We study signalling by cytokine receptors in normal and malignant blood formation. A particular focus of our group is the JAK-STAT signalling pathway that transmits signals from extracellular cytokines to the cell nucleus to influence gene transcription (Figure 1).
Figure 1. Signalling by a prototypic cytokine receptor. The thrombopoietin receptor (TpoR) is physiologically activated by its ligand Tpo. Dimerisation induced by Tpo leads to reciprocal activation of JAK2 tyrosine kinases which phosphorylate (P) cytosolic receptor tyrosines (Y). In turn those attract STAT proteins that become substrates of JAK2, leading to dimers of tyrosine phosphorylated STATs being translocated into the nucleus to regulate gene expression related to proliferation, differentiation and survival. In addition to STATs, the adaptors Shc/Grb2 and p85/p110 that connect receptors to the MAP-kinase (green, right) and PI-3’-kinase (grey, left) pathways, respectively, are bound to tyrosine phosphorylated receptors. The combination of signalling pathways being activated by JAK2 induce survival, proliferation and differentiation of myeloid progenitors.
We contributed to the discovery that a clonal and recurrent activating mutation in Janus kinase 2 (JAK2 V617F mutation) occurs in most patients with Myeloproliferative Neoplasms (MPNs). MPNs are a group of rare, closely related disorders of the bone marrow that result in abnormal levels of red blood cells, white blood cells or platelets.
Of great interest, MPN patients that do not harbour the JAK2 V617F mutation instead carry mutations in either the receptor for thrombopoietin (TpoR), that we co-discovered, or mutations in the calreticulin gene (Figure 2). We have reported that mutant calreticulins activate TpoR in the secretory pathway and at the cell surface, with these mutants being the first example of a chaperone turned into an oncogene by mutations. Both TpoR and calreticulin mutations persistently activate JAK2 to allow proliferation, differentiation and survival of myeloid progenitor cells in the absence of cytokines.
Figure 2. Mutations in MPNs. The hallmark of myeloproliferative neoplasms (MPNs) is the acquisition of somatic mutations in haematopoietic stem cells that lead to persistent phosphorylation (P) and activation of JAK2-STAT5. The most prevalent mutation in MPNs is JAK2 V617F which activates signalling from homodimeric receptors TpoR (right) and also EpoR and GCSFR (not shown). The second most prevalent mutations are calreticulin mutations that induce persistent pathologic activation of TpoR (left). The third type of mutations in MPNs are mutations in TpoR (c-MPL) itself around the transmembrane/juxtamembrane domains (S505N and W515K/L/R/A), which induce persistent activation of JAK2. These mutants allow myeloid progenitors to survive, proliferate and differentiate in the absence of cytokines.
We are investigating how the JAK2 mutation and other driver mutations of MPNs influence chromatin states through the post-translational modifications of epigenetic regulators such as TET2 and DNMT3A, regulating DNA methylation, and EZH2, a histone lysine methyltransferase within the polycomb repressive complex. We aim to understand how chromatin configuration is altered and how switching between states is controlled.
Patients with MPNs are at a higher risk of developing the very severe condition, secondary acute myeloid leukaemia (sAML). Progression is associated with accumulation of additional mutations in epigenetic regulators and 50% of patients harbour mutations in TP53 or alterations of TP53 signalling (Figure 3). These events lead to proliferation of immature blood cells which are blocked in their capacity to differentiate and are highly resistant to therapies. We are studying the interplay between MPN driver mutations, p53 tumour suppressor mutations and chromatin states in this context and aim to determine the molecular basis of the progression from MPNs to sAML.
Figure 3. Progression of myeloproliferative neoplasms (MPNs). Mutations in JAK2, the thrombopoietin receptor or calreticulin in blood stem cells result in MPNs. Heterozygous TP53 mutations and mutations in epigenetic regulators cause MPNs to enter the pre-leukaemic phase. Progression to secondary acute myeloid leukemia (sAML) involves further mutations or defective function of the TP53 pathway (such as TP53 homozygous mutations or MDM4 amplification) in combination with mutations in epigenetic regulators and/or splicing factors (not shown). sAML is different from de novo AML with respect to mutational and gene expression profiles. The chromatin states of MPN, MPN pre-leukaemic cells and sAML blasts are not known.