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Mechanistic Insights of Pomalidomide Activity in Combination with Bortezomib and Dexamethasone in Multiple Myeloma and Immune Cells


 Introduction: Results from the Phase III, randomized, open-label, clinical study OPTIMISMM comparing the combination of pomalidomide, bortezomib and low-dose dexamethasone (PVd) to bortezomib and low-dose dexamethasone (Vd) in patients with relapsed/refractory multiple myeloma who have all received 1-3 prior lines of therapy, demonstrated significant improvement in progression-free survival (PFS) for pomalidomide versus the comparator arm. Each agent exhibits cytotoxic effects on multiple myeloma (MM) cells when given alone, but acts synergistically in combination, due to non-redundant mechanisms of action. Furthermore, pomalidomide has immuno-stimulatory functionality and enhances T cell and NK-cell-mediated immunity. Pomalidomide binds Cereblon and facilitates degradation of Aiolos and Ikaros, conferring direct cytotoxic effects, while stimulating immune activity. MG132, a proteasome inhibitor (PI), can stabilize pomalidomide-mediated degradation of Aiolos and Ikaros at relatively high concentrations in vitro. A key question is whether PIs, at therapeutic levels, inhibit pomalidomide activity. One obstacle to achieving greater understanding of the bortezomib impact on pomalidomide activity is the highly cytotoxic effect of bortezomib on cultured MM cells. Following intravenous bortezomib injection at the maximal tolerated dose, the Cmax in blood is 260-520 nM, 5 minutes after injection, followed by a rapid drop-off. In vitro, bortezomib is cytotoxic at 1-4 nM, limiting the usefulness of the cell culture model. To circumvent this issue, we implemented a pulse method developed by Shabaneh (2013) and identified sublethal concentrations of bortezomib that had synergistic anti-proliferative effects at concentrations within the clinical window.
 Results: Pomalidomide alone had a dose-dependent anti-proliferative effect on all MM cell lines tested and addition of dexamethasone led to further inhibition of proliferation. Bortezomib pulse at concentrations ≥ 40 nM (MM1.S), 70 nM (H929) or 2 uM (LP-1) led to complete inhibition of proliferation at all concentrations of pomalidomide or dexamethasone used. The synergy and/or additivity of drug combinations was analyzed using the Chou-Talalay Method. Synergies of pomalidomide and bortezomib were observed in the 1-4 nM concentration range for continuous treatment, and 50-560 nM for pulsed treatment, with pomalidomide most synergistic around 100nM. Degradation of Ikaros, Aiolos and ZFP91 was confirmed in MM cells treated with pomalidomide alone and in combination with bortezomib, indicating no inhibitory effect of proteasomal inhibition on substrate degradation.
 In peripheral blood mononuclear cells (PBMCs), IL-2 and Granzyme B production was induced by pomalidomide and inhibited by bortezomib. The combination of pomalidomide and bortezomib (PV) also enhanced IL-2 and Granzyme B secretion albeit to a lesser extent than pomalidomide alone. Degradation of Aiolos, Ikaros and ZFP91 was also observed in isolated T-cells and results closely paralleled those of MM cells. T-cell proliferation was also enhanced by pomalidomide, with maximal effects observed in the 100 nM-1 uM concentration range. While bortezomib alone decreased T-cell proliferation by ~3-fold, the proliferation effect was comparable in cells treated with pomalidomide alone and PV.
 Conclusion: Pomalidomide, bortezomib and dexamethasone exert synergistic anti-proliferative effects on tumor cells. Despite inhibiting proteasomal activity, bortezomib does not antagonize pomalidomide-mediated degradation of Cereblon substrates Aiolos, Ikaros and ZFP91 when used at therapeutically relevant concentrations. Pulsing experiments provide the advantage of mimicking bortezomib dosing regimens; under these conditions, pomalidomide-mediated degradation of substrates was still observed. In immune cells, the PV combination has an overall positive impact on T-cell proliferation despite inhibitory activity of single agent bortezomib. Our results suggest that in combination, pomalidomide can overcome the immunosuppressive effects of bortezomib, when bortezomib is used at therapeutic levels.
 
 
 Katz: Celgene: Consultancy. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership. Serbina:Celgene: Employment.
