Summary Clinical trials have shown the high anti‐myeloma activity of the proteasome inhibitor bortezomib. The present study examined the activity of bortezomib combined with PXD101, a histone deacetylase inhibitor, against multiple myeloma (MM) and osteoclastogenesis. Treatment of myeloma cell lines with combinations of bortezomib and PXD101 led to synergistic inhibition of proliferation and induction of cell death. The combination significantly decreased the viability of primary human CD138+ myeloma cells but not of bone marrow mononuclear cells. Further studies showed a dose‐dependent activation of caspases‐3, ‐8 and ‐9 and nuclear fragmentation in myeloma cells. Bortezomib/PXD101 treatment markedly triggered reactive oxygen species (ROS) generation that was accompanied by p53, H2A.X and p38–mitogen‐activated protein kinase phosphorylation. ROS generation could be blocked by the free radical scavenger N‐acetyl‐l‐cysteine. The combination of bortezomib and PXD101 also resulted in synergistic inhibition of osteoclast formation. In conclusion, bortezomib and PXD101 have different molecular targets. The combination induces cell death in myeloma cells via ROS‐mediated DNA damage and also inhibits osteoclastogenesis. Therefore, this study provides the rationale for the clinical evaluation of bortezomib combined with PXD101 in patients with MM.

IntroductionThe major source of morbidity and possible mortality associated with multiple myeloma (MM) is osteolytic bone destruction throughout the axial skeleton. Lytic bone lesions occur in 70% to 80% of these patients, and are frequently associated with severe bone pain and pathologic fractures. 1 Besides MM, osteolytic lesions also occur in connection with bone metastases from solid tumors. Although the exact incidence of bone metastases is unknown, it is estimated that 350 000 people die with bone metastases each year in the United States. 1 Osteolytic lesions represent an extreme of a continuum of dysregulation of the normal bone remodeling process with excessive bone resorption mediated by osteoclasts (OCLs). OCLs are multinucleated cells that derive from monocyte/macrophage lineage cells. [2][3][4][5][6] How commitment to a given lineage occurs among macrophages, OCLs, and dendritic cells has not been fully elucidated, but complex signaling mechanisms are known to control OCL differentiation. 7 These signals include cytokines such as macrophage colony-stimulating factor (CSF-M), granulocytemacrophage colony-stimulating factor (CSF-GM), and interleukin-3, 8 which are macrophage-inducing cytokines, and the receptor activator of NF-B ligand (RANKL), which is a critical factor for late stages of OCL development. 9 In addition to cytokines, several transcription factors are vital for OCL formation; the most important are PU.1 10 and c-fos. 11,12 PU.1 (Spi-1) is essential for the development of myeloid and B-lymphoid cells. [13][14][15] In myeloid cells, PU.1 is known to regulate transcription of both c-fms and CD11b/CD18 (Mac1), both of which are central to the OCL phenotype. 16-18 PU.1-deficient mice show a failure in macrophage differentiation, and osteoclastogenesis is inhibited at a very early stage of differentiation. 10,19 Mice deficient in c-fos exhibit osteopetrosis due to an OCL differentiation defect, while the number of macrophages increases, indicating that c-fos acts on a later stage of OCL development. 10,11 It is clear that any disruption of hematopoiesis can profoundly affect OCL numbers.Thalidomide was originally developed in the 1950s to treat pregnancy-induced morning sickness. Following the discovery of its teratogenic effects, it was subsequently withdrawn from the market in 1961. 20 Since the discovery of thalidomide's antimyeloma activity in the late 1990s, 21 new and more potent immunomodulatory derivatives (IMiDs) of thalidomide such as CC-4047 (Actimid) and CC-5013 (Revlimid) with fewer side effects have been developed. In previous studies, we showed that IMiDs inhibit For personal use only. on May 10, 2018. by guest www.bloodjournal.org From the in vitro and in vivo growth of MM cells. 22,23 In initial clinical phase 1 and 2 trials, single agent CC-4047 and CC-5013 in combination with dexamethasone achieved response rates of over 50% in relapsed and refractory MM. [24][25][26] We have shown that CC-4047 induces a shift in lineage commitment, resulting in increased myeloid cell deve...

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