Targeting Src- Family Kinase Activation and Downstream Molecular Events To Overcome MM Cell Growth, Survival, and Drug- Resistance.

IL-6 induces growth and survival factor in multiple myeloma (MM) via ERK and Akt signaling cascades. Our recent studies difined early signaling events mediating phosphorylation of ERK and Akt-1: specifically, a gp130/Hck- containing complex plays a pivotal role in Hck- mediated phosphorylation of Grb2- associated binder (Gab) family adapter proteins thereby mediating downstream signaling (Podar et al, 2004). We have also shown that IL-6 triggers resistance to dexamethasone via a PI3kinase/ Akt-1 signaling pathway by specific activation of SHP2 phosphatase, withsubsequent blockade of related adhesion focal tyrosine kinase (Chauhan et al, 2000). In the present study we first evaluated AP 23464, a potent adenosine 5′-triphosphate (ATP)- based inhibitor of Src kinase (ARIAD Pharmaceuticals Inc., Cambridge, MA), for its effect on proliferation and survival in MM cells. Our results show that AP23464 markedly inhibits both IL-6- triggered Hck activation and the association of Gab adapter protein Gab1 with SH2- domain containing protein- tyrosine phosphatase - 2 (SHP2). Consequently, AP23464 both inhibited MM cell proliferation, as evidenced by 3H[dT] uptake, and induced apoptosis, evidenced by marked increase in sub-G1 phase cells. In contrast, proliferation of normal donor peripheral blood mononuclear cells remained unchanged. Next we investigated the role of Gab-1- SHP2 association mediating IL-6- induced resistance to dexamethasone, and conversely, the effect of inhibition by AP23464. Specifically, we determined whether IL-6- triggered Gab1- SHP2 binding is required for both RAFTK binding to SHP2 and associated IL-6- mediated dexamethasone resistance in MM cells. Utilizing wild- type Gab1 and SHP2- Gab1 binding mutants, our results show marked reduction in IL-6- triggered SHP2- RAFTK association. Moreover, IL-6- triggered resistance to dexamethasone was markedly inhibited in MM cells transfected with SHP2- Gab1 mutants, but not with wild type- Gab1. Importantly, AP23464 downregulated IL-6- mediated resistance to dexamethasone in a dose- dependent manner. Taken together, the present study shows anti-MM effects of AP23464, including abrogation of IL-6- induced dexamethasone resistance via inhibition of SHP2/ RAFTK- binding to Gab1. These studies provide the preclinical basis for the therapeutic evaluation of AP23464, alone or in combination with dexamethsoneto improve patient outcome in MM.