Introduction: Multiple myeloma (MM) is characterized by the presence of complex karyotypes and chromosome instability, suggesting that cell cycle checkpoints are defective. Filanesib (ARRY-520) is a highly selective, targeted inhibitor of kinesin spindle proteins (KSP), which are required to establish mitotic spindle bipolarity, driving centrosome separation. Filanesib monotherapy has demonstrated clinical activity in heavily pretreated MM patients (Lonial et al, ASH 2013). In this work we aimed to explore the preclinical activity of filanesib alone and in combination with IMiDs.
Methods: In vitro activity of filanesib alone and in combination with IMiDs (thalidomide, lenalidomide and pomalidomide) was evaluated in MM cell lines by MTT assay and Annexin V, Propidium Iodide and DiOC6 analysis by flow cytometry, Western Blot and immunofluorescence. Synergy was quantified with combination indices (CI) by Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice.
Results: Filanesib demonstrated significant activity in a broad panel of 11 MM cell lines, with 48-hour IC50 values ranging between 0.3 and 5 nM. Interestingly, the highest activity was observed in drug-resistant cell lines such as OPM-2 and RPMI-LR5. We next evaluated whether the cell death MoA was dependent on apoptosis or blockade of proliferation. Time response experiments performed in three different cell lines with different sensitivity to filanesib (OPM-2, MM1S and U266) showed accumulation of cells in G2/M, followed by loss of the mitochondrial membrane potential and activation of apoptosis. Accordingly, Western blot analysis demonstrated an activation of the mitotic checkpoint indicated by an increase in Cyclin B1, and activation of apoptosis with PARP, and caspase-3 and -7 cleavage.
Furthermore, filanesib activity was very rapid as 15 minutes of exposure was sufficient to exert all of the apoptotic and cell cycle effects observed at 48 hours. Immunofluorescence microscopy using alpha-tubulin demonstrated that filanesib induces monopolar spindle formation.
The sensitivity of MM to filanesib has been previously correlated with the cell-dependency of the anti-apoptotic protein Mcl-1. We, therefore, studied the basal levels of six Bcl-2 family members (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the 11 cell lines and observed a correlation between the basal levels of these proteins and drug sensitivity. In particular, we confirmed the relationship with the anti-apoptotic proteins Mcl-1 and Bcl-2 and demonstrated that conversely to what is observed with proteasome inhibitors, cells with high basal levels of pro-apoptotic Bax or Bak were more resistant to filanesib. Moreover, treatment with filanesib induced a clear decrease of Mcl-1 in the 3 cell lines analyzed (U266, MM1S and OPM-2) that coincided with a decrease of Bcl-2 in the most sensitive cell line, OPM-2.
Finally, we evaluated the activity of filanesib in combination with IMiDs and dexamethasone. In vitro studies showed a synergistic effect of filanesib with dexamethasone (CI: 0.21), and with all IMiDs, being most pronounced with pomalidomide (CI: 0.09). Of note, this triple combination demonstrated the highest synergistic activity (CI: 0.06). These results were confirmed in vivo where the triple combination of filanesib, dexamethasone, and pomalidomide was also synergistic, with a significant reduction of tumor growth of up to 50 days, which correlated with a statistically significant survival improvement. Mechanistic studies on the combination are ongoing.
Conclusions: Our results demonstrate the potent, rapid activity of filanesib which induces a cell cycle blockade through the inhibition of KSP, leading to apoptosis in MM. It was identified that this activity is dependent on the anti-apoptotic protein Mcl-1 and pro-apoptotic proteins Bax and Bak. Furthermore, in animal xenograft studies, filanesib exhibited robust synergism in combination with dexamethasone and any IMiD, pomalidomide being the most synergistic. These data are the rationale for the clinical trial "Pomdefil" which uses this combination in patients with relapsed refractory MM. The trial will begin soon as a collaboration of the Spanish Myeloma Group (GEM).
This work was supported in part by Array BioPharma.
Humphries:Array Biopharma: Employment. Tunquist:Array Biopharma: Employment. Mateos:Array Biopharma: Honoraria. Ocio:Array Biopharma: Honoraria, Research Funding.
Asterisk with author names denotes non-ASH members.