Filanesib Primarily Initiates the Apoptotic Program By Activating Bax through a Calpain-Dependent Mechanism

Introduction:

Filanesib (ARRY-520) is a highly selective inhibitor of kinesin spindle protein (KSP), a microtubule motor protein active in proliferating cells, which plays an essential role in assembly and maintaining of the bipolar spindle. In cells arrested by KSP inhibition, Mcl-1 is rapidly depleted resulting in cell death, and consequently cells that are dependent on this pro-survival protein, such as myeloma cells, are particularly sensitive to filanesib. We investigated the mechanisms underlying the antimyeloma effect of this agent, focusing on other Bcl-2 family members.

Methods: In vitro action of filanesib, alone and in combination with calpain inhibitor PD150606, was evaluated in multiple myeloma (MM) cell lines by MTT assay, Annexin V staining and cell cycle profile analysis by flow cytometry. MM cells were transiently transfected with non- targeting control short interfering RNA (NT-siRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Expression levels of different proteins were analyzed by Western-Blot.

Results:

We previously showed that all 11 MM cell lines tested were sensitive to filanesib and that sensitivity to this agent correlated with Bax levels. For these experiments, we focused on 3 cell lines with different Bax expression and sensitivity to filanesib: OPM-2 and MM1S (sensitive and high Bax levels) and U266 (less sensitive and low Bax levels). Treatment of MM1S with this agent triggered the translocation to the mitochondria of several proapoptotic Bcl-2 family members such as Noxa, Bim and Bax with several downstream effects. The mitochondrial translocation and activation of Noxa is key in the degradation of Mcl-1 by mediating the translocation of this protein from the cytosol to the mitochondria promoting its degradation. Regarding Bim, filanesib also induced the early (12-24 hours) expression of several proapoptotic isoforms of Bim that also translocated to the mytochondria. As previously reported, Bax is the top determinant of sensitivity to filanesib. In the present study, remarkably, once translocated into the mitochondria, Bax was also cleaved into the very potent proapoptotic 18 kDa fragment. All these events triggered the mitochondrial release of cytochrome C (caspase dependent apoptosis) and AIF (caspase independent apoptosis).

In order to confirm the role of Bax in filanesib-induced apoptosis, we knocked-down Bax in MM1S by using small interfering RNA. This approach clearly decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced only 26% apoptosis in the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib).

Furthermore, treatment with filanesib also induced cleavage of effector caspases (3 and 7) in all cell lines studied (OPM2, MM1S, U266). PARP was also cleaved in these cells, but it was previous to caspase activation in the most sensitive cell lines (OPM2, MM1S) suggesting a caspase-independent mechanism of apoptosis. This was confirmed by pre-treatment with the pan caspase-inhibitor Z-VAD-FMK, which did not rescue OPM2 and MM1S cells from apoptosis.

Interestingly, one potential mechanism that could link both effects is the activity of calpain, a cysteine protease involved in caspase-independent apoptosis. This protein is a well-known caspase-independent way of processing PARP into the 60 kDa fragment, and has also been described as being responsible for Bax cleavage into the 18-kDa fragment. Consistent with this hypothesis, pre-treatment with the calpain inhibitor PD150606 clearly reduced the activity of filanesib in these cells (35 % to 70 % of survival) as assessed by MTT.

Finally, consistent with the previous hypothesis, the less sensitive U266 cell line contained undetectable Bax protein suggesting that filanesib was not able to trigger caspase-independent apoptosis. However, a secondary caspase dependent apoptosis mechanism was confirmed as the pan-caspase inhibitor ZVAD-FMK was able to almost completely abrogate the activity of filanesib.

Conclusions: Our results show that filanesib primarily initiates apoptosis by activating Bax in a caspase-independent manner, probably via calpain, a powerful accelerator of the apoptotic process. In addition, Noxa and BIM appear to be crucial for modulating Mcl-1 proteasomal degradation and Bax activation.

This work was funded in part by the company Array BioPharma.