Reducing Mcl-1 by Homoharringtonine Induces Apoptosis In Chronic Lymphocytic Leukemia Cells
Authors: Chen Rong , Guo Lei , Chen Yuling , Jiang Yingjun , Wierda William G. , Plunkett William 
Published: October 2019

Abstract
Abstract 2896
Homoharringtonine (HHT, omacetaxine) is an inhibitor of translation elongation that is currently in clinical evaluation in chronic myelogenous leukemia. We hypothesized that this compound would be active in inducing apoptosis in chronic lymphocytic leukemia (CLL) based on the following rationale: 1): CLL cells are characterized by their dependence on the overexpression of anti-apoptotic proteins to maintain their survival; 2): one of the key anti-apoptotic proteins, Mcl-1, turns over rapidly. Thus, upon transient exposure to a translation inhibitor, Mcl-1 expression would decrease rapidly and allow the pro-apoptotic proteins to activate the mitochondria-dependent apoptosis. Using tritiated leucine incorporation as an indicator of new protein synthesis, our results in primary CLL cells demonstrated that HHT was more potent at inhibiting translation than cycloheximide. A polysome profiling analysis confirmed that Mcl-1 translation was blocked by HHT. Mcl-1 protein levels were reduced significantly after HHT exposure, measured by an immunoblotting analysis. This led to mitochondrial membrane depolarization and initiation of apoptosis, analyzed by flow cytometry. Sensitivity to HHT varied in different CLL samples. Further studies showed that this heterogeneous response was independent of the CLL prognostic factors such as the mutation status of the IgVH or ZAP70 expression. Rather, it correlated significantly to the baseline Mcl-1 expression in each CLL sample and the extent of reduction of Mcl-1 protein. A detailed time course study showed that Mcl-1 reduction was evident as early as 2 h and continue to decrease in the next 6 to 8 h; cell death started in 2 h and continued to increase for 24-h. The reduction of Mcl-1 was not affected by the pan caspase inhibitor zVAD, indicating that reduction of Mcl-1 was the initiation event of apoptosis, rather than the results of caspase cleavage. On the contrary, the proteosome inhibitor MG-132 caused retention of the Mcl-1 level in the presence of HHT, suggesting that when Mcl-1 synthesis was blocked by HHT, the remaining Mcl-1 in the cells was degraded rapidly in a proteosome-dependent mechanism. As the action of HHT relied largely on the rapid turn-over of Mcl-1 protein, blocking Mcl-1 degradation by the proteosome inhibitor would counteract the effect of HHT. This explained the observed antagonistic reaction of HHT and MG-132 when they were used together. Further, interaction of CLL cells with the microenvironment has been shown to protect CLL cells from chemotherapy. Incubating CLL cells on either a murine or human stromal cell layer in vitro clearly protected CLL cells from the toxicity of fludarabine. There was an induction of both Mcl-1 transcript and protein levels, which could explain this protection. However, this induction was reversed by HHT, overcoming stromal cell-mediated protection. Finally, because of the important role of Mcl-1 in the pathogenesis CLL, we hypothesized a sequential blockade strategy that inhibits both Mcl-1 transcription and translation to completely block Mcl-1 synthesis. Median effect analysis showed that combining the transcription inhibitor SNS-032 together with HHT killed the CLL cells synergistically. An immunoblotting analysis demonstrated that the combination of the two drugs depleted Mcl-1 more completely than either single drug at the same concentration, which correlated to a greater induction of apoptosis than either drug alone. Taken together, our data demonstrated that transiently inhibiting translation and depleting Mcl-1 was the major mechanism of HHT-induced apoptosis in CLL cells. This action overcame stromal cell-mediated protection and was synergistic with inhibitors of transcription. Thus, these data provided rationale for the clinical development of HHT in CLL, and for the design of combination strategies.

Disclosures:
No relevant conflicts of interest to declare.