Bone destruction, a hallmark of multiple myeloma (MM), arises as a consequence of the interactions between MM cells and the bone marrow microenvironment, which lead to an increase in the bone-resorptive activity and number of osteoclasts (OC) and a reduction of the bone-forming activity and differentiation of osteoblasts (OB).
MLN9708, which hydrolyzes to pharmacologically active MLN2238 in aqueous solution, is an investigational proteasome inhibitor (PI) with demonstrated preclinical anti-myeloma activity. However, it is currently not known whether MLN9708, may have a beneficial effect on myeloma-associated bone disease. Here, we have conducted in vitro and in vivo studies to evaluate its ability to promote osteogenic differentiation and to inhibit OC formation and function in the myeloma setting.
The human MM cell lines RPMI-8226 and MM.1S (or RPMI-8226-luc and MM.1S-luc) together with the mesenchymal stem hMSC-TERT cell line were employed. Also, MSCs from BM samples of healthy donors and MM patients were used in OB differentiation studies, whereas PBMCs from healthy volunteers were used to generate OCs. NOD.SCID.IL2Rγ−/− mice were used in the in vivo model of disseminated human MM. MLN2238 and bortezomib (Velcade) were provided by Millennium Pharmaceuticals, Inc.
OB differentiation from MSCs and OB function were investigated by measurement of ALP activity, quantitative mineralization, luciferase reporter assays, siRNA gene silencing and real time RT-PCR. The effect of the new PI on OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells. Measurement of resorbed area, immunofluorescence and flow cytometry were used to further investigate the effect of MLN2238 on OC function. In our in vivo model, bioluminescence imaging, micro-CT analysis and serum levels of Igλ and bone markers were determined.
Physiologic concentrations of MLN2238 were able to stimulate the osteogenic differentiation of MSCs from both myeloma patients and healthy donors in vitro to an extent comparable to bortezomib; this was assessed by increased levels of ALP activity, higher expression of bone formation markers (Runx2, osterix, osteopontin and osteocalcin) and augmented matrix mineralization. The enhanced OB formation and function induced by MLN2238 was at least partly due to induction of T-cell factor 4 (TCF4) transcriptional activity, as well as to activation of the unfolded protein response. A similar range of MLN2238 doses also markedly inhibited OC formation and resorption from human progenitors. Similarly to that described with bortezomib, MLN2238 treatment of human pre-OCs prevented RANKL-induced NF-κB activation, disrupted the integrity of the F-actin ring and also reduced the expression of the αVβ3 integrin, thus contributing to inhibition of OC function. MLN2238 was also able to overcome the growth advantage conferred to MM.1S-luc cells by co-culture with MSCs or OCs.
Oral administration of MLN2238 in a mouse model of disseminated human MM decreased human RPMI-8226-luc tumor burden as assessed by diminished bioluminescence signal and decreased serum levels of Igλ secreted by RPMI-8226-luc cells. In addition, MLN2238 prevented tumor-associated bone loss with significant increases in femoral trabecular bone parameters as compared to vehicle control animals. Serum markers of bone turnover showed that MLN2238 inhibited bone resorption (decreased levels of CTX) while enhancing bone formation (increased levels of P1NP).
MLN2238 in vitro was capable of promoting osteoblastogenesis and OB activity as well as of inhibiting OC formation and function to an extent similar to bortezomib. In a disseminated human MM mouse model, orally administered MLN2238 showed anti-resorptive and bone-anabolic effects in addition to its anti-tumor properties. Given the thus far available data on the preclinical safety and favorable pharmacologic properties of MLN2238, it is conceivable that MLN9708, the clinical formulation of this proteasome inhibitor, may also achieve bone benefits in myeloma patients.
Berger:Millennium Pharmaceuticals, Inc.: Employment. San-Miguel:Millennium Pharmaceuticals, Inc.: Consultancy.
This work was supported by funding from the Spanish MICINN-ISCIII (PI081825), Fundación Española de Hematología (AG-G), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, the Spanish Myeloma Network Program (RD06/0020/0006 and RD06/0020/0041) and Spanish FIS (PS09–01897).
Asterisk with author names denotes non-ASH members.