Abstract B6: Identification of novel mutations in the inhibitory adaptor protein LNK in patients with JAK2 V617F-negative and -positive chronic myeloproliferative neoplasms

Abstract

Abstract Dysregulated JAK-STAT signaling is a hallmark of myeloproliferative neoplasms (MPNs), as evidenced by the identification of activating mutations in JAK2, and the thrombopoietin (TPO) receptor MPL in a subset of MPN patients. Clinical trials with highly specific inhibitors of JAK2 are currently ongoing, and clinical responses have been observed in the majority of MPN patients, validating JAK2 as an important therapeutic target in these patients. In addition, responses have been observed in patients lacking known mutations in JAK2 or MPL, suggesting that other regulatory elements in this pathway are altered. However, the molecular basis for this observation is not well understood. One regulator of JAK-STAT signaling is LNK (SH2B3), a member of a family of adaptor proteins that share several structural motifs, including a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain, an SH2 domain, and a conserved C-terminal tyrosine residue. LNK binds to MPL via its SH2 domain and co-localizes to the plasma membrane via its PH domain. Upon cytokine stimulation with TPO, LNK binds strongly to JAK2 and inhibits downstream STAT activation, thereby providing critical negative feedback regulation. LNK-/- mice exhibit an MPN phenotype, including an expanded hematopoietic stem cell compartment, megakaryocyte hyperplasia, splenomegaly, leukocytosis, and thrombocytosis. We sequenced LNK in a cohort of MPN patients, leading to the identification of novel mutations in 7/159 (4.4%) patients. One patient with JAK2 V617F-negative primary myelofibrosis (PMF) exhibited a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. Six additional patients were found to have point mutations affecting conserved residues in the PH domain. Interestingly, a point mutation leading to an E208Q substitution was found in one JAK2 V617F- negative patient with essential thrombocythemia (ET), as well as one JAK2 V617F-positive ET patient. Similarly, a P242S substitution was also found in both a JAK2 V617F-negative ET patient, as well as a JAK2 V617F-positive patient with post-polycythemic myelofibrosis. These latter findings suggest that even in the presence of the JAK2 V617F mutation, abrogation of LNK function may be a cooperating pathogenetic mutation. TPO-dependent BaF3-MPL cells transduced with the LNK DEL mutant exhibited augmented and sustained TPO-dependent growth and activation of JAK2-STAT3/5. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes. Primary patient samples from MPN patients bearing the LNK DEL and E208Q mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant. The STAT3/5 activation response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Our identification of mutations in LNK, the first reported in human disease, demonstrates that loss of JAK-STAT negative feedback control is a novel mechanism of MPN pathogenesis. As each of these LNK mutations localizes to the PH domain and appears to be heterozygous, mislocalized mutant LNK may exert a dominant negative effect by binding and sequestering wild-type LNK. These findings may also partly explain why some MPN patients lacking JAK2 or MPL mutations respond to treatment with JAK2 inhibitors, and highlight the importance of a more complete understanding of the role of inhibitory pathways in MPN pathogenesis. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B6.