Structural and functional characterisation of mutant calreticulin in chronic myeloproliferative neoplasms

Calreticulin (CALR) is an endoplasmic reticulum (ER)-resident chaperone that is mutated in ~40% of patients with myeloproliferative neoplasms (MPNs). Mutant CALR exerts its effects by binding to and activating the thrombopoietin receptor MPL to instigate hyperactive JAK-STAT signalling and the MPN phenotype. However, several aspects of the mechanism by which mutant CALR interacts with MPL to promote aberrant MPL activation remains unclear. In this work, I present work that identifies critical domains within mutant CALR and within MPL which are essential for malignant transformation. In Chapter 3, I describe experiments that implicate two motifs critical for mutant CALR oncogenic activity: (i) the glycan-binding lectin motif, and (ii) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was required for facilitating mutant CALR homomultimerisation which was a co-requisite for MPL binding, and that depletion of intracellular zinc levels led to decreased CALR-MPL heteromeric complexes. These data implicate zinc as an essential cofactor for mutant CALR oncogenic activity. In Chapter 4, I describe experiments that identify essential signalling motifs within MPL that are required to transmit mutant CALR-induced signalling. Specifically, I identified that mutant CALR does not exert its oncogenic effects by binding to the thrombopoietin binding site on the extracellular domain nor the eltrombopag binding site in the transmembrane domain. Moreover, my data show that a single residue, Tyr626, within the intracellular domain of MPL is critical for mediating mutant CALR signalling. Finally, in Chapter 5, I optimised conditions for purification of wild-type and mutant CALR and undertook preliminary structural analysis and initial protein modelling of purified proteins using electron microscopy to reveal structural differences between wild-type and mutant CALR. These data indicate that mutant CALR is unstable and prone to aggregation, which make their purification challenging. Altogether, these findings reveal new biological insights into the molecular mechanism of action of mutant CALR, which could have therapeutic implications for treatment of CALR-mutated MPN.