Adaptor Protein Lnk Binds to and Inhibits Normal and Leukemic FLT3

The production and lineage commitment of hematopoietic cells is controlled by the actions of a complex network of signaling pathways. Mutations and translocations of tyrosine kinases within these pathways lead to constitutive signaling and enhanced proliferation. Classic examples are BCR-ABL in CML, Janus kinase 2 (JAK2) mutations in MPN, Fms-like tyrosine kinase 3 (FLT3) and c-KIT mutations in AML.

FLT3 is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in about 1/3 of AML patients, mostly by internal tandem duplications (ITD). Adaptor protein Lnk is expressed in hematopoietic cells and is an important negative regulator in cytokine signaling and hematopoiesis. Previously, we and others have shown that Lnk interacts with the JXM domain of c-KIT, PDGFRA, PDGFRB and FMS, all of which share a similar sequence in this domain. The fact that FLT3 harbors a conserved JXM domain prompted us to investigate whether Lnk interacts with FLT3.

Co-immunoprecipitation and GST-pulldown assay showed that Lnk physically interacts with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through its SH2 domain in multiple types of hematopoietic cells. Through affinity fishing assay with immobilized peptides, we identified the tyrosine residues 572, 591 and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Importantly, Lnk itself was tyrosine-phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Functionally, both shRNA-mediated depletion and ectopic expression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Consequently, Lnk inhibited 32D cell proliferation driven by different FLT3 oncogenic variants. Moreover, analysis of primary bone marrow cells from Lnk^−/−mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors, mainly through inhibiting MAPK-ERK activation by FL.

This study reveals that through direct binding to FLT3-WT and FLT3-ITD, Lnk constrains FLT3-WT/ITD-dependent signaling pathways involved in the proliferation and expansion of hematopoietic cells as well as related leukemic cells. Modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic diseases.

No relevant conflicts of interest to declare.