An Acidic Peptide Inhibits the Interleukin-6-Induced Activities of the Src Family Kinases Hck, Lyn and Fyn in Myeloma Cells.

Background: Src Family Kinases (SFKs) play pivotal roles in normal B-cell development and in B-cell neoplasias. Our aim is to elucidate the roles of SFKs in signaling cascades resulting in cell proliferation or anti-apoptosis in multiple myeloma (MM). The pleiotropic cytokine interleukin-6 (IL6) is one of the major growth factors for MM cells. We have previously shown that IL6 induces the activation of the SFKs Hck, Lyn and Fyn and that Hck is associated with the IL6R beta chain (gp130) via an acid domain (AD) in gp130.

Aim: We observed that an 18mer peptide (18AD), which is derived from the AD, inhibited the IL-6-dependent growth of myeloma cells. In order to develop a lead structure for small molecule inhibitors we wish to elucidate the cellular and molecular effects of peptide 18AD.

Results: On the cellular level, 50–100μM of a membrane-permeable myristoylated peptide 18AD inhibited factor-dependent proliferation in 7TD-1 (mouse) and INA-6 (human) cells by ~75%. Taking the percentage of annexinV-stained cells after IL6-withdrawal as the reference value for the level of maximal apoptosis, treatment of 7TD1 and INA-6 cells with peptide 18AD resulted in 100 and 60 % of the respective percentages of apoptotic cells. A control peptide (18sc) with an identical amino acid composition but an arbitrarily scrambled sequence had no effect on proliferation and apoptosis. Similar effects of peptides 18AD and 18sc were observed in Baf-EH cells, which overexpress a chimeric erythropoietin receptor-gp130 and human Hck and grew in an erythropoietin dependent manner. Peptide 18AD had no significant growth inhibiting effects on BaF-HE cells cultured in the presence of IL3 or on factor-independent OKT1 hybridoma cells. In 7TD1 cells the expression of a kinase-inactive Hck mutant lead to a reduced proliferative response to IL6. On the molecular level, immunoprecipitation experiments in 7TD1 cells showed that peptide 18AD decreased the complex formation between Hck and gp130 in a concentration dependent way. Immunocomplex kinase assays in INA-6 cells showed that the IL6 induced activation of Hck, Lyn and Fyn was blocked by peptide 18AD, but not by peptide 18sc.

Conclusion: We characterized peptide 18AD, which inhibits the association of Hck and gp130 as well as IL6-induced SFK activity. Thus, the observed anti-proliferative and pro-apoptotic effects of peptide 18AD may be due to inhibition of SFK-mediated pathways.