Combination of the Proteasome Inhibitor Bortezomib and a Histone Deacetylase Inhibitor PXD101 Results in Synergistic Inhibition of Osteoclastogenesis and Significantly Stronger Inhibition of Multiple Myeloma Growth In Vitro and In Vivo.

Abstract

Bortezomib, a proteasome inhibitor, has been known to have in vitro and in vivo activity against multiple myeloma (MM). PXD101, a novel, low molecular weight histone deacetylase (HDAC) inhibitor recently has been recognized to induce growth arrest and apoptosis in MM cells. We hypothesized that these two agents, with different molecular targets, can be combined with a high probability of additive or synergistic effects without exerting cross-resistance.In this study, we investigated the activity of the combination of PXD101 and bortezomib against osteoclast generation and MM cell growth. To evaluate the effects of bortezomib and PXD101 on the formation of osteoclasts from mononuclear hematopoietic precursors, we generated osteoclasts cultured from human bone marrow cells from healthy donors and MM patients using in vitro RANKL/M-CSF stimulation. Proliferation of MM cell lines treated with PXD101, bortezomib, or their combination was determined by measuring 3H-Thymidine incorporation. Annexin V-FITC/PI staining was used to determine the degree of apoptosis, and trypan blue was used to assess cell viability. We utilized a xenograft murine model to test the in vivo synergistic activity of the two agents against MM tumor formation.We found that even though either drug alone significantly inhibited osteoclast formation at concentrations of PXD101 at 25 nM or bortezomib at 1 nM, the combination resulted in synergistic inhibition in osteoclast assays. Treatment of MM cells lines (MM.1S, RPMI-8226, OPM2) with serial dilutions (10–100 nM) of the combination also led to significantly stronger inhibition of proliferation, apoptosis and cell death than single drug treatment. In addition, the combination demonstrated synergistic induction of cell death in primary human CD138+ myeloma cells isolated from patient samples. In vivo, the combination of PXD101 and bortezomib resulted in significantly higher xenograft tumor inhibition compared to either drug treatment alone and was not more toxic.The current study provides the rationale for clinical evaluation of this novel PXD101-bortezomib combination for patients with MM. The data generated here support the hypothesis that simultaneous targeting of different proliferative and anti-apoptotic pathways both in tumor cells and activated osteoclasts avoids cross-resistance and constitutes a dynamic approach to treat MM.