Structural and Functional Characterisation of the Thrombopoietin Receptor and its Negative Regulator, LNK in Myeloid Malignancies Christine Lee Mei Mei (BSc. Hons, MRes) S44197856 A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 Faculty of Medicine, Diamantina Institute Abstract Thrombopoietin (TPO), by activating its cognate receptor, the thrombopoietin receptor (TPOR, also known as MPL), supports haematopoietic stem cell survival and expansion, megakaryocyte differentiation, as well as platelet and megakaryocyte production. LNK is an SH2-B adaptor protein that negatively regulates lymphohaematopoiesis by modulating various signalling pathways such as those mediated by TPOR. LNK can regulate downstream signalling by direct binding to the receptor and/or JAK2, a tyrosine kinase that is associated with the receptor and responsible for downstream signal transduction. In myeloproliferative neoplasms (MPNs) and leukaemia, various activating (oncogenic) mutations in TPOR and JAK2, as well as inactivating LNK mutations are frequently found. However, the mechanisms by which mutations in TPOR and LNK lead to these diseases is poorly understood. Further elucidation of the mechanism of JAK-STAT signal regulation by TPOR and LNK in normal haematopoiesis and blood cancers such as MPNs and leukaemia will be essential for understanding their role in disease and for developing novel targeted therapeutics. Particularly, the functional changes that occur in TPOR and the structural mechanisms that are used by LNK to regulate JAK-STAT signalling are key to this understanding. This thesis employed a multi-disciplinary approach to gain a greater understanding of the mechanisms of activation and regulation of JAK-STAT and mitogen-activated protein kinases (MAPK) signalling by TPOR and LNK. Novel mutations in the transmembrane domain (TMD) of TPOR that result in constitutive signalling were characterised. Further, a peptide antagonist against the crucial signal- modulating domain (the extracellular domain-juxtamembrane (ECD-JM)/TMD) of TPOR was discovered using phage display and its functionality analysed in cell-based assays. By using single- particle tracking photoactivated localisation microscopy (sptPALM), the spatiotemporal dynamics of TPOR in live cell membranes was determined. The current study also determined the regions of LNK that may be important in contributing to inhibition of STAT and MAPK signalling, and revealed a novel role in regulating receptor levels. In addition, expression and purification of LNK for structural and biochemical assays were performed. By employing sptPALM, the effect of cytokine stimulation on wild type TPOR and TPOR mutations (W515K and Box1Δ) on the lateral diffusion of TPOR were investigated. In the basal and stimulated states, TPOR exhibited bimodal mobility (fast and slow-moving receptor subpopulations). Upon ligand stimulation of TPOR or the TPOR mutant W515K that results in constitutive receptor activation, the mobility of the fast-moving subpopulation was significantly reduced and a fraction of 1 these receptors transitioned into the slow-moving subpopulation. This reduction in mobility may be related to the initiation or attenuation of signalling and this finding may elucidate the relationship between the mobility patterns of TPOR and different phases of cell signalling. As part of a collaborative effort, every amino acid residue in the TPOR TMD was evaluated for their ability to result in constitutive proliferation in Ba/F3 cells in the absence of cytokine by using the deep mutational scanning approach. Four novel mutants were found to result in constitutive signalling (L498W, V501A, V501S, and L502S) indicating that these mutations may potentially drive myeloid malignancies. Indeed, some of these mutations were identified for the first time in patient samples by collaborators. In order to develop antagonists targeting constitutive active oncogenic TPOR mutants, peptides against the ECD-JM/TMD were discovered via phage display screening. The functionality of the peptides was assessed via cell-based assays using Ba/F3 TPOR WT and W515K mutant cell lines. Results suggest that peptide V may be acting as a peptide antagonist against the TPO/TPOR- mediated signalling pathway. Confirmation of peptide specificity to this pathway will allow for the peptide to be developed into a useful therapeutic agent by itself or to be used as a scaffold for the development of small molecules. This discovery may facilitate the development of improved targeted drugs to treat patients suffering from myeloid malignancies as a result of activating TPOR mutations. In order to investigate the role of individual domains of LNK in regulating JAK2, various LNK domain deletions and point mutations were generated. All LNK expression constructs retained the SH2 domain required for JAK2 phosphorylated tyrosine binding. A novel function of the LNK proline dimerisation domain (Pro/DD) was found in mediating constitutive binding to TPOR with the LNK SH2 acting as the main anchor to bind to JAK2. These interactions prevent downstream signalling through a mechanism that has yet to be fully elucidated. The findings of the current study act as an important foundation for future work to increase the understanding regarding the structure-function of this important negative regulator of JAK2 signalling. As the crystal structure of LNK is not currently available, there is still a lack of understanding towards its role as a negative regulator, in particular, for JAK signalling. Therefore, expression and purification of LNK for structural and biophysical studies were performed. Although LNK was able to be purified to a high level, sufficient quantities of soluble human LNK fractions that could be used for crystallisation studies were not able to be obtained. In the absence of LNK crystals, the purified protein could also be used for protein interaction studies and kinase activity assays. These investigations will allow better understanding regarding how LNK mutations may contribute to the different diseases. 2 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. 3 Publications included in this thesis No publications included. 4 Submitted manuscripts included in this thesis No manuscripts submitted for publication Publications during candidature Peer-reviewed paper Bridgford, J.L., Lee S.M., Lee C.M.M., Guglielmelli P., Rumi E., Pietra D., Wilcox S., Chhabra Y., Rubin A.F., Cazzola M., Vannucchi A.M., Brooks A.J., Call M.E., Call M.J. (2019). Novel Drivers and Modifiers of MPL-dependent Oncogenic Transformation Identified by Deep Mutational Scanning, Blood. Review article Dehkhoda, F., Medina, J.J., Lee, C.M.M., Brooks, A.J. (2017). The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects. Frontiers in Endocrinology, 9:35. 5 Conference presentations Presenting author underlined Christine M.M. Lee, Yash Chhabra, Andrew Perkins, and Andrew J. Brooks. Specific domains of LNK (SH2B3) bind and regulate TPOR stability and signalling. Poster session presented at the 48th Annual Scientific Meeting International Society for Experimental Hematology, Brisbane, Australia, 2019. Christine M.M. Lee, Rob Luetterforst, Melissa J. Call, Matthew E. Call, Raphael Trenker, and Andrew J. Brooks. Development of peptides targeting constitutive active oncogenic thrombopoietin receptor (TPOR) mutants. Poster session presented at the 2018 TRI Translational Research Symposium, Brisbane, Australia, 2018. Christine M.M. Lee, Rob Luetterforst, Melissa J. Call, Matthew E. Call, Raphael Trenker, and Andrew J. Brooks. Development of peptides targeting constitutive active oncogenic thrombopoietin receptor (TPOR) mutants. Poster session presented at the 8th International Conference on Myeloproliferative Neoplasms, Dublin, Ireland, 2018. Christine M.M. Lee, Rob Luetterforst,