Mechanisms Underlying the Synergistic Interaction of Filanesib with Pomalidomide and Dexamethasone (FPD) in Multiple Myeloma

Introduction: Filanesib (ARRY-520) is a novel inhibitor of the "kinesin spindle protein" (KSP), which has demonstrated efficacy in heavily pretreated patients with refractory MM, (Lonial et al, ASH 2013). Our preliminary studies demonstrated synergy with standard anti-MM agents, especially with pomalidomide and dexamethasone. This set the stage for a recently activated trial being run by the Spanish MM group investigating FPD in relapsed MM patients. In this abstract we investigate the mechanisms underlying the synergy of the combination.

Methods: In vitro action of FPD was evaluated in MM cell lines by MTT assay, bioluminescence, Annexin V staining, cell cycle profile analysis and TMRE staining by flow cytometry. Synergy was quantified with the Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice (The Jackson Laboratory, Bar Harbor, ME, USA). The mechanism of action was analyzed by Western blot, flow cytometry, genomic techniques, immunohistochemistry and immunofluorescence techniques.

Results: The triple combination of FPD resulted in clear synergy in multiple myeloma cell lines (MM1S, OPM2, and RPMI8226) with combination indices between 0.4-0.7, and abrogated the effect of the soluble cytokines IL-6 and IGF-I and the protective effect of the adhesion of plasma cells to BMSCs, HS-5 and TERT cells.

FPD caused cell cycle arrest in G2/M and specific apoptosis of cells arrested in these proliferative phases (with apoptosis percentage of 5, 23, 58 and 88 for control, poma+dexa, filanesib and FPD, respectively) demonstrated by flow cytometry with DRAQ5 and Annexin-V. Thus, FPD and filanesib in monotherapy treatments induced a similar effect on the cell cycle profile (arrest in G2/M) with a concordant increase of cyclin B1 and phosphorylated Histone H3. Although a secondary increase of KSP protein levels would be expected, pomalidomide and dexamethasone induced a decrease of the levels of this protein, which was still present in the triple combination (FPD). This fact could be contributing to the potentiation observed with the combination. Attending to apoptosis mechanism, proapoptotic stimulus from the extrinsic and intrinsic apoptotic pathways were promoted by pomalidomide and dexamethasone and filanesib, and converged in the triple combination. In this regard, a decrease of MCL-1 (antiapoptotic protein) and a significant increase of the proapoptotic BCL2 family members of the intrinsic pathway like NOXA and BIM_EL BIM_L, BIM_S(this last one being the most potent proapoptotic isoform), tBID (extrinsic pathway) and Bax protein were observed. We confirmed that all these proteins were translocated into the mitochondria, resulting in a decrease of the mitochondrial membrane potential by TMRE, increase of permeability and a release of cytochrome C and AIF.

These results were confirmed in vivo in a model of subcutaneous plasmacytoma in small (70 mm3) and large (2000 mm3) tumors. In this model we observed a significant reduction of tumor growth, which was correlated with a statistically significant improvement in survival. Changes induced by FPD in the gene expression profile were concordant with the in vitro results as several overexpressed genes belonging to the previous pathways were identified, such as spindle assembly checkpoint (CENP-E and CENP-F) and apoptosis (BCL2L11, gene that codifies BIM protein). Furthermore, IHC of tumors treated with FPD showed more apoptosis by TUNEL and a significant increase of monopolar spindles (2, 0, 53 and 140 per 10 high-power fields, for control, poma+dexa, filanesib and FPD, respectively).

Conclusions: The synergy observed with filanesib in combination with pomalidomide and dexamethasone is the result of several coincidental mechanisms: a potentiation of the KSP inhibition with a subsequent increase in monopolar spindle formation and a simultaneous activation of the intrinsic and extrinsic pathways of apoptosis. In this regard, NOXA, BIM, BAX and tBID are probably the central players that, through different mechanisms, inhibit antiapoptotic proteins (MCL-1, BCL2 and BCL-XL) and promote mitochondrial outer membrane permeabilization and the release of apoptogenic factors such us cytochrome C and AIF.

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