Orientation-Specific Signalling of Human TpoR with JAK2 WT or JAK2 V617F Mutant in Myeloproliferative Neoplasms

Details

Supervisors

Constantinescu, Stefan N.

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

The thrombopoietin receptor (TpoR) is pivotal in haematopoietic stem cell renewal, megakaryocyte differentiation and platelet formation. Yet, it is also the central player in myeloproliferative neoplasms (MPNs), malignancies characterised by excessive number of progenitors and mature blood cells, currently lacking selective treatment options. Janus kinase 2 (JAK2) V617F, their main driver mutant, induces cytokine-independent dimerization and activation of TpoR. Our group has previously shown that murine TpoR (mTpoR) and human TpoR (hTpoR) require distinct dimeric interfaces for activation, mainly due to a human-specific histidine at the outset of the transmembrane domain. While some of these interfaces are common between JAK2 wild-type (WT) and V617F, one prevents activation by JAK2 V617F and another can exclusively be activated by JAK2 V617F. Assessing signalling and autonomous proliferation of IL3-dependent Ba/F3 cell lines expressing dimerization-imposing hTpoR fusion proteins and endogenous JAK2 WT we confirm the physiologically active dimeric orientations. Employing crosslinking assays, we refine the interfaces induced in physiological conditions by thrombopoietin (Tpo). In pathologic context, we provide further evidence for JAK2 V617F-specific active hTpoR dimeric interface and inactive interface. We identify ways to impede JAK2 V617F oncogenic activity while preserving Tpo-induced signalling. Using bioluminescence resonance energy transfer (BRET) and flow cytometry assays, we observe that specific inhibition of JAK2 V617F-driven hTpoR activation does not prevent the the chaperone functions of JAK2 WT/V617F and the JAK2 V617F-induced dimerization of the receptor. To validate these findings in vivo, a murine model of JAK2 V617F-driven MPN must be engineered where mTpoR is humanized to faithfully reproduce the characteristics of hTpoR. Here, we identify a humanizing mutant background in mTpoR that is sufficient to model the inhibitory effects of the identified mutants. Finally, we mutagenize several mTpoR transmembrane residues and characterize their impact on the surface level differences between mTpoR and hTpoR. Taken together, this work offers a basis for exploiting the modulation of hTpoR dimeric orientation as a therapeutic strategy in MPN and paves the way for relevant murine models to faithfully recapitulate JAK2 V617F-driven MPN phenotypes observed in patients.