Targeting the Ubiquitin-proteasome System for the Treatment of Multiple Myeloma and Other Human Diseases

Abstract

The ubiquitin-proteasome-degradation system plays a key role in multiple cellular functions. Its deregulation is associated with the initiation and progression of human diseases including not only solid and hematologic malignancies but also neurologic and autoimmune disorders. This article discusses several novel mechanistic aspects of the ubiquitin-proteasome system. Moreover, it focuses on the development, mechanisms of action, and clinical experience with Bortezomib, the first in-class-proteasome inhibitor to enter the clinics. Finally, it summarizes novel approaches to specifically target distinct components within the highly complex and dynamic ubiquitin-proteasome machinery to ultimately further increase drug activity, as well as reduce drug resistance and adverse side effects.

Introduction

Newly synthesized proteins are not static but undergo a finely tuned process of synthesis and destruction. Utilizing isotopic tagging of amino acids, it was Rudolf Schoenheimer who first found that proteins were in a state of continuous turnover and thereby prepared the foundation for the era of biosynthesis in the latter half of the 20th century which shaped the development of modern chemistry.^1,2 Lysosomes, acidic- and protease-filled membrane-bound organelles with vacuolar structures responsible for protein destruction, were discovered by Christian Rene de Duve, for which he was awarded the Nobel Prize for Physiology or Medicine in 1974.^3,4 However, it soon became apparent that lysosomes predominantly degrade extracellular and transmembrane proteins. The existence of an ATP-dependent system that degrades intracellular proteins remained elusive.^5–7 In 1964 Rabinovitz and Fisher reported the inhibition of hemoglobin synthesis in rabbit reticulocytes, which do not contain lysosomes, by threo-α-amino-β-chlorobutyric acid. ⁸ In 1977, Etlinger and Goldberg were the first to identify a soluble non-lysosomal proteolytic system responsible for the energy-dependent degradation of abnormal hemoglobin in reticulocytes. ⁹ The components of this complex were subsequently characterized, purified, and analyzed in greater detail, leading to the description of an elegant molecular mechanism by which an ATP-dependent proteolytic complex (the 26S proteasome complex) degrades intracellular proteins after covalent attachment (conjugation) of one or more ubiquitin (also called ATP-dependent proteolysis factor 1, AFP-1) (ubiquitylation).^10–19 For their pioneering work Ciechanover, Hershko, and Rose received the Nobel prize in Chemistry in 2004.

After summarizing up-to-date knowledge of structure, assembly, and molecular functions of the ubiquitin-proteasome machinery both under physiologic and pathologic conditions, we here present the past and ongoing preclinical and clinical development of proteasome inhibitors, focusing on the first-in-class proteasome inhibitor Bortezomib in particular. Moreover we describe novel therapeutic approaches to specifically target distinct components of this highly complex and dynamic machinery.

Proteasome Structure

The functionally active 26S proteasome (1,500 to 2,000 kD) is an ATP-dependent barrel-like proteolytic complex located in the cytoplasm and nucleus of eukaryotic cells. It consists of a core 20S (also 20S core particle, CP) catalytic complex (~700 kD) and a 19S (also PA700, regulatory particle, RP) regulatory complex (~900 kD). The 20S proteasome is composed of 14 nonidentical subunits comprising four stacked rings of seven subunits each.^20,21 The outer rings each consist of seven alpha subunits and form a “gate” through which proteins enter the barrel under the control of “cap” structures formed by the regulatory particles (see below). The inner rings each consist of seven beta-subunits. In mammals subunits β1, β2, and β5 are catalytic with peptidylglutamyl-like (PG-L), tryptic-like (T-L), and chymotrypticlike (CT-L) activities, respectively, located on the inner surface of the cylinder where protein substrates bind.^22–27

In vertebrates four additional catalytic β-subunits are encoded: three interferon (IFN)γ-inducible sub-units, β1i (low-molecular mass protein (LMP) 2), β2i multicatalytic endopeptidase complex subunit-1 (MECL-1), β5i (LMP7) immunosubunits; and one thymus-specific β5t subunit. These subunits are coordinately incorporated into nascent proteasomes, thereby replacing their constitutive homologues β1, β2, and β5 and forming immunoproteasomes, proteasomes with altered catalytic activities^28–35 and thymoproteasomes.^36,37 In addition to standard/ constitutive proteasomes (c20S) which are constitutively expressed in all cells, immunoproteasomes are present in cells of hematopoietic origin or in cells exposed to inflammatory cytokines. The immunoproteasome has increased CT-L and T-L activities mediating the production of antigenic peptides that bind to the groove of MHC I molecules.^38–40 They play a pivotal role in proteasome-dependent antigen processing, the generation of the naive CD8+ T-cell repertoire, and in shaping cytotoxic T-cell responses.^41–43 The β5i variant subunit (5βt) is exclusively expressed in the thymus, thereby forming a thymus-specific thymoproteasome. The reduced CT-L activity of thymoproteasomes may trigger the production of the unique repertoire in the thymus.^39,44 The functional role of thymo-proteasomes is still elusive.

The 19S subunit consists of at least 19 different subunits forming the base and the lid. The base consists of six different triple-A ATPase subunits; three non-ATPase subunits; regulatory particles non-ATPase1 (RPN1), RPN2, and RPN13; and regulatory particle tiple-A 1 (RPT1) to RPT6. These subunits are required for substrate unfolding and channel opening. The lid consists of nine non-ATPase subunits including RPN3, RPN5–9, RPN11–12 and RPN15 (also SEM1 or DSS1). The lid de-ubiquitinates captured substrates, thereby facilitating their degradation. Taken together, the 19S subunit selects proteasomal substrates through the recognition of polyubiquitin chains, deubiqitinates them, and translocates them in an ATP-dependent manner into the proteolytic 20S proteasome, which efficiently degrades the proteins. While processes involving the assembly of the 20S subunit are well understood, the 19S subunit assembly remains still elusive.^45,46

Proteasome Function: Ubiquitination and Degradation

The highly complex, temporally controlled, and tightly regulated process of proteolysis of cellular proteins plays a major role in controlling a broad array of cellular processes including the cell cycle, apoptosis, response to cellular stress (infection, heat shock, oxidative stress), development, differentiation, regulation of transcription, antigen presentation, signal transduction, receptor-mediated endocytosis, and modulation of diverse metabolic pathways.

The first step in labelling a specific protein for degradation by the 26S proteasome is the ATP-dependent activation of ubiquitin, a highly conserved, ubiquitiously expressed 76aa heat-stable regulatory protein, by the ubiquitin-activating enzyme (E1). Activated ubiquitin is then transferred to one of the various ubiquitinconjugating enzymes (E2). Finally, both E2 and the specific target protein are bound to ubiquitin ligase (E3), which transfers the activated ubiquitin to specific lysine residues in the target protein.^47,48 Finally an additional conjugation factor, named E4 is required for efficient multiubiquitination needed for proteasomal targeting of a model substrate.^49,50 Ubiquitin contains seven acceptor lysines mediating functionally distinct types of ubiquitin conjugation. For example, conjugation through lysine 48 leads to proteasomal degradation whereas conjugation through lysine 63 triggers signalling and trafficking events. ⁵¹ Importantly, deciphering the human genome highlighted the existence of hundreds of distinct E3 enzymes, confirming the complexity of the ubiquitin-proteasome system.

After multiple rounds of this transfer, the protein is “tagged” with a polyubiquitin chain recognized by the 26S proteasome. Ubiquitin-tagged proteins are then recognized by the 19S regulatory complex. Exact mechanisms by which a polyubiquitinated protein is targeted to the proteasome are still elusive and involve ubiquitin-receptor proteins with ubiquitin-associated (UBA) domains, which are recognized by the 19S proteasome caps. ⁵² After removal of the ubiquitin, protein substrates are unfolded in an ATP-dependent way, and thereafter fed into the inner catalytic 20S cylinder.^28,53 Indeed, energy from ATP-hydrolysis is required only for substrate unfolding, but not for complex assembly, gate opening, translocation, and proteolysis.^26,54 Within the inner chamber, the proteins are hydrolyzed into small polypeptides ranging from 3 to 22 aa in length. ⁵⁵ After degradation, ubiquitin is released from the resultant peptides by de-ubiquitinating enzymes and can be reused.

Ubiquitination accounts for the stability, functionality, localization, and interactive capabilities of a variety of proteins including cyclins, IκB, p21, p27, p15, p16, p18, p19, p53, Bax, c-Myc, N-Myc, JNK, and topoisomerase IIα.^25,56 For example, extracellular stimuli (e.g. cytokines) trigger phosphorylation of the signal response domain of IκB via activated IKK (IκB kinase), which leads to IκB ubiquitination by ubiquitin ligases and its degradation by the 26S proteasome, thereby releasing NFκB to activate gene transcription. ⁵⁷

Besides its proteolytic function, ubiquitin also has non-proteolytic functions. For example, the protooncoprotein Hdm2 (a E3-ubiquitin ligase) interacts with Tat and mediates its ubiquitination thereby triggering Tat-mediated transactivation of the HIV-1 promoter. ⁵⁸ Similarly, HectH9 (a E3 ubiquitin ligase)-induced ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. Interestingly, HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. ⁵⁹

In addition to ubiquitination, ubiquitin-like protein (Ubl) modifications including sumoylation (small ubiquitin-related modifier; SUMO), neddylation (NEDD8), isgylation (interferon-stimulated gene 15; ISG15), and fatylation (FAT10) regulate 26S proteasomal protein degradation; ⁶⁰ and are also involved in non-proteolytic functions including sublocalization of proteins and protection of other proteins from ubiquitination. ⁷

Ubiquitin-independent Protein Degradation

Although the degradation of the majority of proteasomal substrates requires prior ubiquitination, some proteins are degraded in a ubiquitin-independent manner. For example, ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. ⁶¹ Moreover, tumor suppressors p53 and p73 are regulated both by an Mdm2/ubiquitin-dependent, as well as by a NAD(P)H quinone oxidoreductase 1 (NQO1)-dependent Mdm2/ubiquitin-independent, 26S proteasomal pathway.^62,63 Surprisingly, a recent report demonstrated that two degradation pathways, an ubiquitin-dependent and an ubiquitin-independent pathway, control the level of IκBα. Specifically, free IκBα degradation is not controlled by IKK or ubiquitination, but rather intrinsically by the C-terminal PEST domain. NFκB binding to IκBα masks the PEST domain from proteasomal recognition, precluding its ubiquitin-independent degradation. Conversely, bound IκBα requires IKK phosphorylation and ubiquitination for slow basal degradation. Indeed, alteration of free IκBα degradation dampens NFκB activation. ⁶⁴

Targeting the Proteasome for the Treatment of Human Diseases

Aberrations in the ubiquitin-proteasome system have been implicated not only in several malignancies, but also in inflammatory, autoimmune, and neurodegenerative disorders. New drugs, e.g. the proteasome inhibitor Bortezomib, play a pivotal role in treatment of cancers including multiple myeloma (MM) and mantle cell lymphoma. However, the mechanism of selective cytotoxicity to cancer cells compared to normal cells remains still elusive. For example, it has been hypothesized that increased susceptibility of cancer cells to proteasome inhibition might be due to: 1) the accumulation of defective proteins at a higher rate than in normal cells, resulting in cellular dysfunction and cell death; 2) a reversal or bypass of mutational effects in cell-cycle and apoptotic checkpoints, which would lead to tumorigenesis; and 3) abnormal regulation of the NFκB pathway. ⁵⁵

Dysregulation of the ubiquitin-proteasome system is also associated with several neural diseases including astrocytoma, Parkinson's disease, as well as cognitive disorders such as autism, muscle wasting diseases, and myocardial infarction.^65–69 Studies to investigate the therapeutic potential of proteasome inhibition in these diseases are ongoing. Promising data also indicate a therapeutic role of proteasome inhibitors in autoimmune diseases including systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome, and rheumatoid arthritis (RA);^70,71 proteasome inhibition also reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model. ⁷² Finally the proteasome may also be a novel target to combat asthma. ⁷³

Although ongoing research is predominantly focused on proteasome inhibition, studies in neurodegenerative diseases e.g. Huntingon's disease as well as antiaging strategies investigate a role of proteasome activators including oleuropein.^74–76

Preclinical Evaluation of Proteasome Inhibitors

The first agent found to inhibit proteasome activity was lactacystine; a natural metabolite of Streptomyces bacterium which induced apoptosis in human monoblast U937 cells. ⁷⁷ Other initial proteasome inhibitors were reported by Shinohara et al, ⁷⁸ Tanimoto et al, ⁷⁹ Drexler, ⁸⁰ and Orlowski et al; ⁸¹ however, these inhibitors lacked specificity.^25,56

Bortezomib

One of the most recent successes in cancer treatment was the identification of the ubiquitin-proteasome system as a new therapeutic target and the development, introduction, and clinical approval of the small molecule the first in class proteasome inhibitor Bortezomib. Bortezomib was generated by substituting the pharmacophore of its aldehyde with boronic acid, and identified within a panel of synthetic boronic acid proteasome inhibitors which were screened for their activity in the 60 NCI human tumor cell line panel. Based on its activity in a broad spectrum of different tumor types, Bortezomib, previously named PS-341 (N-pyrazinecarbonyl-L-phenylalanaine-L-leucine boronic acid), was chosen for further studies. It showed remarkable preclinical and clinical activity first in MM and later in mantle cell lymphoma and was the first-in-class proteasome inhibitor approved by the Food and Drug Administration (FDA) in 2003, 2005, and 2008 for treatment of relapsed/refractory, relapsed, and newly diagnosed MM, respectively, and in 2006 for treatment of patients with mantle cell lymphoma who have received at least one prior therapy.

Bortezomib induces apoptosis in drug-resistant MM cells, and inhibits both binding of MM cells in the BM microenvironment, as well as production and secretion of cytokines that mediate MM cell growth and survival. The initial rationale to use bortezomib in MM was its inhibitory effect on NFκB activity via blockade of the proteasomal degradation of IκBα.^82,83 However, recent studies demonstrate that bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NFκB activity in MM cells. Indeed, in MM cells bortezomib surprisingly triggers canonical NFκB activation via IκBα phosphorylation. Conversely, IKKβ inhibitors block downregulation of IκBα and NFκB activity and enhance Bortezomib-induced cytotoxicity. The cytotoxic activity of Bortezomib must therefore depend on molecules other than NFκB. ⁸⁴

Alternative mechanisms of Bortezomib-induced apoptosis include: induction of p53 and MDM2 expression; induction of the phosphorylation (Ser15) of p53; activation of JNK; cleavage of DNA-PKCs and ATM; ⁸⁵ caspase-dependent downregulation of gp130; ⁸⁶ generation of reactive oxygen species; release of cytochrome C/Smac from mitochondria into the cytosol and activation of caspase-9 and caspase-3; activation of Bim and caspase 8; blockade of DNA repair; inhibition of MM-BMSC interactions and sequelae including activation of MAPK and PI3K signaling pathways; as well as accumulation of improperly folded proteins and subsequent ER stress; and the blockade of the unfolded protein response (UPR) by preventing stress-induced phosphorylation of IRE1 and the resultant splicing of Xbp-1. ⁸⁷ In addition, IL-6-triggered phosphorylation of ERK, but not of STAT3, is blocked by Bortezomib. ⁸⁸

The anti-angiogenic effect of Bortezomib is another potential mechanism of its anti-MM activity, as demonstrated in a plasmacytoma xenograft mouse model. ⁸⁹ Specifically, Bortezomib both downregulates caveolin-1 expression and inhibits caveolin-1 tyrosine phosphorylation, which are required for VEGF-mediated MM cell migration on fibronectin; as well as blocks VEGF-induced tyrosine phosphorylation of caveolin-1 in HUVECs, thereby inhibiting ERK-dependent endothelial cell proliferation. ⁹⁰

Dendritic cells (DCs) of MM patients are functionally defective due, at least in part, to IL-6-, VEGF- or β2-microglobulin-triggered inhibition of DC maturation.^91–94 In addition, direct MM cell-DC interaction enhances MM clonogenicity and confers drug resistance.^95,96 Ongoing preclinical studies are evaluating a variety of anti-MM agents for their ability to normalize DC function and inhibit their supportive role on MM cell growth. For example, Bortezomib enhances DC-mediated induction of immunity to MM via exposure of cell surface HSP-90 on dying tumor cells; ⁹⁷ but also disrupts tumor-DC interactions in MM and lymphoma. ⁹⁸ HSP-90-mediated protein folding within DCs is additionally required for DC-specific function. Conversely, HSP90 inhibition disrupts DC function. ⁹⁹

Importantly, selective inhibitors of the osteoblast (OBL) proteasome stimulate bone formation via BMP2 expression, ¹⁰⁰ and Bortezomib stimulates new bone formation in mouse calvarian cultures. ¹⁰¹ Indeed, elevated serum levels of bone-specific alkaline phosphatase and osteocalcin were seen in MM patients responding to Bortezomib therapy ¹⁰² but also in non-responders. ¹⁰³ Functionally, Bortezomib induces decreased DKK1 (an OBL inhibitor), increased RUNX2 (a OBL-specific transcription factor) and stabilizes β-catenin. ¹⁰⁴ Whether β-catenin stabilization occurs in a Wnt-dependent or independent manner is still controversial.^105–108 Mesenchymal stem/progenitor cells (MSCs), which differentiate in a context-specific manner into muscle, bone, fat, and other cell types, represent another potential therapeutic target of Bortezomib in MM.

BM MSCs isolated from MM patients, as compared to normal MSCs, produce high levels of IL-6, DKK1, as well as factors associated with angiogenesis and osteogenic differentiation. ¹⁰⁹ Moreover they have decreased ability to inhibit T-cell proliferation. ¹¹⁰ Bortezomib induces MSCs to preferentially undergo osteoblastic differentiation in mice, in part by modulation of the bone-specifying transcription factor Runt-related transcription factor 2 (Runx-2). Mice implanted with MSCs showed increased ectopic ossicle and bone formation after treatment with Bortezomib. Treatment with Bortezomib also increased bone formation and rescued bone loss in a mouse model of osteoporosis. ¹¹¹ These results are consistent with the therapeutic benefits of Bortezomib on MM bone disease. ¹⁰⁴

Despite its success, sixty-five percent of patients with relapsed or refractory MM do not respond to Bortezomib. Resistance is mediated via upregulation of heat shock protein Hsp27 upregulation;^112,113 as well as Mcl-1. ¹¹⁴ Several preclinical and more than 130 clinical studies combining Bortezomib with other conventional and novel therapies are ongoing to further improve the activity of Bortezomib and overcome development of drug resistance. For example targeted therapies in combination Bortezomib include: Lenalidomide or IMiD; Flavopiridol (NSC649890) which targets CDK; Sorafenib (BAY43-9006), a small-molecule inhibitor which targets a multitude of kinase (e.g. Raf, VEGFR1, VEGFR2); Bevacizumab which targets VEGF; Perifosine which predominantly targets Akt; ATN-224, which targets superoxide dismutase 1; tipifarnib (R115777), a FTI; CNTO 328, which targets IL-6; Mapatumumab, which targets TRAIL receptor; 17AAG, which targets HSP90; CCI-779/temsirolimus which targets mTOR; and HDAC inhibitors, which block aggresomal degradation of proteins. ¹¹⁵ Of note, Bortezomib has already been combined with dexamethasone (Dex) and with melphalan and prednisone as initial therapy for transplant candidates and elderly patients, respectively, and achieved enhanced extent and frequency of response, as well as prolonged survival.^116,117

Importantly, there remains a paucity of data in identifying patients who are most likely achieve meaningful clinical response to bortezomib treatment. A recent study indicates that patients who express cyclin D1 are more likely to achieve a response. In contrast, patients who express p16(INK4A), cytoplasmic p53, and the highest intensity of Bcl-2 staining have a poor response. Patients who achieved a response to bortezomib and those patients who expressed cyclin D1 at baseline showed a significant survival advantage. Patients who expressed FGFR3, a poor prognostic marker, responded equally well and had similar outcomes with bortezomib compared with FGFR3-negative patients. Ongoing studies are aiming to further improve our knowledge about clinical and immunohistochemical features associated with a response to bortezomib in patients with MM. ¹¹⁸

Promising preclinical antitumor activity of Bortezomib has also been shown in a variety of other malignancies including adult T cell lymphoma; ¹¹⁹ lung and breast cancer;^120,121 prostate cancer;^56,122,123 pancreatic cancer;^124,125 head and neck cancer; ¹²⁶ colon cancer; ¹²⁷ and melanoma. ¹²⁸

Carfilzomib

Based on the success of Bortezomib in MM, the potential therapeutic activity of other proteasome inhibitors was subsequently evaluated with the aim to further increase potency, while decreasing adverse side effects and facilitating the route of administration. Carfilzomib, former PR-171 (Proteolix, USA), is a potent irreversible inhibitor of the CT-L activity of the proteasome. ¹²⁹ Based on these promising preclinical data, several clinical trails have now been initiated testing carfilzomib, alone or in combination with lenalidomide and Dex, in relapsed/and or refractory MM and relapsed solid tumors (http://clinicaltrials.gov).

Salinosporamidine A

Salinosporamidine A (NPI-0052) is an orally bioavailable inhibitor of all three catalytic activities of the proteasome. ¹³⁰ Importantly, bortezomib and NPI-0052 induce synergistic anti-MM activity in vitro against both MM cell lines or patient CD138+ MN cells, as well as in vivo in a human plasmocytome xenograft mouse model. ¹³⁰ NPI0052 is now being investigated in patients with relapsed or relapsed/refractory MM, as well as in patients with advanced solid or hematologic malignancies (http://clinicaltrials.gov). Antitumor activity of NPI0052 has also been observed in colon cancer, chronic lymphatic leukaemia (CLL), and prostate cancer.^131–134

Based on these promising data, other proteasome inhibitors are now under preclinical and clinical evaluation including the orally bioavailable CEP-18770. ¹³⁵

Others

The LMP-7-selective peptide ketoepoxide proteasome inhibitor PR-957, which is related to carfilzomib, blocks presentation of LMP-7-specific, MHCI-restricted antigens in vitro and in vivo, as well as production of IL-23 and IFNγ by activated monocytes, and IL-2 by T cells. PR-957 treatment resulted in reductions in cellular infiltration, cytokine production, and autoantibody levels in a murine model of rheumatoid arthritis. ¹³⁶ The anti-HIV compound ritonavir, besides blocking free proteases, inhibits CT-L-proteasome activity, thereby modulating antigen presentation and T cell response.^137,138 Preclinical data indicate a role for ritonavir in the treatment of glioblastoma. ¹³⁹

Clinical Evaluation of Proteasome Inhibitors

Bortezomib in patients with relapsed and relapsed/refractory MM

Based on its comprehensive preclinical evaluation in MM and encouraging results from a phase I trial, ¹⁴⁰ bortezomib was tested in two multicenter phase II clinical trials for relapsed and refractory MM patients. In the SUMMIT trial, 202 patients received 1.3 mg/m² of Bortezomib twice-weekly i.v. for up to eight cycles; oral Dex (20 mg/day) was then added to Bortezomib in patients with suboptimal response on the day of treatment and the day after. More than 90% of patients had at least 3 lines of previous therapy and were refractory to their last regimen, with a median of 6 lines of prior therapy overall. Response rates were 35% minimal response (MR) or better, and partial response (PR) or better in 28%, including 12% complete response/near complete response (CR/nCR). The median overall survival (OS) was 16 months, with a median time to progression (TTP) of 7 months. ¹⁴¹ The CREST trial comparing two doses of Bortezomib (1.0 or 1.3 mg/m²) in 53 patients, showed greater response rates but also higher toxicity in the high-dose cohort. ¹⁴² An updated survival analysis showed a median OS of 27 months for the lower dose and 60 months for higher dose Bortezomib-treated groups. ¹⁴³ Based on the results of these two phase II trials, Bortezomib was approved by the US Food and Drug Administration (FDA) for the treatment of relapsed and refractory MM in 2003 and by the European Medicines Agency (EMEA) in 2005.

The subsequent international, randomized phase III APEX trial compared Bortezomib monotherapy versus high dose Dex in relapsed MM patients who had received 1–3 previous lines of therapy. ¹⁴⁴ An updated analysis of the APEX trial with extended follow-up (median 22 months) revealed a superior response rate and prolonged median OS for patients treated with Bortezomib compared to high dose Dex. This benefit was seen despite the fact that 62% of the patients on the high-dose Dex arm crossed over to receive Bortezomib after interim analysis demonstrated significant benefit to Bortezomib. ¹⁴⁵ Bortezomib is therefore the only single agent to provide survival benefit and high overall response rate of 43% (PR + CR) in the setting of relapsed MM. These results led to its FDA approval in 2005 for the treatment of patients in first relapse and beyond.

Bortezomib in newly diagnosed transplant candidates with MM

Results of the large randomized Intergroup Francophone du Myelome (IFM) 2005/01 clinical trial compared bortezomib versus VAD in newly diagnosed patients. Improved overall and extent of response to Bort/Dex induction before and after high dose melphalan and autologous SCT were demonstrated when compared to VAD, with a decreased need for second transplantation and no additive role for consolidative chemotherapy with DCEP. ¹⁴⁶ Similarly positive results and superiority to Thal/Dex alone were subsequently demonstrated upon combination of Bort/Thal/Dex (VTD) prior to transplantation. ¹⁴⁷ Importantly, at least 60% of the patients receiving VTD achieved a very good partial response (VGPR) versus 25% (P < 0.001) in the group receiving Thal/Dex, and a CR/nCR rate of 38% versus 7% (P = 0.007), and VTD overcame the potential effect of adverse cytogenetics on response. Moreover, Bortezomib induction treatment in combination with Dex or liposomal (PEGylated-) doxorubicin is highly active in the upfront setting, demonstrating an ORR over 80% and CR/nCR of 18% to 32%.^{117,148–152} Finally, very recent results from a large multicenter phase I/II study suggest that 100% of patients respond, with 71% VGPR or better, including 36% nCR/CR, when Len/Bort/Dex is used as induction therapy, with minimal toxicity. ¹⁵³ Bortezomib gained EMEA sanction as first line therapy for MM in 2008.

Bortezomib in MM patients not eligible for transplant

Melphalan in combination with Prednisone has been the standard in MM therapy for the last four decades; however, data from recent randomized clinical trials show that the combination of thalidomide with MP (MPT) as well as of lenalidomide (Revlimid, MPR) is superior to MP alone.^154–158 Similarly, MP with bortezomib (Velcade™, MPV) is associated with a remarkable ORR and CR rate and a significant improvement of EFS and OS.^159,160 Importantly, MPV can safely be administered in patients with renal insufficiency and overcomes the impact of adverse cytogenetics. Striking advantages in PFS, TTP, OS, ORR and CR were demonstrated in a randomized phase III trial (VISTA) comparing MPV versus MP confirmed the superiority of MPV. Consequently, Bortezomib used in this combination was FDA approved for initial MM treatment in June 2008. ¹¹⁶

To reduce the risk of deep vein thrombosis (DVT) in patients who are at higher risk, MPV is a preferred option based upon its reduced thrombogenicity; while for patients with increased risk of peripheral neuropathy (PN), MPR is a potential choice, with phase III trials of this combination ongoing. ¹⁶¹

Recently, a Phase III trial comparing Bortezomib and pegylated liposomal doxorubicin with Bortezomib alone showed significant improvement of the time to progression, a superior 15-month survival rate, and an increased duration of response for the combination versus Bortezomib alone treatment, independent of the number of prior lines of therapy.^150,162 Importantly, patients who had previously progressed on anthracycline-containing regimens benefited from this combination, consistent with pre-clinical data suggesting that Bortezomib can sensitize MM cells to chemotherapy. ¹⁶³ These results were the basis for the FDA approval of this combination regimen in 2007 for the treatment of MM patients who have not previously received Bortezomib and have received at least one prior line of anti-MM therapy.

Bortezomib in patients with relapsed and relapsed/refractory MCL and follicular lymphoma

In mantle cell lymphoma, five independent studies including the PINNACLE study used Bortezomib in first relapse and beyond. Based on overall response rates ranging from 30%–45% and durable remissions despite inclusion of many chemotherapy-resistant patients, the FDA granted approval to Bortezomib for treatment of patients who had received at least one prior therapy. Bortezomib is given twice a week for two weeks followed by a 10 day gap; cycles are repeated until there is disease progression or there is a need to stop due to adverse side effects.^164–168 Moreover, 47% responses, including 5 complete and 14 partial responses which were durable, were observed in patients with chemotherapy-refractory MCL. Remarkably, PFS was identical to patients with relapsed disease, indicating response to Bortezomib irrespective of the prior exposure to cytotoxic chemotherapy. ¹⁶⁹ Finally, Bortezomib also shows promising activity in follicular lymphoma. ¹⁷⁰ Clinical trials of bortezomib in combination with rituximab have also shown responses in relapsed as well as newly diagnosed Waldenstrom's macroglobulinemia.^171,172

Other clinical trials

Other clinical trials evaluating Bortezomib alone or in combination with conventional chemotherapies are ongoing in solid tumors including prostate cancer, non-small cell lung cancer, broncho-alveolar cancer, breast cancer, renal cancer, ovarian cancer, cervical cancer, gastric cancer, adrenal cancer, head and neck cancer, colorectal cancer, bladder cancer, sarcoma, pancreatic cancer, peritoneal cavity cancer, malignant mesothelioma, and esophageal cancer. In hematologic malignancies, it is being studied in AML, Waldenstroms Macroglobulinemia, Hodgkin's lymphoma, myelodysplasia, MALT lymphoma, amyloidosis, B-cell lymphoma, CLL, and Castleman's disease (http://clinicaltrials.gov/).

Adverse side effects of Bortezomib

The most common adverse side effects of Bortezomib include fatigue, gastrointestinal toxicity, and polyneuropathy as well as thrombocytopenia, neutropenia, anemia and rash. At least in part these side effects may be attributed to the accumulation of immature proteins within cells. Bortezomib-associated peripheral neuropathy originates in the dorsal root ganglion neurons, probably by changes in mitochondria-mediated dysregulation of calcuim homeostasis and neurotrophins. ¹⁷³ Quantitative sensory analysis in patients with Bortezomib-induced pain demostrated deficits in all three fiber types. ¹⁷⁴ Grade 1 neuropathy is treated by dose reduction to 1.0 mg/m² instead of 1.3 mg/m²), whereas grade 2 and 3 neuropathy requires treatment pause until resolution and reinstitution at a dose of 0.7 mg/m². Discontinuation is required in patients with grade 4 neuropathy. ¹⁷⁵ Of note, baseline MM-associated neuropathy seems more common than previously reported, and Bortezomib-associated neuropathy, although a common toxicity, is reversible in most patients. ¹⁷⁶ Bortezomib-induced thrombocytopenia occured in 26% of patients in the APEX trial, with 4% of patients having clinically significant bleeding, and may be due to inhibition of NFκB-dependent budding of platelets from megakaryocytes.^177,178

Previous clinical studies of Bortezomib, mostly in the United States and Europe, have shown low incidences of pulmonary adverse effects. However, a Japanese study study showed that Bortezomib can cause serious lung injury, suggesting that its incidence might vary among different ethnicities. ¹⁷⁹ The EMEA therefore recommended in March 2008 that Bortezomib should not be used in patients with certain severe pulmonary or heart problems (acute diffuse infiltrative pulmonary and pericardial disease).

Concluding Remarks and Future Perspectives

Based on preclinical studies, several clinical trials are now ongoing to test bortezomib and the newer generation of proteasome inhibitors in combination with other conventional and novel therapies to further enhance efficacy, overcome resistance and reduce toxicity in patients with MM and MCL, as well as other hematologic and solid malignancies. Moreover, modulation of proteasome function also represents a novel treatment strategy in neurologic disorders, heart disease, collagenoses, and other non-malignant disorders. Importantly, targeting the immuno- and/or thymoproteasome may also be a novel strategy to modulate the immune response i.e. asthma.

The success of Bortezomib in MM and MCL, as well as promising preclinical and clinical data using next-generation proteasome inhibitors, has triggered further investigations to delineate molecular mechanisms leading to the assembly of the proteasome, as well as detailed function of the ubiquitin/proteasome system. For example, fluorescent inhibitors as well as atomic force microscopy of the proteasome have been developed to investigate ubiquitination and proteasome assembly and function.^{45,69,180–182}

This increased knowledge has provided the basis for new therapeutic strategies that are not only aiming to block selective proteasomal catalytic activities, but also specifically target: 1) molecular mechanisms that are involved in the assembly of the proteasome; 2) the recognition of specific substrate proteins; 3) de-ubiquitination of specific substrate proteins by the 19S base; and 4) translocation of de-ubiquitinated substrate proteins. Inhibitors of E3 ligases, E1, and de-ubiquitinating enzymes; as well as drugs that target Ubl modifiers are already under intense preclinical and clinical evaluation.^60,183

E3 ligases mediating ubiquitin conjugation play a key role in cancer development, e.g. RING-type E3 enzyme Mdm2, a negative regulator of p53; and the multisubunit SCF ligases. The small molecule HLI98 inhibits E3 ligase activity of Mdm2 and thereby selectively blocks p53 ubiquitination. ¹⁸⁴ Importantly, dependent on substrate specificity, E3 ligases not only act as tumor promoters, but also on suppressors. For example, agonists of SCF (Fbxw7/hCdc4), which functions as interchangeable substrate recognition component of the SCF ubiquitin ligases, trigger increased proteasomal degradation of c-Myc, c-Jun, cyclin E1, and Notch. ¹⁸⁵

Nutlins and reactivation of p53 and induction of tumor cell apoptosis (RITA, 2,5-bis(5-hydroxymethyl-2-thienyl)furan) target protein-protein interactions between p53 and Mdm2, rather than influence their enzymatic function. Specifically, nutlins (nutlin-1, -2, -3) are cis-imidazoline analogs discovered by screening a chemical library, which was designed to identify compounds that bind Mdm2 in the p53-binding pocket. Consequently, nutlins activate the p53-pathway and induce cell cycle arrest, apoptosis, and growth inhibition in p53-wildtype cancer cells in vitro and in vivo. ¹⁸⁶ Nutlin-3 in particular has demonstrated promising preclinical activity. ¹⁸⁷ RITA binds the amino terminus of p53, thereby promoting growth arrest. ¹⁸⁸

Deubiquitinating enzymes (DUBs) recognize ubiquitinated proteins and remove their ubiquitin tag. Forming five subfamilies (ubiquitin-specific proteases, Usp; ubiquitin carboxy-terminal hydrolases, UCH; ovarian tumor-like proteases, OTU; JAMM/MPN metalloproteases; and Machado-Jakob-disease proteases, MJD), DUBs are highly investigated as another class of targets for novel compounds.^189,190

Another therapeutic and likely the most challenging approach is to modulate the binding of ubiquitin and ubiquitin-binding domains (UBDs), small modular domains which recognize different types of ubiquitin modifications (monoubiquitination, and polyubiquitination of distinct lenghts and linkage types). For example, ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Moreover, they prevent the recognition of ubiquitinated substrates by UBDs present in the proteasome receptors RPN10 and RPN13, as well as the shuttling receptor RAD23.^191,192

In summary, efforts to identify inhibitors/modulators of protein-protein interaction systems including ubiquitin ligases, DUBs, and UBDs hold great promise to increase drug specificity and thereby decrease adverse side effect profiles of current compounds.

Disclosures

This manuscript has been read and approved by all authors. This paper is unique and is not under consideration by any other publication and has not been published elsewhere. The authors report no conflicts of interest.