Abstract #1815: Homoharringtonine down-regulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia cells

Homoharringtonine (HHT) is a plant alkaloid with antileukemic activity that is currently in clinical trials for the treatment of chronic myelogenous leukemia (CML). Its mechanism of action in CML is likely inhibition of protein synthesis and the consequent depletion of Bcr-Abl. Because anti-apoptotic proteins, such as Mcl-1, are important in the survival of chronic lymphocytic leukemia (CLL) cells, we hypothesized that inhibition of protein synthesis by HHT would decrease the expression of Mcl-1, induce apoptosis in CLL, and that HHT may act synergistically with transcription inhibitors. We first studied the cytotoxicity of HHT in primary CLL cells incubated with 50-400 nM HHT for 6 to 24 hr; apoptosis was quantified by Annexin V-propidium iodide staining. HHT at concentrations as low as 50 nM induced significant apoptosis in CLL cells after 12 hr of treatment. The IC₅₀ of HHT for 24 hr of treatment was 222 nM, indicating that HHT is a potent agent in killing CLL cells. The inhibition of protein synthesis was studied by [³H]leucine incorporation. HHT (10 \#956;M) inhibited protein synthesis to 50 to 60% of control levels in 3 hr. Furthermore, the actions of HHT on anti-apoptotic proteins were investigated by immunoblotting. HHT (50 nM) decreased Mcl-1 levels by 60% in 12 hr. This response was concentration-dependent and increased with time of exposure. The cleavage of PARP accompanied this effect, indicating the initiation of apoptosis. In contrast, no change in Bcl-2 protein levels was detected, probably because of its prolonged protein half-life. To determine whether the disappearance of Mcl-1 was associated with inhibition of protein synthesis or with cleavage by a caspase, CLL cells were incubated with HHT alone or in the presence of the pan-caspase inhibitor zVAD. The addition of zVAD did not affect the decrease in Mcl-1, although PARP cleavage was largely blocked. These results suggested that the inhibition of protein synthesis by HHT directly contributed to the diminished Mcl-1 protein levels rather than this being a secondary effect of the initiation of apoptosis by a different mechanism. The actions of HHT and SNS-032, a transcription inhibitor, would likely put the actions of the agents in series, mechanistically. Low concentrations (50 nM) of each compound appeared to induce additive cell killing in CLL cells. In summary, HHT inhibits protein synthesis, down-regulates Mcl-1, and induces apoptosis in CLL cells. These results provide a rationale for clinical trials in CLL as a single agent or in combination with transcription inhibitors

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1815.