Abnormal Expression and Function of the Lnk Adaptor Protein in Myeloproliferative Neoplasms (MPN).

We have shown in a deficient mouse model that the adaptor protein Lnk had an important role as negative regulator of cytokine signaling during hematopoiesis (Velazquez et al., J. Exp Med 2002). Lnk-/- animals display abnormal megakaryopoiesis sharing many features with that found in MPN patients. This phenotype is due to loss of Lnk inhibition of thrombopoietin (TPO)-mediated JAK2 activation (Tong et al., J Exp Med 2004). Recent studies have shown that Lnk, when expressed in hematopoietic cell lines, could bind and regulate two mutant proteins found in MPNs, JAK2V617F and MPLW515L. However, the role of Lnk in MPN pathogenesis is still unclear. In the present study, we studied in detail both Lnk expression and function in MPNs.

The study included a total of 82 MPN patients (pts), including 41 essential thrombocytemia (ET), 29 polycythemia vera (PV) and 12 primary myelofibrosis (PMF). Lnk expression was assessed by quantitative RT-PCR. Biochemical and cellular analyses of Lnk and JAK2 interaction were carried out using co-immunoprecipitation, GST pull-down and proliferation assays on primary hematopoietic cells and cell lines expressing either wild-type (WT) or mutant forms of both Lnk and JAK2.

Lnk mRNA was clearly overexpressed in platelets and CD34+ cells of most MPN pts compared to controls (P=0.005 and P=0.03, respectively). Moreover, this increased Lnk expression strongly correlated with JAK2V617F allele burden (P=0.02). In contrast, Lnk mRNA levels were reduced in the 18 pts treated with interferon-α compared to the 34 pts treated with hydroxyurea (P=0.04).

TPO specifically upregulated Lnk expression at both mRNA and protein levels in both primary and UT7/Mpl megakaryocytic (MK) cells. Analysis of TPO-stimulated platelets from ET patients revealed the existence of a new interaction site between Lnk and JAK2 located in the N-terminal region of Lnk, in addition to the previously known interaction mediated by Lnk SH2 domain. This interaction resulted in Lnk phosphorylation. In JAK2V617F expressing platelets or cell lines, we observed both increased phosphorylation of Lnk, and stronger binding of JAK2 to the N-terminal region of Lnk compared to WT-JAK2 cells.

Overexpression of Lnk in JAK2V617F cells showed a dose dependent growth inhibition, as seen in JAK2 WT cells. In addition, overexpression of various mutant forms of Lnk showed that this inhibition required a fully functional SH2 domain. Finally, expression of either WT or mutant forms of Lnk also demonstrated the crucial role of Lnk SH2 domain in growth inhibition of myeloid and MK progenitors in Lnk-/- hematopoietic cells.

This first study of a large cohort of 82 patients allowed us to investigate the role of Lnk in MPN: (1) Lnk mRNA was found to be significantly overexpressed in MPN derived platelets and CD34+ cells, and correlated with JAK2V617F allele burden. (2) Lnk expression is upregulated by TPO, an effect likely mediated by JAK2 activation. (3) The Lnk SH2 domain plays a major role in the down-regulation of both normal and MPN-derived hematopoiesis. (4) We describe here a novel interaction site between the N-terminal region of Lnk and JAK2. Stronger interaction of the JAK2V617F mutant form with this N-terminal binding site may account for the dysregulated hematopoiesis observed in MPN patients despite Lnk overexpression.

No relevant conflicts of interest to declare.