Bortezomib has become one of the most important novel agents in the current therapy for multiple myeloma (MM). This agent has demonstrated substantial activity in the setting of recurrent/refractory disease and, more recently, in the frontline setting; in the latter setting, it was shown to be superior to conventional treatments in both the transplant and nontransplant protocols.[1-10]
Bortezomib exerts its potent antimyeloma activity in 2 predominant ways: it directly impacts myeloma cell survival through the downregulation of growth/survival signaling pathways and the upregulation of molecules implicated in proapoptotic cascades, and it acts on the bone marrow microenvironment by interfering with the adhesion of MM cells to bone marrow stromal cells. This inhibits the production of cytokines in the bone marrow and restricts the development of tumor-associated blood vessels.[11-13] In addition, data are accumulating that indicate that the effects of bortezomib extend beyond the MM cell and that the agent influences other processes, such as bone metabolism. Indeed, the proteasome plays a crucial role in the degradation of regulatory proteins such as cell cycle and tumor suppressor proteins, transcription factors, and mutant or damaged proteins. It is therefore fundamentally involved in the activation and inactivation of many cellular processes and the maintenance of cellular homeostasis. Inhibition of the proteasome results in growth arrest and cell death, believed to be due to the induction of an apoptotic cascade as a result of the rapid accumulation of regulatory proteins within the cell. The transcription factor nuclear factor kappa B (NF-κB) is one of the proteins that is regulated by proteasomal activity. In the cytoplasm, bound to its inhibitor IκB, NF-κB is inactive. However, in response to stimuli, IκB is degraded by the proteasome, thereby releasing NF-κB, which, at the time of translocation into the nucleus, initiates the transcription of a wide range of genes encoding proteins involved in cell survival, cell adhesion, and cytokine signaling. NF-κB has been found to be constitutively active in some cancer cells and to be associated with resistance to anticancer therapy. The constitutive activation of NF-κB has been shown to promote the expression of cytokines with proinflammatory and proangiogenic activities. In patients with MM, NF-κB activation has been shown to promote the growth, survival, and drug resistance of MM cells in the bone marrow microenvironment.[18, 19] Therefore, NF-κB has been identified as an attractive target for anticancer agents and as a target in patients with MM in particular. Conversely, NF-κB is also a key regulator of proteins involved in immune or inflammation responses.[20, 21] The inhibitory action of bortezomib is mediated in part through the stabilization of the NF-κB inhibitor IκB, resulting in the inhibition of NF-κB.[11, 16] However, it is recognized that bortezomib also affects additional cell survival pathways, such as the p44/42 mitogen-activated protein kinase pathway. Furthermore, inhibitory effects on interleukin-6, tumor necrosis factor-α, and vascular endothelial growth factor have also been demonstrated.[12, 23, 24]
Thus, the multifaceted effects of bortezomib provide the rationale for the investigation of the agent in patients with malignant disease and particularly in those with MM. The objective of the current review was to address the effects of bortezomib outside the tumor cell.
Myeloma-Associated Bone Disease Pathophysiology
Bone disease is a hallmark of MM. Myeloma bone disease is due to an imbalance in bone remodeling characterized by increased osteolytic bone destruction that is not compensated for by new bone formation. This imbalance in bone metabolism arises from the activity of myeloma cells, which promote osteoclast function, thereby mediating bone destruction while simultaneously suppressing osteoblast activity, resulting in the prevention of new bone formation.[25-27] The regulation of bone remodeling is in large part controlled by a cytokine system consisting of receptor activator of NF-κB ligand (RANKL) and its cellular receptor RANK, as well as the decoy receptor for RANKL, osteoprotegerin (OPG). Maturation and activation of osteoclasts is initiated by the binding of RANKL to RANK, which is expressed on the surface of osteoclasts. Binding of OPG to RANKL neutralizes the ligand, thereby preventing osteoclast maturation and the accompanying bone resorption. The balance between RANKL and OPG, which exists in healthy bones, is disturbed in patients with MM in favor of RANKL, which is produced by myeloma cells themselves, as well as bone marrow stromal cells as a result of stimulation through myeloma cells. Furthermore, myeloma cells inhibit the production of OPG by stromal cells, further contributing to the imbalance between the 2 factors. Other factors produced by myeloma cells, such as macrophage inflammatory protein-1α and interleukin-6, contribute to increased osteoclast activity, further adding to the promotion of bone resorption. This in turn causes the release of cytokines and growth factors that promote myeloma cell growth. The result is a vicious cycle of bone destruction and myeloma cell growth.
In addition, myeloma cells inhibit osteoblasts, an effect that is mediated by dickkopf-1 (DKK-1), an inhibitor of the wingless-type (Wnt) signaling pathway, which is important in osteoblast maturation. Furthermore, other factors important to this process, such as secreted frizzle-related protein-2, another Wnt signaling pathway inhibitor, and the transcription factor runt-related transcription factor 2 (Runx2)/core-binding factor subunit alpha-1 (Cbfa1) are believed to be affected by myeloma cells and further contribute to a reduction in osteoblast activity.
Bortezomib and Bone Disease: Preclinical Data
The first indication of a potential role for proteasome inhibitors in patients with bone disease from MM was provided by Garrett et al, who observed that the treatment of murine bone tissue with proteasome inhibitors increased osteoblast differentiation and bone formation in vitro. These investigators found a strong correlation between the magnitude of stimulation of bone formation activity and the degree of proteasome inhibition of the different agents. Bone formation was found to be mediated through the stimulation of bone morphogenetic protein-2 production in osteoblasts. These data indicated that the ubiquitin-proteasome pathway is involved in the regulation of osteoblast differentiation and bone formation. Furthermore, Zavrski et al found that the ubiquitin-proteasome pathway also plays an important role in the regulation of osteoclast development. In preclinical experiments, the investigators observed that proteasome inhibitors impeded osteoclast differentiation and function and that the decrease in resorption correlated with the extent of NF-κB binding capacity.
Bortezomib has been found to exert a direct inhibitory effect on the differentiation and activity of osteoclast precursors because of inhibition of NF-κB and p38 mitogen-activated protein kinase pathways.[30-32] In addition, nuclear factor of activated T cells and TRAF6 have been identified as targets of bortezomib in the inhibition of osteoclast maturation and function.[33, 34] Preclinical experiments have also suggested that bortezomib has a direct stimulatory effect on osteoblast development, both increasing the number of osteoblasts and stimulating their differentiation.[35-37] The inhibitory activity of bortezomib on the ubiquitin-proteasome pathway is suggested to be of central importance in explaining the effect of bortezomib on osteoblast development.[28, 32, 35] For example, the induction of osteoblast differentiation was found to be due to bortezomib significantly increasing the activity of the osteoblast transcription factor Runx2/Cbfa1 in human osteoblast progenitors.[35, 36] Furthermore, bortezomib treatment has been found to result in an increase in Osterix RNA and in enhanced activity of bone morphogenetic protein-2. In addition, the stimulatory effect of bortezomib on bone formation was found to be due to its inhibition of DKK-1 expression.[39, 40]
A recent report has suggested that bortezomib may also stimulate osteogenic differentiation independently of proteasome inhibition through an inhibitory effect on fibroblast growth factor-2, which is found at high levels in patients with MM, and which inhibits a transcriptional coactivator that induces differentiation of mesenchymal cells into osteoblasts. With regard to second-generation proteasome inhibitors, preclinical data have also suggested an effect on bone disease. Indeed, a recent study by Hurchla et al demonstrated that carfilzomib and its orally available analog oprozomib directly inhibited osteoclast formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Furthermore, in a mouse model, carfilzomib and oprozomib were found to decrease bone resorption and enhance bone formation.
Taken together, these studies provided the rationale for the investigation of bortezomib in patients with myeloma-associated bone disease.
Bortezomib and Myeloma-Associated Bone Disease: Clinical Data
In the human setting, initial clinical evidence to suggest that bortezomib may play a role in promoting osteoblastic bone formation was obtained in the phase 3 APEX trial, in which Zangari et al observed a significant increase in serum alkaline phosphatase (ALP) levels indicative of an increase in osteoblast activity in a patient who responded to treatment with bortezomib. This increase in ALP was due to bortezomib increasing the levels of bone-specific ALP (bALP), indicating a direct and specific effect on osteoblastic activity. In contrast, no increases in ALP levels were observed in patients responding to dexamethasone treatment, suggesting that the increase in ALP as a result of bortezomib treatment is not due to an antimyeloma effect alone. Increases in bALP as well as osteocalcin (OC) levels after treatment with bortezomib have since been observed in several clinical studies investigating the drug as a single agent or in combination.[35, 44-46] In addition, clinical studies have shown that bortezomib effectively reduces levels of DKK-1.[45, 46] In some of the reports, the increase in osteoblast number and activity was restricted to responders,[35, 47] whereas in others, both responders and nonresponders demonstrated signs of increased osteoblast activity, thereby supporting the hypothesis that a direct stimulatory effect on the bone formation process may occur during treatment with bortezomib.[44, 45, 48] In addition, the inhibitory effect of bortezomib on osteoclasts observed in the preclinical setting has been confirmed in clinical studies. Bortezomib treatment was shown to result in a significant reduction in RANKL levels, with concomitant reductions in osteoclast function and bone resorption, as assessed by the following markers of bone resorption: C-telopeptide of type I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b), and urinary N-telopeptide.[45, 46, 48, 49]
To the best of our knowledge, Terpos et al were the first to demonstrate a reduction in DKK-1 and RANKL serum levels as a result of treatment with bortezomib, leading to the normalization of bone remodeling. Patients with recurrent MM were treated with bortezomib either as monotherapy or in combination with dexamethasone. Treatment resulted in a significant reduction in the levels of DKK-1, sRANKL, CTX, and TRACP-5b after 4 cycles, a reduction that was maintained after 8 cycles (Table 1). In addition, marked increases in bALP and OC levels were noted. Although the increase in bALP was greatest in patients who achieved complete remission (CR) or very good partial remission (VGPR) after 4 cycles of bortezomib, bALP was also found to be elevated over baseline in patients who did not achieve at least a VGPR as well as in 3 of 4 nonresponders. For the other bone markers (OC, DKK-1, sRANKL, CTX, and TRACP-5b) no correlation with response was observed, suggesting that the effect of bortezomib on bone occurs irrespective of treatment response.
|Parameter||Baseline||After 4 Cycles of Bortezomib||P (Versus Baseline)||After 8Cycles of Bortezomib||P (Versus Baseline)|
|Markers of bone resorption|
|Markers of bone formation|
In several recent studies, bortezomib treatment was found to result in increases in bone mineral density (BMD) and bone matrix deposition.[50-53] Notably, bone healing has been observed in a small number of cases.[51, 53] Terpos et al studied the effect of treating patients with MM at the time of first recurrence with bortezomib plus dexamethasone and zoledronic acid on BMD using dual-energy x-ray absorptiometry. After 8 cycles of therapy, a significant increase in BMD was observed in the lumbar spine, but not in the femoral neck. The investigators noted that the increase in BMD was noted soon after the initiation of treatment (within approximately 6 months). In addition, urinary N-telopeptide levels were found to be significantly reduced, whereas levels of bALP and OC were markedly increased over baseline levels.
In a recent case report, the effect of bortezomib on myeloma bone lesions was assessed using technetium-99m (99mTc)-methyl-diphosphonate bone scans in 2 patients. An increased uptake of the radiopharmaceutical by osteoblasts is associated with rebuilding activity. After 3 cycles of bortezomib monotherapy in a patient with recurrent disease and combination therapy with bortezomib, doxorubicin, and dexamethasone in a patient with newly diagnosed disease, bone scan images revealed markedly increased osteoblastic function as indicated by increased isotope uptake, which may suggest improvements in lesions. However, these results require confirmation in larger series and using other imaging techniques, such as x-ray or computed tomography (CT) scans. In another prospective study, Zangari et al investigated the effect of single-agent bortezomib on bone structure in patients with recurrent or refractory disease using micro-CT scans, tetracycline labeling, and morphometric analysis.52 Treatment with bortezomib was shown to induce significant increases in osteoblast activity; moreover, it also resulted in increases in bone architectural parameters in responding patients. In responders, a rapid increase in serum parathormone levels was found to precede the osteoblastic response, suggesting that the rise in parathormone levels may trigger osteoblastic activation and bone anabolism. The authors concluded that the results of their study indicate a central role of proteasome function in bone metabolic disorders in addition to the treatment of MM.
In another prospective study, Lund et al treated patients with newly diagnosed MM who had not received any prior bisphosphonate therapy with bortezomib, initially as monotherapy and subsequently in combination with glucocorticoids. Not only did the treatment yield an increase in osteoblast activity, but it also led to enhanced bone matrix deposition in responding patients, as witnessed by a significant increase in the novel bone marker procollagen type I N-terminal propeptide (PINP). PINP is released as collagen is deposited and becomes insoluble during the formation of the organic bone matrix and may therefore serve as a marker for ongoing bone formation. Both bALP and PINP were found to be increased after only 1 week of treatment. Furthermore, CT scans revealed signs of bone healing in 2 cases at only 3 months after the initiation of therapy. It is interesting to note that it was also found that the increase in PINP was inhibited by the addition of glucocorticoids. The investigators suggest that the observation of an inhibitory effect of glucocorticoids on bortezomib-induced bone formation may have implications for treatment strategies involving the combination of bortezomib and glucocorticoids. It may indicate that after initial use in combination, glucocorticoids should be discontinued to maximize the positive effect of bortezomib on bone.
Finally, Delforge et al conducted a post hoc analysis of the phase 3 VISTA trial to examine clinical bone disease events during treatment. In the trial, patients with previously untreated myeloma who were not eligible for transplantation were randomized to receive either bortezomib plus the combination of melphalan and prednisone (VMP) or MP alone. The analysis revealed that treatment with VMP was associated with lower rates of bisphosphonate use during treatment, a lower rate of disease progression due to worsening bone disease, and a lower requirement for subsequent radiotherapy compared with MP. Importantly, analysis of prebaseline and postbaseline radiologic data, which were available at the data cutoff for 11 patients receiving VMP, revealed evidence of bone healing in 6 of these patients, with the following myeloma responses: 3 CRs, 1 PR, and 2 cases of stable disease. For the 3 patients achieving a CR, an analysis of CT or x-ray images demonstrated the improvement or resolution of lesions. In contrast, no evidence of bone healing was observed on the MP arm, even in those patients with similar responses to treatment. Another important finding is the reduction in DKK-1 from baseline to day 4 with VMP treatment, whereas an increase in DKK-1 levels was observed when MP therapy was administered. The rapid effect of a single dose of bortezomib on DKK-1 levels suggests that an early reduction of DKK-1 may be an important first step in the subsequent changes in bone remodeling brought about by bortezomib. The investigators concluded that despite the post hoc nature of the analysis, it provides important data regarding the effect of bortezomib on bone disease in a large patient group.
Conclusions and Future Directions
Taken together, the findings discussed above suggest that bortezomib may combine potent antimyeloma activity with significant effects on bone. Studies are currently ongoing to further examine the effect of bortezomib on bone metabolism and how the beneficial effect may be used in the clinic. Thus, two phase 2 studies evaluated the effect of bortezomib on BMD using dual-energy x-ray absorptiometry (NCT01286077 and NCT00972959; www.clinicaltrial.gov), whereas another study used ALP to evaluate osteoblast activation induced by bortezomib (NCT01062230; www.clinicaltrial.gov). The expected results will most likely influence current treatment guidelines. Indeed, open questions remain, such as whether the agent should be used as monotherapy or in combination, whether the beneficial effect is similar in both the frontline and disease recurrence settings, and how the administration of concomitant bisphosphonates should be managed. In addition, it is of interest to investigate whether the positive effects on bone may also exist in other malignancies.