Abstract 239: Mutation of the inhibitory adaptor protein LNK drives potentiated JAK-STAT signaling in patients with JAK2 V617F-negative myeloproliferative neoplasms
Authors: Oh Stephen T. , Simonds Erin F. , Jones Carol , Hale Matthew B. , Goltsev Yury , Gibbs Kenneth D. , Merker Jason D. , Zehnder James L. , Nolan Garry P. , Gotlib Jason 
Published: February 2011

Dysregulated JAK-STAT signaling via activating mutations in tyrosine kinases (e.g. JAK2 and MPL) is a hallmark of chronic myeloproliferative neoplasms (MPNs). Even in the absence of mutations in JAK2 or MPL, JAK-STAT activation can be demonstrated, suggesting that alterations of other regulatory elements in this pathway may contribute to MPN pathobiology. One regulator of JAK-STAT signaling is LNK (SH2B3), an adaptor protein that binds to MPL via it SH2 domain and co-localizes to the plasma membrane via its pleckstrin homology (PH) domain. Upon cytokine stimulation, LNK binds strongly to JAK2 and dampens or terminates downstream STAT activation. LNK−/- mice exhibit features consistent with an MPN phenotype, including splenomegaly, leukocytosis, and thrombocytosis.

We therefore sequenced LNK in 34 JAK2 V617F-negative MPN patients, and report the identification of novel mutations in exon 2 of LNK in two patients. In a patient with primary myelofibrosis, a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains was identified. A second patient with essential thrombocythemia exhibited a missense mutation leading to an E208Q substitution in the PH domain. DNA isolated from cultured skin fibroblasts revealed wild-type (WT) sequence, confirming that these mutations were somatic.

TPO-dependent BaF3-MPL cells were transduced with WT and mutant LNK. While WT LNK inhibited TPO-dependent growth and activation of JAK2-STAT3/5, the DEL mutation led to loss of these negative feedback properties, thereby permitting augmented and sustained JAK-STAT activation in response to TPO stimulation. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes.

In peripheral blood samples obtained from MPN patients, stimulation with TPO or G-CSF revealed a unique phosphorylated STAT3/5 (pSTAT3+/5+) subpopulation that was increased in DEL compared with normal donor samples. A similar pSTAT3+/5+ subpopulation was seen with JAK2 V617F and MPL W515L-positive samples, suggesting that this may be a shared feature of MPNs. E208Q cells exhibited STAT3/5 phosphorylation in response to TPO, but not G-CSF, indicating that a partial loss of LNK function may generate differential STAT activation profiles in response to specific cytokines. The cytokine-responsive pSTAT3+/5+ cells from DEL were primarily CD34+, and the DEL mutation was detected in this subset, suggesting that LNK mutations arise in a hematopoietic stem or progenitor cell. Finally, the pSTAT3+/5+ response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Thus, mutations in LNK, the first reported in human disease, lead to loss of LNK negative feedback function and represent a novel mechanism of MPN pathogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 239.