Comparative Mechanisms of Action of Proteasome Inhibitors

The proteasome has emerged as an important target for therapeutic intervention. In preclinical studies, proteasome inhibitors (PIs) induced apoptosis and inhibited tumor growth, supporting their potential role in the treatment of various tumor types, especially hematologic malignancies. Bortezomib (Velcade), the first clinically validated PI, reversibly binds to the chymotrypsin-like (ChT-L) active sites in the 20S proteasome and potently inhibits cell growth and proliferation in human tumor cell lines and in multiple myeloma (MM) and mantle cell lymphoma. However, the adverse event profile and intravenous administration of bortezomib have underscored the need for the development of PIs with selective actions on different proteasome subunits, which would have different binding kinetics and routes of administration. The most advanced next-generation PI is carfilzomib, an epoxyketone that differs structurally and mechanistically from bortezomib. In preclinical studies, carfilzomib demonstrates sustained inhibition of proteasomal ChT-L activity and greater selectivity than bortezomib. It is thought that the selectivity of carfilzomib for the β5 subunit contributes to its greater cytotoxic response and improved tolerability profile relative to bortezomib. Furthermore, in preclinical studies, carfilzomib did not exhibit the same magnitude of off-target activity against non-proteasomal proteases that is observed with bortezomib. Variations in the binding profiles of some of the next-generation PIs may translate into key differences in pharmacokinetic and toxicity profiles, and thus may be clinically relevant in the treatment of MM.

Introduction

The proteasome is an important therapeutic target in the treatment of a small but increasing number of diseases- most notably, B cell malignancies. Multiple myeloma (MM) is one of the first diseases for which proteasome inhibitors (PIs) have been validated and in which they have found widespread use. Following the launch of bortezomib (Velcade) in 2003, other PIs have been developed that have different chemical structures and proteasome-binding profiles. This article reviews the biology of the proteasome and its involvement in MM and compares the physicochemical profiles of various PIs, examining how specific differences in their mechanisms of action and binding characteristics may affect their clinical profiles.

Proteasome: Biology and Normal Function

FIGURE 1

Structure of the 26S Proteasome

The ubiquitin proteasome system (UPS) plays a key role in maintaining normal cellular homeostasis in eukaryotic cells.[1] The majority of intracellular proteins are regulated by ubiquitin-mediated proteolysis, including those controlling cell-cycle progression, apoptosis, DNA repair, and the stress response.[2,3] A key step in this process is the "tagging" of proteins targeted for degradation with multiple molecules of ubiquitin, a 76–amino acid protein whose primary sequence is highly conserved in organisms ranging from yeast to mammals.[4] Once polyubiquitinated, proteins targeted for degradation bind to the 26S proteasome.

The 26S eukaryotic proteasome is a large ATP-dependent complex consisting of a barrel-shaped 20S proteolytic core, the ends of which are covered by two 19S regulatory cap complexes (Figure 1).[5-8] The 19S regulatory complex is an additional feature of the eukaryotic proteasome consisting of a lid and a base that binds to the 20S particle to form the 26S proteasome holoenzyme.[9] The 19S regulatory complex opens a gated channel into the 20S proteasome core, access to which is blocked in the free 20S proteasome.[10] While the lid recognizes ubiquitinated protein substrates with high fidelity, the base (which caps the end of the 20S proteasome core) unfolds protein substrates and threads them into the catalytic chamber of the 20S particle.[11] The structure of the 20S core is well conserved in virtually all organisms ranging from archaebacteria and yeast to humans[8]; it is essentially a hollow barrel shape consisting of four stacked heptameric rings. The two outer rings, composed of α-subunits (α1 through α7), and two inner rings, composed of β-subunits (β1 through β7), form a central chamber within the stack that runs from top to bottom.[9]

Unlike typical proteases, the 20S proteasome in eukaryotic cells exhibits multiple proteolytic activities, resulting in cleavage of protein targets at multiple sites. Distinct β-subunits are associated with the specific catalytic activities of the proteasome, which can be characterized as chymotrypsin-like (ChT-L; β5), trypsin-like (T-L; β2), and peptidylglutamyl-peptide hydrolyzing (also known as caspase-like [C-L; β1]).[9,12] All three types of activities contribute significantly to proteolysis, although their relative importance varies widely according to the specific primary amino acid sequence of the protein substrate.[7] Of the three catalytically active β-subunits, particular interest has focused on the ChT-L activity of the β5 subunit.[5] Increased levels of ChT-L activity and the resultant protein breakdown have been implicated in various disease states, including malignancies.[5] Inhibition of ChT-L activity results in accumulation of ubiquitin-protein conjugates and induction of apoptosis, particularly in transformed cell lines.[12] Two major isoforms of the proteasome include the constitutive form, which is present in most cells, and the immunoproteasome, which is primarily expressed in cells of lymphoid origin.[13]

The Proteasome as a Therapeutic Target in Multiple Myeloma

Because of its involvement in the regulation of key functions necessary for cell growth and viability in both normal and transformed cells, protein degradation appeared to be an unlikely and somewhat counterintuitive target for pharmaceutical intervention. Nonetheless, there are a number of reasons why the proteasome presents such an important target for therapeutic intervention.[12] The UPS controls the abundance and activity of oncogene and tumor suppressor gene products, transcription factors, and other signaling molecules, such as nuclear factor kappa B (NFκB) and the tumor suppressor protein p53, and it has been shown to play an important role in the formation of metastases.[1] Cancer cells generally have higher levels of proteasome activity compared with normal well-differentiated cells-possibly due to higher levels of oxidative stress and the secretion of cytokines or growth factors-making the proteasome a potential therapeutic target in oncology.[1] Preclinical research with PIs demonstrated their ability to induce apoptosis and inhibit tumor growth, supporting the potential role of PIs in the treatment of various tumor types, especially hematologic malignancies.[7,14,15]

Chemical Classes of Proteasome Inhibitors

FIGURE 2

Chemical Structure of Bortezomib and Next-Generation Proteasome Inhibitors

Because of evidence demonstrating the key role of proteasomes in regulating a variety of critical proliferative pathways in transformed cells, both synthetic and naturally derived PIs have been intensely pursued as therapeutic agents. PIs may have varying specificities, affinities, and avidities for the different catalytic sites within the proteasome core and can be broadly classified into three distinct categories based on their chemical structures (Figure 2): boronates, epoxyketones, and lactams (cyclic amides).[1] The synthetic PIs include a peptide moiety with a reactive pharmacophore group (eg, boronate, aldehyde).

Boronates

Bortezomib was the first clinically validated boronate-based dipeptide PI approved for use in cancer-specifically, relapsed/refractory MM (R/R MM). Bortezomib was approved in the United States in 2005 for the treatment of patients with MM who have received at least one prior therapy, and in 2008 for frontline treatment of patients with MM in combination with melphalan and prednisone.[9] Two other boronate-based analogs (CEP-18770 and MLN9708) are currently in the early phases of clinical development.[5,9,16]

Epoxyketones

The synthetic epoxyketone inhibitors are related to the natural product epoxomicin and include carfilzomib (previously PR-171; currently in phase III clinical trials in MM) and its orally bioavailable analog ONX 0912 (formerly PR-047). Carfilzomib is structurally similar to epoxomicin but differs structurally and mechanistically from the boronate-based PI bortezomib.[17]

Salinosporamides

Lactacystin is a Streptomyces lactacystinaeus metabolite that was discovered because of its ability to induce neurite outgrowth in a murine neuroblastoma cell line.[18] It was subsequently shown to target the 20S proteasome via irreversible modification of the N-terminal threonine of β-subunits.[19] The naturally occurring lactacystin analog salinosporamide A (NPI-0052; marizomib), a β-lactone derived from the marine bacteria Salinospora tropica, is currently in phase I clinical development for treatment of various hematologic malignancies and solid tumors.[9]

Molecular Targets and Mechanisms of Action of Specific Classes of PIs

TABLE

Properties of Bortezomib vs Second-Generation Proteasome Inhibitors

In general, PIs demonstrate reversible or sustained binding to active sites in the 20S proteasome, primarily the ChT-L site.[1,9] However, different chemical classes have different β-subunit binding profiles that are thought to account for differences in pharmacokinetics and toxicity among PIs (Table).[9]

It is presumed that peptide boronates form a stable, slowly reversible tetrahedral adduct with the active site threonine.[1] Bortezomib reversibly binds to the 20S subunit, demonstrates high affinity for the proteasomal ChT-L active site and significant affinity for the C-L active site, and has no or minimal effect on T-L activity.[1] Like other PIs, bortezomib targets numerous pathways by inhibiting the proteasome and controlling key transcription factors (eg, NFκB).[1] In in vivo human tumor cell lines, bortezomib potently inhibited cell growth and proliferation.[20] Biochemical studies indicate that bortezomib triggers pleiotropic signaling pathways, and a number of signaling events contribute to its antitumor activity.[9] The apoptotic activity of bortezomib results from inhibition of NFκB activity, disruption of cyclin-dependent kinase activity, stabilization of c-Jun N-terminal kinases leading to Fas upregulation, stabilization of p53, and a shift of the proapoptotic and antiapoptotic balance in the Bcl-2 family of proteins.[1,9] The recovery of activity following bortezomib inhibition in vitro and in vivo probably takes place primarily by de novo synthesis of new proteasomal proteins rather than by reversibility of the boronate tetrahedral complex.[21,22]

The adverse event profile and intravenous administration of bortezomib, along with concerns regarding the potential for resistance, have underlined the need for next-generation PIs that have different molecular and chemical characteristics. These characteristics would include selectivity for various proteasome subunits, binding kinetics, and route of administration.[9]

Both of the boronic acid PIs under development, MLN9708 and CEP-18770, are reversible inhibitors, primarily of proteasomal ChT-L activity, with potencies similar to that of bortezomib.[9] In preclinical studies, CEP-18770 was a potent promoter of apoptosis in human MM cell lines and patient-derived cells while displaying a favorable cytotoxicity profile for normal epithelial cells, bone marrow progenitors, and bone marrow stromal cells.[16] In MM cell lines, CEP-18770 exhibited concentration-dependent inhibition of NFκB activity, resulting in a reduction in proinflammatory cytokines (eg, tumor necrosis factor α, interleukin-1β), intracellular adhesion molecules, and the proangiogenic vascular endothelial growth factor (VEGF).[16] CEP-18770 displayed similar potency to bortezomib in two hematologic tumor cell lines and in bone marrow aspirates from relapsed MM patients. Complete cell death was observed at 20 nanomolar (nM) with both agents.[16]

Epoxomicin and its analogs are made up of a peptide that selectively binds with high affinity in the substrate binding pocket(s) of the proteasome and an epoxyketone pharmacophore that irreversibly interacts with the catalytic threonine residue to inhibit enzyme activity.[1] Epoxyketones form a dual covalent morpholino adduct with the N-terminal threonine of the β5 subunit and react with both the hydroxyl and amino groups of the threonine residue at the active site.[23] Carfilzomib demonstrates potent and sustained inhibition of proteasomal ChT-L activity (> 80% inhibition at ≥ 10 nM doses) in both in vivo and in vitro cellular models of MM by binding to the β5 subunits of the constitutive proteasomes and immunoproteasomes.[17,22,24] In ex vivo treated blood from normal human subjects and in samples derived from patients with MM, carfilzomib demonstrated greater selectivity for the β5 subunit compared with bortezomib.[24] While bortezomib also displayed significant activity for the β1 subunit, carfilzomib had minimal affinity for the β1 and β2 subunits (at doses up to 100 nM).[17] It is thought that the selectivity of carfilzomib for the β5 subunit contributes to its improved tolerability profile compared with bortezomib.

Irrespective of cell type, brief exposure (1 hour) to carfilzomib resulted in greater cytotoxicity (particularly to hematologic tumor cell lines) in terms of cell viability, apoptosis, and cell cycle progression, compared with bortezomib.[22] Sustained inhibition of the proteasome with carfilzomib may explain the greater cytotoxic response.[22] It is important to note that in vitro studies confirm that the sustained binding of carfilzomib to the proteasome does not significantly affect the rate of recovery of proteasomal activity (50% to 100% recovery within 24 hours in all tissues), which was only moderately slower than with bortezomib, suggesting that proteasomal recovery involves de novo protein synthesis.[22] However, these results for carfilzomib cannot be generalized to whole blood; less than 50% recovery was observed after 1 week, compared with complete recovery after 48 hours in bortezomib-treated samples.[22]

The binding profile of carfilzomib results in accumulation of ubiquitin-protein conjugates and proteasome substrates, as well as inhibition of myeloma cell proliferation via induction of apoptosis. Carfilzomib demonstrated potent antiproliferative and pro-apoptotic effects in myeloma cell lines and in patient-derived models of myeloma.[17] Like bortezomib, carfilzomib-induced apoptosis occurs through intrinsic and extrinsic caspase pathways that converge on the effector caspase-3. However, in preclinical models of MM, carfilzomib demonstrated increased activity against MM cell lines, enhanced JNK phosphorylation, and greater potency in increasing caspase-3, caspase-8, and caspase-9 activity (1.5-fold, 1.8-fold, and 2.0-fold increases, respectively), compared with bortezomib.[17] Of note, in both cell-line models and clinical samples, carfilzomib overcame primary and secondary resistance to bortezomib.[17] The researchers suggest that this may be due to the sustained inhibition of the ChT-L subunit, which potentially requires the cell to synthesize and reassemble new proteasomes. This may overcome the cell’s attempt at resistance by overproduction of proteasome subunits and other ubiquitin-proteasome pathway proteins-a characteristic displayed by cells resistant to bortezomib.

The tripeptide epoxyketone ONX 0912 demonstrated sustained inhibition of ChT-L activity in vitro comparable to that observed with carfilzomib, and it induced tumor cell death across multiple tumor types with a half-maximal inhibitory concentration (IC50) of < 100 nM.[25]

Salinosporamides bind to all three catalytic subunits of the proteasome and may also inhibit other cellular proteases, albeit at higher concentrations.[26] NPI-0052, a non-peptide PI, provides sustained inhibition of the β5 subunit.[27] In human erythrocyte 20S proteasomes and in MM cell lines, NPI-0052 inhibits ChT-L and T-L activities at lower concentrations than bortezomib; however, higher concentrations are needed to inhibit C-L activity.[27] In contrast to bortezomib and carfilzomib, NPI-0052-induced apoptosis is predominantly mediated through caspase-8 (extrinsic) and not caspase-9 (intrinsic) pathways.[27] NPI-0052 is a more potent inhibitor of NFκB and related cytokine secretion, and it is a significantly more potent inducer of apoptosis in MM cell lines compared with bortezomib (P < .005). It also significantly inhibits VEGF-induced migration of MM cells (P < .05). NPI-0052 appears to have a wider therapeutic index than bortezomib, with little reported effect on normal lymphocytes at therapeutic doses.[27]

Rationales for Dosing Regimens

Bortezomib is limited by toxicities such as peripheral neuropathy (PN) and thrombocytopenia that restrict the clinical dosing regimen to a biweekly day 1/day 4 schedule, allowing recovery of proteasome activity between doses.[28,29] In an effort to improve tolerability in patients with relapsed/refractory MM, a recent analysis evaluated the feasibility of a single weekly dose of bortezomib in the setting of combination therapy. The majority of response parameters, including overall response rate and progression-free survival, were similar, while the safety profile was improved (with a PN incidence of 8% vs 28% in patients receiving twice-weekly dosing).[30] However, long-term clinical experience with this dosing schedule has been limited thus far. A separate phase III study examined the effect of subcutaneous dosing of bortezomib on safety and efficacy parameters. The findings of this study were also promising and showed similar results for efficacy, with moderate improvements in the safety profile (PN ≥ grade 3 = 6% vs 16% in patients receiving intravenous dosing).[31]

In human tumor xenograft models, carfilzomib administered on either 2 or 5 consecutive days at doses resulting in > 80% proteasome inhibition demonstrated increased antitumor activity with acceptable toxicity compared with the dosing regimen of bortezomib.[22] Phase I dose-escalation studies of two dose-intensive schedules of carfilzomib (a 2-week cycle with dosing on 5 consecutive days followed by 9 days rest in one study, and a 4-week cycle with dosing on 2 consecutive days weekly for 3 weeks followed by 12 days rest in another) confirmed proteasome inhibition levels exceeding 75% in whole blood 1 hour after the first dose, an absence of dose-limiting toxicities, and no PN ≥ grade 3.[32]

The development of an oral PI will improve both flexibility of administration and patient convenience.[25] Orally administered ONX 0912 demonstrated proteasome inhibition > 80% at doses four to ten times less than the maximum tolerated dose.[25] In human tumor xenograft models of MM, ONX 0912 displayed antitumor activity at least equivalent to carfilzomib at well-tolerated doses.[25]

Off-Target Activities

Although both carfilzomib and bortezomib potently inhibit the ChT-L activity of the proteasome, bortezomib exhibits off-target activity directed at several additional serine proteases. This off-target activity is presumably due to the fact that bortezomib was originally identified as a potential inhibitor of serine proteases and still retains some activity against those targets.[33] However, the extent to which off-target inhibition contributes to the toxicities of bortezomib, especially PN, remains to be determined. In preclinical studies, carfilzomib did not exhibit the off-target activity against non-proteasomal proteases that is observed with bortezomib.[34,35] Comparative data from an in vivo model of peripheral nerve degeneration demonstrated that bortezomib is ten times more potent at inducing neurodegeneration and five times more cytotoxic to neuroblastoma cells than carfilzomib.[34] While equivalent proteasome inhibition was reported for the two agents, only bortezomib inhibited nonproteasomal targets, including the mitochondrial serine protease HtrA2 (which protects neurons from stress-induced apoptosis)[34] and such serine hydrolases as cathepsin G, cathepsin A, dipeptidyl peptidase-4, and chymase.[35] In contrast, carfilzomib displays minimal off-target activity, primarily because it is substantially more selective in its binding to N-terminal threonine proteases. When given to animals or humans in daily intravenous doses for up to 5 days (the maximum number of daily doses tested), carfilzomib produced extremely high levels (> 80%) of prolonged proteasome inhibition without dose-limiting PN.[22,36] Bortezomib-induced grade 3 to 4 PN occurs in up to 30% of patients with recurrent disease, potentially necessitating dose modifications that may lead to suboptimal levels of proteasome inhibition.[37] Research is currently focused on further characterization of the nonproteasomal targets of bortezomib and the clinical consequences of their inhibition.

One recent psychophysical study of PN in 11 patients with MM treated with bortezomib in the front-line setting and 26 control subjects found persistent neurophysiological changes associated with bortezomib treatment.[38] These changes were characterized by decreased skin temperature in the area of pain, diminished touch and sharpness detection, and decreased sensitivity to skin heating.[38] Tandem immunohistochemical analyses of patient skin biopsies revealed significant decreases in the density of epidermal nerve fibers and a complete loss of Meissner corpuscles. While such PN can be a painful and debilitating consequence of therapy with bortezomib, pilot studies of complementary medical approaches, including acupuncture, have shown some promise in reducing the intensity of these effects. A recent study involving 20 patients with bortezomib-induced or thalidomide-induced PN evaluated the effect of electroacupuncture over a period of 9 weeks.[39] Improvements were seen in Functional Assessment of Cancer Therapy/Gynecologic Oncology Group (FACT/GOG)-neurotoxicity scores, pain severity, self-reported physical well-being, and coin test scores.[39] Larger controlled studies are needed to better characterize the nature of bortezomib-induced PN and the effects of complementary therapies.

Conclusions

Over the past decade, proteasomal inhibition has emerged as an important therapeutic strategy, and PIs represent a promising treatment option for the management of MM. Variations in the binding profiles of different PIs may translate into key differences in pharmacokinetic and toxicity profiles that may prove clinically relevant in the treatment of MM. Further clinical data from emerging classes of PIs are awaited with interest.

Acknowledgments:The author would like to thank Anna Battershill, MSc, and Brian E. Szente, PhD, of Fishawack Communications for their assistance with manuscript development. Their editorial support was funded by Onyx Pharmaceuticals.

References:

REFERENCES

1. Jung T, Catalgol B, Grune T. The proteasomal system. Mol Aspects Med. 2009;30:191-296.

2. Ciechanover A. Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat Rev Mol Cell Biol. 2005;6:79-87.

3. Rolfe M, Chiu MI, Pagano M. The ubiquitin-mediated proteolytic pathway as a therapeutic area. J Mol Med. 1997;75:5-17.

4. Wilkinson KD. Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Semin Cell Dev Biol. 2000;11:141-8.

5. Dorsey BD, Iqbal M, Chatterjee S, et al. Discovery of a potent, selective, and orally active proteasome inhibitor for the treatment of cancer. J Med Chem. 2008;51:1068-72.

6. Groll M, Ditzel L, Lowe J, et al. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 1997;386:463-71.

7. Kisselev AF, Callard A, Goldberg AL. Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate. J Biol Chem. 2006;281:8582-90.

8. Kopp F, Hendil KB, Dahlmann B, et al. Subunit arrangement in the human 20S proteasome. Proc Natl Acad Sci U S A. 1997;94:2939-44.

9. Dick LR, Fleming PE. Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy. Drug Discov Today. 2010;15:243–9.

10. Baumeister W, Walz J, Zuhl F, Seemuller E. The proteasome: paradigm of a self-compartmentalizing protease. Cell. 1998;92:367-80.

11. Braun BC, Glickman M, Kraft R, et al. The base of the proteasome regulatory particle exhibits chaperone-like activity. Nat Cell Biol. 1999;1:221-6.

12. Orlowski M, Wilk S. Catalytic activities of the 20 S proteasome, a multicatalytic proteinase complex. Arch Biochem Biophys. 2000;383:1-16.

13. Griffin TA, Nandi D, Cruz M, et al. Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFN-gamma)-inducible subunits. J Exp Med. 1998;187:97-104.

14. Orlowski RZ, Eswara JR, Lafond-Walker A, et al. Tumor growth inhibition induced in a murine model of human Burkitt's lymphoma by a proteasome inhibitor. Cancer Res. 1998;58:4342-8.

15. Shinohara K, Tomioka M, Nakano H, et al. Apoptosis induction resulting from proteasome inhibition. Biochem J. 1996;317(pt 2):385-8.

16. Piva R, Ruggeri B, Williams M, et al. CEP-18770: a novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib. Blood. 2008;111:2765-75.

17. Kuhn DJ, Chen Q, Voorhees PM, et al. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood. 2007;110:3281-90.

18. Omura S, Fujimoto T, Otoguro K, et al. Lactacystin, a novel microbial metabolite, induces neuritogenesis of neuroblastoma cells. J Antibiot (Tokyo). 1991;44:113-6.

19. Fenteany G, Standaert RF, Lane WS, et al. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science. 1995;268:726-31.

20. Adams J. Development of the proteasome inhibitor PS-341. Oncologist. 2002;7:9-16.

21. Meiners S, Heyken D, Weller A, et al. Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes. J Biol Chem. 2003;278:
21517-25.

22. Demo SD, Kirk CJ, Aujay MA, et al. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res. 2007;67:6383-91.

23. Groll M, Kim KB, Kairies N, et al. Crystal structure of epoxomicin:20S proteasome reveals a molecular basis for selectivity of α', β'-epoxyketone proteasome inhibitors. J Am Chem Soc. 2000;122:1237-38.

24. Levitsky K, Lee SJ, Shenk KD, et al. Inhibition of proteasome subunits by carfilzomib in phase Ib clinical trial measured using a novel subunit-specific ELISA. 14th Congress of the European Haematology Association; June 4-7, 2009; Berlin, Germany. Haematologica; 2009. Abstract 0947.

25. Muchamuel T, Aujay M, Bennett MK, et al. Preclinical pharmacology and in vitro characterization of PR-047, an oral inhibitor of the 20S proteasome. 50th Annual Meeting of the American Society of Hematology; December 6-9, 2008; San Francisco, CA. Blood; 2008. Abstract 3671.

26. Genin E, Reboud-Ravaux M, Vidal J. Proteasome inhibitors: recent advances and new perspectives in medicinal chemistry. Curr Top Med Chem. 2010;
10:232-56.

27. Chauhan D, Catley L, Li G, et al. A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from bortezomib. Cancer Cell. 2005;8:407-19.

28. Orlowski RZ, Stinchcombe TE, Mitchell BS, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies.
J Clin Oncol. 2002;20:4420-7.

29. Mohty B, El-Cheikh J, Yakoub-Agha I, et al. Peripheral neuropathy and new treatments for multiple myeloma: background and practical recommendations. Haematologica. 2010;95:311-9.

30. Bringhen S, Larocca A, Rossi D, et al. Efficacy and safety of once-weekly bortezomib in multiple myeloma patients. Blood. 2010;116:4745-53.

31. Moreau P, Pylypenko H, Grosicki S, et al. Subcutaneous versus intravenous administration of bortezomib in patients with relapsed multiple myeloma: a randomised, phase 3, non-inferiority study. Lancet Oncol. 2011;12:431-40.

32. O'Connor OA, Orlowski RZ, Alsina M, et al. Multicenter phase I studies to evaluate the safety, tolerability, and clinical response to intensive dosing with the proteasome inhibitor PR-171 in patients with relapsed or refractory hematological malignancies. 48th Annual Meeting of the American Society of Hematology; December 9-12, 2006; Orlando, FL. Blood; 2006. Abstract 2430.

33. Arastu-Kapur S, Anderl JL, Kraus M, et al. Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events. Clin Cancer Res. 2011;17:2734-43.

34. Arastu-Kapur S, Ball AJ, Anderl JL, et al. Neurodegeneration induced by bortezomib exposure in vitro occurs via proteasome independent mechanisms. 51st Annual Meeting of the American Society of Hematology; December 5-8, 2009; New Orleans, LA. Blood; 2009. Abstract 2859.

35. Arastu-Kapur S, Shenk K, Parlati F, Bennett MK. Non-proteasomal targets of proteasome inhibitors bortezomib and carfilzomib. 50th Annual Meeting of the American Society of Hematology; December 6-9, 2008; San Francisco, CA. Blood; 2008. Abstract 2657.

36. O'Connor OA, Stewart AK, Vallone M, et al. A phase 1 dose escalation study of the safety and pharmacokinetics of the novel proteasome inhibitor carfilzomib (PR-171) in patients with hematologic malignancies. Clin Cancer Res. 2009;15:7085-91.

37. Argyriou AA, Iconomou G, Kalofonos HP. Bortezomib-induced peripheral neuropathy in multiple myeloma: a comprehensive review of the literature. Blood. 2008;112:1593-9.

38. Boyette-Davis JA, Cata JP, Zhang H, et al. Follow-up psychophysical studies in bortezomib-related chemoneuropathy patients. J Pain. 2011 Jun 22 [Epub ahead of print].

39. Wang M, Garcia K, Zhou Y, et al. Electroacupuncture is an effective therapeutic modality for bortezomib- or thalidomide-induced peripheral neuropathy in patients with multiple myeloma: a pilot study. 13th International Myeloma Workshop; May 3-6, 2011; Paris, France. Haematologica; 2011. Abstract P-367.

40. Orlowski RZ, Kuhn DJ. Proteasome inhibitors in cancer therapy: lessons from the first decade. Clin Cancer Res. 2008;14:1649-57.

41. Wang M, Fayad L, Wagner-Bartak N, et al. Oral lenalidomide plus 4 doses of rituximab induced prolonged remissions in relapsed/refractory mantle cell lymphoma: a completed phase 1/2 clinical trial. 11th International Conference on Malignant Lymphoma; June 15-18, 2011; Lugano, Switzerland. Ann Oncol; 2011. Abstract 109.

42. Marteijn JA, Jansen JH, van der Reijden BA. Ubiquitylation in normal and malignant hematopoiesis: novel therapeutic targets. Leukemia. 2006; 20:1511-8.