Treatment with a proteasome inhibitor, bortezomib, for thalidomide-resistant multiple myeloma

A proteasome inhibitor with a new molecular target (PS-341: bortezomib) was recently developed, and its efficacy in the treatment of refractory multiple myeloma has been reported in the United States. Here, we present a 54-year-old Japanese male patient with refractory multiple myeloma resistant to thalidomide. In 1998, the patient was diagnosed as having multiple myeloma (IgG-kappa) and underwent chemotherapy, autologous hematopoietic cell transplantation and interferon therapy, but the disease recurred. In December 2002, thalidomide and high-dose dexamethasone therapy was initiated, and while this combination therapy was effective at first, the multiple myeloma became unresponsive. On 23 June 2003, bortezomib therapy with the following regime was therefore started: 2.2 mg (1.3 mg/m2) of bortezomib was injected intravenously on days 1, 4, 8 and 11, and after a one-week break, another cycle was performed. Starting on day 8 of the administration, the serum total protein, IgG, serum calcium and LDH levels decreased rapidly, and after day 45 of the administration, blood transfusion was no longer needed. Since this is the first report of the use of bortezomib in the treatment of refractory multiple myeloma in Japan, further monitoring of this patient will provide extremely valuable information for developing a therapy against this disease.     

NPI-0052, a novel proteasome inhibitor, induces caspase-8 and ROS-dependent apoptosis alone and in combination with HDAC inhibitors in leukemia cells

The proteasome has been successfully targeted for the treatment of multiple myeloma and mantle cell lymphoma; however, in other hematologic malignancies, bortezomib has been less effective as a single agent. Here, we describe effects of NPI-0052, a novel proteasome inhibitor, in leukemia model systems. In cell lines, NPI-0052 inhibits all 3 proteolytic activities associated with the proteasome: chymotrypsin-, trypsin-, and caspase-like. NPI-0052 also induces DNA fragmentation in leukemia lines and in mononuclear cells from a Ph + acute lymphoblastic leukemia (ALL) patient. Caspase-3 activation by NPI-0052 was seen in wild-type Jurkat cells, but was significantly lessened in Fas-associated death domain (FADD)-deficient or caspase-8-deficient counterparts. NPI-0052-induced apoptosis was further probed using caspase-8 inhibitors, which were more protective than caspase-9 inhibitors. N-acetyl cysteine (NAC) also conferred protection against NPI-0052-induced apoptosis, indicating a role for oxidative stress by NPI-0052. In support of the drug's in vitro activities, biweekly treatment with NPI-0052 lessened total white blood cell (WBC) burden over 35 days in leukemic mice. Interestingly, combining NPI-0052 with either MS-275 or valproic acid (VPA) induced greater levels of cell death than the combination of bortezomib with these histone deacetylase inhibitors (HDACi). These effects of NPI-0052, alone and in combination with HDACi, warrant further testing to determine the compound's clinical efficacy in leukemia.     

Combining prenylation inhibitors causes synergistic cytotoxicity, apoptosis and disruption of RAS-to-MAP kinase signalling in multiple myeloma cells

The high incidence of activating RAS mutations, coupled with accumulating evidence linking RAS to multiple myeloma (MM) pathogenesis, indicate that novel therapies utilising inhibitors of RAS prenylation and signalling may be successful in the management of this disease. While preclinical studies investigating prenylation inhibitors, such as lovastatin, farnesyltransferase inhibitors (FTI) and geranylgeranyltransferase inhibitors (GGTI), have been promising, recent phase I/II clinical trials with FTI R115777 were disappointing, suggesting resistance to FTI monotherapy. To address this issue, the effects of FTI, GGTI and lovastatin alone and in combination were analysed in MM cell lines and primary cells. FTI treatment blocked H-RAS processing, but was ineffective at inhibiting K- and N-RAS prenylation because of alternative geranylgeranylation of these isoforms. However, combinations of FTI and GGTI or lovastatin were found to synergistically inhibit MM cell proliferation, migration, K- and N-RAS processing, RAS-to-mitogen-activated protein kinase signalling and to induce apoptosis. In contrast to FTI, lovastatin and some GGTI were found to cause intracellular accumulation of Rho proteins. Our results suggest that clinical efficacy of prenylation inhibitors in MM are limited by alternative prenylation of several small G-proteins, such as RhoB, K- and N-RAS. Furthermore, strategies combining FTI with GGTI or statins may provide greater efficacy in MM treatment.     

Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma

We have shown that the proteasome inhibitor bortezomib (formerly known as PS-341) triggers significant antitumor activity in multiple myeloma (MM) in both preclinical models and patients with relapsed refractory disease. Recent studies have shown that unfolded and misfolded ubiquitinated proteins are degraded not only by proteasomes, but also by aggresomes, dependent on histone deacetylase 6 (HDAC6) activity. We therefore hypothesized that inhibition of both mechanisms of protein catabolism could induce accumulation of ubiquitinated proteins followed by significant cell stress and cytotoxicity in MM cells. To prove this hypothesis, we used bortezomib and tubacin to inhibit the proteasome and HDAC6, respectively. Tubacin specifically triggers acetylation of alpha-tubulin as a result of HDAC6 inhibition in a dose- and time-dependent fashion. It induces cytotoxicity in MM cells at 72 h with an IC50 of 5-20 microM, which is mediated by caspase-dependent apoptosis; no toxicity is observed in normal peripheral blood mononuclear cells. Tubacin inhibits the interaction of HDAC6 with dynein and induces marked accumulation of ubiquitinated proteins. It synergistically augments bortezomib-induced cytotoxicity by c-Jun NH2-terminal kinase/caspase activation. Importantly, this combination also induces significant cytotoxicity in plasma cells isolated from MM patient bone marrow. Finally, adherence of MM cells to bone marrow stromal cells confers growth and resistance to conventional treatments; in contrast, the combination of tubacin and bortezomib triggers toxicity even in adherent MM cells. Our studies therefore demonstrate that tubacin combined with bortezomib mediates significant anti-MM activity, providing the framework for clinical evaluation of combined therapy to improve patient outcome in MM.     

The bortezomib/proteasome inhibitor PS-341 and triterpenoid CDDO-Im induce synergistic anti-multiple myeloma (MM) activity and overcome bortezomib resistance

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) induces apoptosis in leukemic cells. Here we show that CDDO and its new derivative CDDO-imidazolide (CDDO-Im) trigger apoptosis in multiple myeloma (MM) cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40), and dexamethasone (MM.1R, U266, RPMI 8226) without affecting the viability of normal cells. CDDO-IM also triggers apoptosis in bone marrow stromal cells (BMSCs) and decreases interleukin-6 (IL-6) secretion induced by MM cell adhesion to BMSCs. Moreover, CDDO-Im-induced apoptosis in MM cells is not blocked by IL-6 or insulin growth factor-1 (IGF-1). Importantly, CDDO-Im and bortezomib/proteasome inhibitor PS-341 trigger synergistic apoptosis in MM cells associated with loss of mitochondrial membrane potential, superoxide generation, release of mitochondrial proteins cytochrome c/second mitochondria-derived activator of caspases (cytochrome c/Smac), and activation of caspase-8, -9, and -3. Conversely, the pancaspase inhibitor Z-VAD-fmk abrogates the CDDO-Im + bortezomib-induced apoptosis. Low doses of CDDO-Im and bortezomib overcome the cytoprotective effects of antiapoptotic proteins Bcl2 and heat shock protein-27 (Hsp27) as well as nuclear factor-kappa B (NF-kappaB)-mediated growth/survival and drug resistance. Finally, combining CDDO-Im and bortezomib induces apoptosis even in bortezomib-resistant MM patient cells. Together, these findings provide the framework for clinical evaluation of CDDO-Im, either alone or in combination with bortezomib, to overcome drug resistance and improve patient outcome in MM.     

The proteasome inhibitor, bortezomib suppresses primary myeloma and stimulates bone formation in myelomatous and nonmyelomatous bones in vivo

Multiple myeloma (MM), a hematologic malignancy of terminally differentiated plasma cells is closely associated with induction of osteolytic bone disease, induced by stimulation of osteoclastogenesis and suppression of osteoblastogenesis. The ubiquitin-proteasome pathway regulates differentiation of bone cells and MM cell growth. The proteasome inhibitor, bortezomib, is a clinical potent antimyeloma agent. The main goal of this study was to investigate the effect of bortezomib on myeloma-induced bone resorption and tumor growth in SCID-rab mice engrafted with MM cells from 16 patients. Antimyeloma response of bortezomib, which was evident in >50% of 16 experiments and resembled clinical response, was associated with significant increased bone mineral density (BMD) and osteoblast numbers, and reduced osteoclast numbers in myelomatous bones. This bone anabolic effect, which was also visualized on X-ray radiographs and confirmed by static and dynamic histomorphometric analyses, was unique to bortezomib and was not observed in hosts responding to melphalan, a chemotherapeutic drug widely used to treat MM. Bortezomib also increased BMD and osteoblasts number and reduced osteoclasts number in nonmyelomatous implanted bones. In vitro bortezomib directly suppressed human osteoclast formation and promoted maturation of osteoblasts. We conclude that bortezomib promotes bone formation in myelomatous and nonmyelomatous bones by simultaneously inhibiting osteoclastogenesis and stimulating osteoblastogenesis. As clinical and experimental studies indicate that bone disease is both a consequence and necessity of MM progression our results suggest and that bortezomib's effects on bone remodeling contribute to the antimyeloma efficacy of this drug.     

Combination of the mTOR inhibitor rapamycin and CC-5013 has synergistic activity in multiple myeloma

Previous studies have demonstrated the in vitro and in vivo activity of CC-5013 (Revlimid), an immunomodulatory analog (IMiD) of thalidomide, in multiple myeloma (MM). In the present study, we have examined the anti-MM activity of rapamycin (Rapamune), a specific mTOR inhibitor, combined with CC-5013. Based on the Chou-Talalay method, combination indices of less than 1 were obtained for all dose ranges of CC-5013 when combined with rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or adherence to bone marrow stromal cells (BMSCs). Combining rapamycin and CC-5013 induced apoptosis of MM cells. Differential signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase/Akt kinase (PI3K/Akt) pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies, therefore, provide the framework for clinical evaluation of mTOR inhibitors combined with IMiDs to improve patient outcome in MM.     

多发性骨髓瘤细胞遗传学变化及其蛋白酶体抑制剂的疗效观察

目的分析多发性骨髓瘤(MM)的细胞遗传学变化及其与蛋白酶体抑制剂治疗的关系。方法对2005年1月至2006年7月北京大学人民医院29例初诊的MM患者行染色体核型检查,采用骨髓细胞24h短期培养,用常规方法制备染色体,G显带进行核型分析。22例MM患者采用传统化疗即长春新碱联合阿霉素和地塞米松(VAD)方案或马法兰与泼尼松(MP)方案;7例患者应用蛋白酶体抑制剂(硼替佐米)为主的化疗方案。比较两组患者的有效率及缓解率。结果29例MM患者中异常核型检出率为37.9%,其中有复杂和高度复杂畸变的占81.8%。采用VAD或MP方案化疗的核型正常组的有效率为81.2%,核型异常组有效率为0,差异有显著性意义(P<0.05)。应用硼替佐米为主的化疗方案中核型异常组5例均有效,核型正常组2例均有效。核型异常组硼替佐米有效率与传统化疗组相比,差异有显著性意义(P<0.05)。结论染色体核型异常的患者,应首选蛋白酶体抑制剂硼替佐米等进行治疗。     

Aggresome induction by proteasome inhibitor bortezomib and alpha-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells

Histone deacetylase (HDAC) inhibitors have shown cytotoxicity as single agents in preclinical studies for multiple myeloma (MM) cells. LBH589 is a novel hydroxamic acid derivative that at low nanomolar concentrations induces apoptosis in MM cells resistant to conventional therapies via caspase activation and poly-(ADP-ribose) polymerase (PARP) cleavage. Significant synergistic cytotoxicity was observed with LBH589 in combination with bortezomib against MM cells that were sensitive and resistant to dexamethasone (Dex), as well as primary patient MM cells. LBH589 at low nanomolar concentrations also induced alpha-tubulin hyperacetylation. Aggresome formation was observed in the presence of bortezomib, and the combination of LBH589 plus bortezomib induced the formation of abnormal bundles of hyeracetylated alpha-tubulin but with diminished aggresome size and apoptotic nuclei. These data confirm the potential clinical benefit of combining HDAC inhibitors with proteasome inhibitors, and provide insight into the mechanisms of synergistic anti-MM activity of bortezomib in combination with LBH589.     

Targeting mitochondria to overcome conventional and bortezomib/proteasome inhibitor PS-341 resistance in multiple myeloma (MM) cells

Bortezomib (PS-341), a selective inhibitor of proteasomes, induces apoptosis in multiple myeloma (MM) cells; however, prolonged drug exposure may result in cumulative toxicity and the development of chemoresistance. Here we show that combining PK-11195 (PK), an antagonist to mitochondrial peripheral benzodiazepine receptors (PBRs), with bortezomib triggers synergistic anti-MM activity even in doxorubicin-, melphalan-, thalidomide-, dexamethasone-, and bortezomib-resistant MM cells. No significant cytotoxicity was noted in normal lymphocytes. Low-dose combined PK and bortezomib treatment overcomes the growth, survival, and drug resistance conferred by interleukin-6 or insulin growth factor within the MM bone marrow milieu. The mechanism of PK + bortezomib-induced apoptosis includes: loss of mitochondrial membrane potential; superoxide generation; release of mitochondrial proteins cytochrome-c (cyto-c) and Smac; and activation of caspases-8/-9/-3. Furthermore, PK + bortezomib activates c-Jun NH2 terminal kinase (JNK), which translocates to mitochondria, thereby facilitating release of cyto-c and Smac from mitochondria to cytosol. Blocking JNK, by either dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor SP600125, abrogates both PK + bortezomib-induced release of cyto-c/Smac and induction of apoptosis. Together, these preclinical studies suggest that combining bortezomib with PK may enhance its clinical efficacy, reduce attendant toxicity, and overcome conventional and bortezomib resistance in patients with relapsed refractory MM.     

Community experience with bortezomib in patients with multiple myeloma

Community practice experience allows a nonselective care of patient using information derived from a more controlled clinical trial environment. We present our community experience with multiple myeloma patients with advanced age, long disease duration since diagnosis, advanced stage, multiple prior therapies including stem cell transplantation, co-morbidities, and other poor prognostic features, such as low albumin, high B-2 microglobulin, renal failure, and the presence of poor risk chromosomal abnormalities. Our response rates are comparable to those from clinical trials. Bortezomib is well tolerated in this population of multiple myeloma patients with the exception of infection adverse events that are generally mild grade 1-2.     

In vivo and in vitro cytotoxicity of R-etodolac with dexamethasone in glucocorticoid-resistant multiple myeloma cells

Glucocorticoids have been widely used in the treatment of multiple myeloma (MM) both as single agents and in combination with other drugs. However, primary or acquired glucocorticoid resistance occurs in most cases. It was recently reported that R-etodolac induced in vitro cytotoxicity in MM cell lines and in primary MM cells, as well as synergistically enhanced dexamethasone (Dex)-induced apoptosis in Dex-sensitive MM.1S cells. This study examined the in vitro and in vivo effects of combination treatment with R-etodolac and Dex on Dex-resistant OPM1 cells. Treatment with R-etodolac and Dex was found to enhance cytotoxicity, inhibit nuclear factor kappaB activity via upregulation of IkappaBalpha, as well as enhance Dex-induced caspase activation and poly (ADP)-ribose polymerase cleavage in OPM1 cells. R-etodolac also enhanced Dex cytotoxicity in patient MM cells that were resistant to glucocorticoids. The in vivo anti-tumour effect of this combination on MM cells was evaluated by using severe combined immunodeficient mice engrafted with OPM1. Treatment with R-etodolac or Dex alone did not induce a significant reduction of tumour volume; in contrast, combination treatment with R-etodolac and Dex induced significant synergistic inhibition of tumour growth. These data indicate that R-etodolac overcomes resistance to Dex in glucocorticoid-resistant MM cells, providing the framework for clinical trials of R-etodolac combined with Dex, to improve patient outcome in MM.     

Retrospective matched-pairs analysis of bortezomib plus dexamethasone versus bortezomib monotherapy in relapsed multiple myeloma

Bortezomib-dexamethasone is widely used for relapsed myeloma in routine clinical practice, but comparative data versus single-agent bortezomib are lacking. This retrospective analysis compared second-line treatment with bortezomib-dexamethasone and bortezomib using 109 propensity score-matched pairs of patients treated in three clinical trials: MMY-2045, APEX, and DOXIL-MMY-3001. Propensity scores were estimated using logistic regression analyses incorporating 13 clinical variables related to drug exposure or clinical outcome. Patients received intravenous bortezomib 1.3 mg/m² on days 1, 4, 8, and 11, in 21-day cycles, alone or with oral dexamethasone 20 mg on the days of/after bortezomib dosing. Median bortezomib cumulative dose (27.02 and 28.60 mg/m²) and treatment duration (19.6 and 17.6 weeks) were similar with bortezomib-dexamethasone and bortezomib, respectively. The overall response rate was higher (75% vs. 41%; odds ratio=3.467; P<0.001), and median time-to-progression (13.6 vs. 7.0 months; hazard ratio [HR]=0.394; P=0.003) and progression-free survival (11.9 vs. 6.4 months; HR=0.595; P=0.051) were longer with bortezomib-dexamethasone versus bortezomib, respectively. Rates of any-grade adverse events, most common grade 3 or higher adverse events, and discontinuations due to adverse events appeared similar between the groups. Two patients per group died of treatment-related adverse events. These data indicate the potential benefit of bortezomib-dexamethasone compared with single-agent bortezomib at first relapse in myeloma. The MMY-2045, APEX, and DOXIL-MMY-3001 clinical trials were registered at, respectively, clinicaltrials.gov identifier: 00908232, 00048230, and 00103506.     

Acute severe cardiac failure in a myeloma patient due to proteasome inhibitor bortezomib

We present here a case of severe congestive cardiac failure, in a 47-year-old patient with myeloma who had no prior cardiac history, after receiving bortezomib. Bortezomib is a boron-containing molecule, which reversibly inhibits the proteasome, an intracellular organelle, which is central to the breakdown of ubiquitinated proteins and consequently crucial for normal cellular homeostasis. Phase II clinical trials demonstrate that it is effective for the treatment of relapsed refractory myeloma. Acute development of congestive cardiac failure associated with bortezomib therapy occurs very rarely or may be underestimated. Inhibition of proteasome activity may impair cardiac function due to accumulation of unfolded, damaged and undegraded proteins in myocytes. Patients with or without cardiac disease or previously received anthracycline-containing regimes should be closely monitored when being subjected to treatment with bortezomib.     

硼替佐米联合地塞米松方案治疗多发性骨髓瘤的临床疗效

目的探讨硼替佐米联合地塞米松方案治疗多发性骨髓瘤(MM)的临床疗效。方法选取该院诊治的51例MM患者作为研究对象,按治疗方案分为两组,观察组31例采用硼替佐米联合地塞米松治疗,对照组20例接受长春新碱、吡柔比星、地塞米松和左旋苯丙氨酸氮芥(马法兰)治疗。比较两组患者临床疗效、不良反应等情况。结果观察组总有效率为80.65%(25/31),对照组总有效率65.00%(13/20)差异显著(P<0.05);观察组对初治病例有效率为86.36%(19/22),难治复发病例有效率为66.67%(6/9);对肾功能正常病例有效率为90.00%(18/20),对肾功能损害病例的有效率为63.64%(7/11)差异显著(P<0.05);观察组总不良反应发生率为51.61%,对照组为65.00%,差异显著(P<0.05)。结论硼替佐米联合地塞米松治疗MM有效率高,不良反应轻,对初治患者疗效最显著,值得推广应用。