Abstract
Anaplastic large cell lymphomas (ALCLs) are a unique subgroup of high grade non-Hodgkin lymphomas. Approximately 60% to 70% of ALCLs contain a t (2:5) (p23; q35) chromosomal rearrangement, generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK encodes a constitutively activated tyrosine kinase that has been demonstrated to be directly involved in the pathogenesis of ALCLs. Currently there is no optimal therapeutic regimen for ALK positive ALCLs. Even with high dose CHOP (cyclophophamide, doxorubicin, vincristine and prednisone)-based chemotherapy, a substantial number of patients with ALK positive ALCLs have very poor outcome, either failing to enter remission or relapsing within a few months from the start of treatment. This prompted a search for small molecule ALK inhibitors as potential therapeutic agents for these patients. In this report, we describe the identification and characterization of a potent small molecule ALK inhibitor, cmpd-1. Cmpd-1 inhibited the ALK kinase activity in an in vitro enzymatic assay with an IC50 value of 4 nM. In NPM-ALK transfected BaF3 cells and ALCL-derived cell lines, cmpd-1 potently inhibited the tyrosine phosphorylation of NPM-ALK with a cellular IC50 value of 10–30 nM. Treatment with cmpd-1 inhibited IL-3 independent proliferation of BaF3 cells expressing constitutively active NPM-ALK (more than 95% inhibition at 100 nM) and the addition of IL-3 rescued the growth of these cells, while cmpd-1 had little effect on the parental BaF3 cells. Inhibition of NPM-ALK activity by cmpd-1 in ALK positive ALCL-derived cell lines, Karpas-299, Sudhl-1, Sup-M2 and Sr-786, resulted in inhibition of cell proliferation (50–90% inhibition at 100–300 nM) and induction of apoptotic cell death, while having marginal effects on the proliferation and survival of K562, an ALK negative leukemia cell line. These data suggest that the inhibition of cell proliferation and induction of apoptosis by cmpd-1 is an ALK-dependent event. Several putative downstream targets of NPM-ALK, including ERK1/2, Stat3 and Akt, were hypo- or unphosphorylated when the ALK positive cells were treated with cmpd-1. With the potent ALK inhibitory activity, cmpd-1 can be used as a molecular and pharmacological tool to study the biological roles of ALK and to dissect ALK-mediated signaling pathways in ALCLs and possibly other tumor types in which ALK is implicated in their pathogenesis.
The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling
