher crizotinib resistance than RU cells and show no crizotinib—ALK binding. The absence of crizotinib—ALK binding in RR cells can be attributed to their high β-catenin expression, since siRNA knockdown of β-catenin restored the crizotinib—ALK binding and lowered the crizotinib IC50 to the level of RU cells. Importantly, enforced expression of β-catenin in RU cells resulted in the opposite effects. Therefore, high expression of β-catenin in the stem-like NB cells contributes to their crizotinib resistance. 3) The mechanisms by which the expression of full-length ALK (wild-type or mutant), which is detectable in the majority of NB cases, are regulated are not well understood. I have identified a novel ALK transcript characterized by the retention of the entire sequence of intron 19 (i.e. ALK-I19). ALK-I19 was detected in the four NB cell lines, used in the previous section, and in one-third of patient samples. In this study, I provided a description of this ALK variant. Specifically, the functional significance of ALK-I19 was determined by specific siRNA knockdown of this transcript, which resulted in substantially decreased expression of the fully-spliced ALK transcripts (i.e. FS-ALK) and ALK protein, as well as a significant reduction in cell growth. Clinically, ALK-I19 expression correlated with undifferentiated histology and strong ALK protein expression, detectable by immunohistochemistry in >50% of the tumors. Importantly, patients with tumors that did not express ALK-I19 and lacked MYCN amplification had an excellent clinical outcome, with 19/19 patients survived at 5-years as compared to only 10/18 patients with tumors carrying ALK-I19 and/or MYCN amplification. This data suggests that the absence of ALK-I19 and MYCN amplification is a useful prognostic marker for NB patients. To conclude, this thesis has provided insights into the molecular mechanism underlying the biology of ALK and a novel mechanism of resistance, which may provide valuable therapeutic targets in neuroblastoma and other ALK-positive cancers.

  • Subjects / Keywords
  • Graduation date
    2017-11:Fall 2017
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3NZ81414
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Medical Sciences-Laboratory Medicine and Pathology
  • Supervisor / co-supervisor and their department(s)
    • Lai, Raymond (Laboratory Medicine and Pathology)
  • Examining committee members and their departments
    • Turner, Suzanne (Pathology, Cambridge University)
    • Sergi, Consolato (Laboratory Medicine and Pathology)
    • Fu, Yangxin (Oncology)
    • Ingham, Robert (Medical Microbiology and Immunology)
    • Tyrrell, Gregory (Laboratory Medicine and Pathology)