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.     

2-Methoxyestradiol overcomes drug resistance in multiple myeloma cells

2-Methoxyestradiol (2ME2) an estrogen derivative, induces growth arrest and apoptosis in leukemic cells and is also antiangiogenic. In this study, we demonstrate that 2ME2 inhibits growth and induces apoptosis in multiple myeloma (MM) cell lines and patient cells. Significantly, 2ME2 also inhibits growth and induces apoptosis in MM cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40 and Dox-6), and dexamethasone (MM.1R). In contrast to its effects on MM cells, 2ME2 does not reduce the survival of normal peripheral blood lymphocytes. Moreover, 2ME2 enhances Dex-induced apoptosis, and its effect is not blocked by interleukin-6 (IL-6). We next examined the effect of 2ME2 on MM cells in the bone marrow (BM) milieu. 2ME2 decreases survival of BM stromal cells (BMSCs), as well as secretion of vascular endothelial growth factor (VEGF), and IL-6 triggered by the adhesion of MM cells to BMSCs. We show that apoptosis induced by 2ME2 is mediated by the release of mitochondrial cytochrome-c (cyto-c) and Smac, followed by the activation of caspases-8, -9, and -3. Finally, 2ME2 inhibits MM cell growth, prolongs survival, and decreases angiogenesis in a murine model. These studies, therefore, demonstrate that 2ME2 mediates anti-MM activity directly on MM cells and in the BM microenvironment. They provide a framework for the use of 2ME2, either alone or in combination with Dex, to overcome drug resistance and to improve outcome in MM.     

Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Heat shock protein 27 (Hsp27) negatively regulates another mitochondrial protein, cytochrome c, during apoptosis; however, the role of Hsp27 in modulating Smac release is unknown. Here we show that Hsp27 is overexpressed in both dexamethasone (Dex)-resistant multiple myeloma (MM) cell lines (MM.1R, U266, RPMI-8226) and primary patient cells. Blocking Hsp27 by an antisense (AS) strategy restores the apoptotic response to Dex in Dex-resistant MM cells by triggering the release of mitochondrial protein Smac, followed by activation of caspase-9 and caspase-3. Moreover, AS-Hsp27 overcomes interleukin-6 (IL-6)-mediated protection against Dex-induced apoptosis. These data demonstrate that Hsp27 inhibits the release of Smac, and thereby confers Dex resistance in MM cells.     

Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia

Caspase-9 is critical for cytochrome c (cyto-c)-dependent apoptosis and normal brain development. We determined that this apical protease in the cyto-c pathway for apoptosis resides inside mitochondria in several types of cells, including cardiomyocytes and many neurons. Caspase-9 is released from isolated mitochondria on treatment with Ca2+ or Bax, stimuli implicated in ischemic neuronal cell death that are known to induce cyto-c release from mitochondria. In neuronal cell culture models, apoptosis-inducing agents trigger translocation of caspase-9 from mitochondria to the nucleus, which is inhibitable by Bcl-2. Similarly, in an animal model of transient global cerebral ischemia, caspase-9 release from mitochondria and accumulation in nuclei was observed in hippocampal and other vulnerable neurons exhibiting early postischemic changes preceding apoptosis. Loss of mitochondrial barrier function during neuronal damage from ischemia or other insults therefore may play an important role in making certain caspases available to participate in apoptosis.     

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.     

Inhibition of heat shock protein 90 (HSP90) as a therapeutic strategy for the treatment of myeloma and other cancers

Heat shock protein 90 (HSP90) is a molecular chaperone that is induced in response to cellular stress and stabilizes client proteins involved in cell cycle control and proliferative/anti-apoptotic signalling. HSP90 is overexpressed in a range of cancers, and may contribute to tumour cell survival by stabilizing aberrant signalling proteins and by interfering with apoptosis. Tanespimycin, an HSP90 inhibitor, reduces tumour cell survival in vitro. In multiple myeloma (MM), HSP90 inhibition affects multiple client proteins that contribute to tumour cell survival, including the IGF1 receptor and the IL-6 receptor, and elements of the PI3/Akt, STAT3, and MAPK signalling pathways. HSP90 inhibition also abrogates the protective effect of bone marrow stromal cells and inhibits angiogenesis and osteoclastogenesis. Tanespimycin acts synergistically with the proteasome inhibitor bortezomib in MM cells and tumour explants, possibly reducing their ability to resist bortezomib-induced stress to the endoplasmic reticulum. The combination of tanespimycin and bortezomib has demonstrated significant and durable responses with acceptable toxicity in a phase I/II study in patients with relapsed and relapsed/refractory MM. HSP90 inhibition is a promising strategy in MM especially in combination with bortezomib; additional studies will further evaluate optimal dosings of candidate drugs and schedules, as well as confirm efficacy in comparative phase III trials.     

Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma

The proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose- and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM.     

Synergistic anti‐myeloma activity of the proteasome inhibitor marizomib and the IMiD® immunomodulatory drug pomalidomide

The proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that the novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action and effects on proteasomal activities, and that it can overcome bortezomib resistance. Pomalidomide, like lenalidomide, has potent immunomodulatory activity and has been approved by the US Food and Drug Administration for the treatment of RRMM. Here, we demonstrate that combining low concentrations of marizomib with pomalidomide induces synergistic anti‐MM activity. Marizomib plus pomalidomide‐induced apoptosis is associated with: (i) activation of caspase‐8, caspase‐9, caspase‐3 and PARP cleavage, (ii) downregulation of cereblon (CRBN), IRF4, MYC and MCL1, and (iii) suppression of chymotrypsin‐like, caspase‐like, and trypsin‐like proteasome activities. CRBN‐siRNA attenuates marizomib plus pomalidomide‐induced MM cells death. Furthermore, marizomib plus pomalidomide inhibits the migration of MM cells and tumour‐associated angiogenesis, as well as overcomes cytoprotective effects of bone marrow microenvironment. In human MM xenograft model studies, the combination of marizomib and pomalidomide is well tolerated, inhibits tumour growth and prolongs survival. These preclinical studies provide the rationale for on‐going clinical trials of combined marizomib and pomalidomide to improve outcome in patients with RRMM.     

Combined treatment with the checkpoint abrogator UCN-01 and MEK1/2 inhibitors potently induces apoptosis in drug-sensitive and -resistant myeloma cells through an IL-6-independent mechanism

The effects of combined exposure to the checkpoint abrogator UCN-01 and pharmacologic MEK1/2 inhibitors were examined in human multiple myeloma (MM) cell lines. Treatment of RPMI8226, NCI-H929, and U266 MM cells with a minimally toxic concentration of UCN-01 (150 nM) for 24 hours resulted in mitogen-activated protein (MAP) kinase activation, an effect that was blocked by coadministration of the MEK1/2 inhibitor PD184352. These events were accompanied by enhanced activation of p34(cdc2) and a marked increase in mitochondrial damage (loss of DeltaPsim; cytochrome c and Smac/DIABLO (direct IAP binding protein with low pI) release), poly(ADP-ribose) polymerase (PARP) cleavage, and apoptosis. PD184352/UCN-01 also dramatically reduced clonogenic survival in each of the MM cell lines. In contrast to As(2)0(3), apoptosis induced by PD184352/UCN-01 was not blocked by the free-radical scavenger N-acetyl-L-cysteine. Whereas exogenous interleukin 6 substantially prevented dexamethasone-induced lethality in MM cells, it was unable to protect them from PD184352/UCN-01-induced apoptosis despite enhancing Akt activation. Insulinlike growth factor 1 (IGF-1) also failed to diminish apoptosis induced by this drug regimen. MM cell lines selected for a high degree of resistance to doxorubicin, melphalan, or dexamethasone, or displaying resistance secondary to fibronectin-mediated adherence, remained fully sensitive to PD184352/UCN-01-induced cell death. Finally, primary CD138(+) MM cells were also susceptible to UCN-01/MEK inhibitor-mediated apoptosis. Together, these findings suggest that simultaneous disruption of cell cycle and MEK/MAP kinase signaling pathways provides a potent stimulus for mitochondrial damage and apoptosis in MM cells, and also indicate that this strategy bypasses the block to cell death conferred by several other well-described resistance mechanisms.     

Bortezomib-induced rhabdomyolysis in multiple myeloma

Although multiple myeloma (MM) remains an incurable disease, its treatment has improved over the past decade. This improvement has been at least in part due to the introduction of novel antimyeloma agents with new mechanisms of action, including those that target both myeloma cells and the tumor microenvironment, with antiangiogenic and immunomodulatory properties. Among these drugs, bortezomib (Velcade), a selective proteasome inhibitor, has been approved for the treatment of relapsed and refractory MM patients after one line of therapy. The toxicity profile of bortezomib includes gastrointestinal symptoms, fatigue, thrombocytopenia, peripheral neuropathy, postural hypotension, as well as some uncommon events. A patient with relapsed MM who developed recurrent bortezomib-induced rhabdomyolysis is reported. To our knowledge, this adverse event has not been previously described is this context.     

General aspects and mechanisms of peripheral neuropathy associated with bortezomib in patients with newly diagnosed multiple myeloma

Introduction of the proteasome inhibitor bortezomib (Velcade, Millennium Pharmaceuticals, The Takeda Oncology Company, Cambridge, MA) has substantially improved outcomes for patients with multiple myeloma (MM), and has become one of the cornerstones of current anti-myeloma treatment regimens. However, with the introduction of bortezomib it has become clear that peripheral neuropathy (PN) is one of the most frequent, potentially disabling, nonhematologic complications of bortezomib, often requiring dose modification or discontinuation, with a potential negative impact on clinical endpoints and quality of life. To find a balance between maximal benefit of bortezomib treatment, while maintaining quality of life, it is necessary to minimize toxicity. Here, we discuss all aspects of bortezomib-induced peripheral neuropathy (BiPN), and elaborate on the mechanisms underlying the development of BiPN.     

Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138(+) MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib-induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-kappaB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.     

Targeting mitochondrial factor Smac/DIABLO as therapy for multiple myeloma (MM)

Second mitochondria-derived activator of caspases (Smac) promotes apoptosis via activation of caspases. Here we show that a low-molecular-weight Smac mimetic LBW242 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. Examination of purified patient MM cells demonstrated similar results, without significant cytotoxicity against normal lymphocytes and bone marrow stromal cells (BMSCs). Importantly, LBW242 abrogates paracrine MM cell growth triggered by their adherence to BMSCs and overcomes MM cell growth and drug-resistance conferred by interleukin-6 or insulinlike growth factor-1. Overexpression of Bcl-2 similarly does not affect LBW242-induced cytotoxicity. Mechanistic studies show that LBW242-induced apoptosis in MM cells is associated with activation of caspase-8, caspase-9, and caspase-3, followed by PARP cleavage. In human MM xenograft mouse models, LBW242 is well tolerated, inhibits tumor growth, and prolongs survival. Importantly, combining LBW242 with novel agents, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the proteasome inhibitors bortezomib and NPI-0052, as well as with the conventional anti-MM agent melphalan, induces additive/synergistic anti-MM activity. Our study therefore provides the rationale for clinical protocols evaluating LBW242, alone and together with other anti-MM agents, to improve patient outcome in MM.     

PI3KCA plays a major role in multiple myeloma and its inhibition with BYL719 decreases proliferation,synergizes with other therapies and overcomes stroma‐induced resistance

The phosphatidylinositide 3‐kinase (PI3K) pathway is activated and correlated with drug resistance in multiple myeloma (MM). In the present study we investigated the role of PI3KCA (PI3K‐α) in the progression and drug resistance in MM. We showed that the gene expression of PI3KCA isoform was higher in MM compared to normal subjects. BYL719, a novel and specific PI3KCA inhibitor inhibited the survival of primary MM cells and cell lines but not normal peripheral blood mononuclear cells. BYL719 induced the apoptosis of MM cells and inhibited their cell cycle by causing G1 arrest. BYL719 inhibited PI3K signalling, decreased proliferation and cells cycle signalling, and induced apoptosis signalling in MM cells. Finally, BYL719 synergized with bortezomib and carfilzomib, and overcame drug resistance induced by bone marrow stroma. These results were confirmed using in silico simulation of MM cell lines, BYL719 and bortezomib, and showed similar trends in survival, proliferation, apoptosis, cell signalling and synergy with drugs. In conclusion, PI3KCA plays a major role in proliferation and drug resistance of MM cells, the effects of which were inhibited with BYL719. These results provide a preclinical basis for a future clinical trial of BYL719 in MM as a single agent or in combination with other drugs.     

Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria.

Sphingosine 1-phosphate (S-1P) has been implicated as a second messenger preventing apoptosis by counteracting activation of executioner caspases. Here it is reported that S-1P prevents apoptosis and executioner caspase-3 activation by inhibiting the translocation of cytochrome c and Smac/DIABLO from mitochondria to the cytosol induced by anti-Fas, tumor necrosis factor-alpha (TNF-alpha), serum deprivation, and cell-permeable ceramides in the human acute leukemia Jurkat, U937, and HL-60 cell lines. Furthermore, the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, which stimulates sphingosine kinase, the enzyme responsible for S-1P production, also inhibits cytochrome c and Smac/DIABLO release. In contrast, dimethylsphingosine (DMS), a specific inhibitor of sphingosine kinase, sensitizes cells to cytochrome c and Smac/DIABLO release triggered by anti-Fas, TNF-alpha, serum deprivation, or ceramide. DMS-induced mitochondrial apoptogenic factor leakage can likewise be overcome by S-1P cotreatment. Hence, S-1P, likely generated through a protein kinase C- mediated activation of sphingosine kinase, inhibits the apoptotic cascade upstream of the release of the mitochondrial apoptogenic factors, cytochrome c, and Smac/DIABLO in human acute leukemia cells.     

Honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis

Honokiol (HNK) is an active component purified from magnolia, a plant used in traditional Chinese and Japanese medicine. Here we show that HNK significantly induces cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM. Neither coculture with bone marrow stromal cells nor cytokines (interleukin-6 and insulin-like growth factor-1) protect against HNK-induced cytotoxicity. Although activation of caspases 3, 7, 8, and 9 is triggered by HNK, the pan-caspase inhibitor z-VAD-fmk does not abrogate HNK-induced apoptosis. Importantly, release of an executioner of caspase-independent apoptosis, apoptosis-inducing factor (AIF), from mitochondria is induced by HNK treatment. HNK induces apoptosis in the SU-DHL4 cell line, which has low levels of caspase 3 and 8 associated with resistance to both conventional and novel drugs. These results suggest that HNK induces apoptosis via both caspase-dependent and -independent pathways. Furthermore, HNK enhances MM cell cytotoxicity and apoptosis induced by bortezomib. In addition to its direct cytotoxicity to MM cells, HNK also represses tube formation by endothelial cells, suggesting that HNK inhibits neovascurization in the bone marrow microenvironment. Taken together, our results provide the preclinical rationale for clinical protocols of HNK to improve patient outcome in MM.