The Role of the Ubiquitin Proteasome System in Lymphoma

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Critical Reviews in Oncology/Hematology 87 (2013) 306–322

The role of the ubiquitin proteasome system in lymphoma

a,∗ b a c

K. Stephen Suh , Takemi Tanaka , Sreeja Sarojini , Ginah Nightingale ,

a a a

Rajendra Gharbaran , Andrew Pecora , Andre Goy

John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ 07601, United States

Thomas Jefferson University, Philadelphia, PA 19107, United States

Jefferson School of Pharmacy, Thomas Jefferson University, Philadelphia, PA 19130, United States

Accepted 14 February 2013

Contents

1. Introduction ...... 306

2. Signaling pathways regulated by the UPS ...... 308

3. Current therapeutic options for lymphoma ...... 309

3.1. Class A compounds ...... 310

3.2. Class B compounds ...... 310

3.3. Class C compounds ...... 314

3.4. Class D compounds ...... 315

3.5. Class E compounds ...... 315

4. Unfolded protein response (UPR) ...... 316

5. Apoptosis ...... 316

6. Conclusion ...... 318

Reviewers ...... 318

References ...... 318

Biographies...... 322

Abstract

The ubiquitin-proteasome system (UPS) maintains the integrity of cellular processes by controlling protein degradation pathways. The

role of the UPS in proliferation, cell cycle, differentiation, DNA repair, protein folding, and apoptosis is well documented, and a wide range

of protein activities in these signaling pathways can be manipulated by UPS inhibitors, which include many anti-cancer agents. Naturally

occurring and synthetic drugs designed to target the UPS are currently used for hematological cancers, including lymphoma. These drugs

largely interfere with the E1 and E2 regions of the 26S proteasome, blocking proteasomal activity and promoting apoptosis by enhancing

activities of the extrinsic (death receptors, Trail, Fas) and intrinsic (caspases, Bax, Bcl2, p53, nuclear factor-kappa B, p27) cell death programs.

This review focuses on recent clinical developments concerning UPS inhibitors, signaling pathways that are affected by down-regulation of

UPS activities, and apoptotic mechanisms promoted by drugs in this class that are used to treat lymphoma.

Keywords: Ubiquitin proteasome system; Lymphoma; Bortezomib; Carﬁlzomib; Marizomib; CEP-18770; Clinical trials-UPS inhibitors

1. Introduction

The homeostasis of cellular proteins is important for

∗

Corresponding author at: The Genomics and Biomarkers Program, John

maintaining the integrity and health of the cell, and active

Theurer Cancer Center, Hackensack University Medical Center, 40 Prospect

protein degradation by the ubiquitin-proteasome system

Avenue, Hackensack, NJ 07601, United States. Tel.: +1 201 996 8214;

(UPS) is the major pathway through which this cellular

fax: +1 201 336 8776.

E-mail address: [email protected] (K.S. Suh). balance is achieved (Fig. 1). This process is pivotal in

K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322 307

Fig. 1. Protein degradation pathway by ubiquitin-proteasome system.

cell cycle control, transcriptional regulation, signal trans- inhibitors that have been developed directly target the 20S

duction, antigen presentation, inﬂammation, ER-mediated proteasome.

degradation, membrane trafﬁcking, receptor endocytosis, The UPS plays an important role in regulating the cell

apoptosis, and development [1–4]. Ubiquitin (Ub), a 76- cycle. Proteins such as cyclins A, B, D, and E, CDK inhibitor

residue protein, is attached to substrate proteins via multiple p27, p21, transcription factor E2F, retinoblastoma (Rb), and

adenosine-5 -triphosphate (ATP)-dependent processes by E1 tumor suppressor p53 are regulated by proteasome-mediated

(Ub-activating), E2 (Ub-conjugating), and E3 (Ub-ligating) proteolysis. The blockage of cell cycle progression with pro-

enzymes. These enzymes act in harmoniously coordinated teasome inhibitors is currently used against various forms of

␬

fashion via the ubiquitin moiety to achieve speciﬁc targeting cancer. NF- B, which participates in immune and inﬂam-

and regulation of oligomerization, degradation, and post- matory responses, apoptosis, and cell proliferation, is also

translational modiﬁcation [5]. Proteasomes are abundant in regulated by the UPS. UPS-mediated protein degradation also

the cytosol, and consist of multiple proteins that form the generates antigenic peptides for presentation on MHC class I

20S and 19S subcomponent complexes. Proteasomal pro- molecules [7]. Consistent with this function, the aldehyde

tein degradation generally requires polyubiquitination, and class of proteasome inhibitors partly inhibits presentation

ubiquitinated proteins are ﬁrst recognized by the 19S cap of antigenic peptides [8]. The UPS is also manipulated

complex, which unfolds the substrate in an ATP-dependent and co-opted by certain viruses, including avian leuko-

process and translocates it into the proteolytic chamber of sis virus, human immunodeﬁciency virus type 1, simian

the 20S complex to be degraded [6]. The ubiquitin conju- immunodeﬁciency virus, Moloney murine leukemia virus,

gation cascade begins with activation of the carboxyl group and Epstein–Barr virus [9–11]. UPS is also involved in viral

of Gly-76 of ubiquitin by E1. Activated ubiquitin binds to assembly; proteolytic viral maturation is inhibited upon treat-

E1 and is transferred to a Cys residue of E2, which either ment of infected cells with proteasome-speciﬁc inhibitors

singly or in cooperation with E3 shuttles ubiquitin to a pro- [12,13]. Proteasome inhibitors are broadly categorized into

tein substrate. Subsequently, different combinations of E2 two groups (synthetic analogs and natural products), and their

and E3 enzymes add a polyubiquitin chain to provide selec- structural properties and mechanisms of action have been

tive tagging for degradation (Fig. 1). This process is used reviewed in detail.

to degrade misfolded or aged proteins, to regulate the cell Given the importance of proteasomal protein degradation

cycle through cyclin degradation, and to promote immune in various intracellular processes, inhibitors of this pathway

responses through antigenic peptide processing. Thus, com- will continue to serve as both molecular probes of major

ponents of the UPS pathway are attractive molecular targets cellular networks as well as potential therapeutic agents

for therapeutic intervention. For a variety of reasons, most for various human diseases [14]. This review focuses on

308 K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322

proteasome inhibitory mechanisms that are related to the cells in vitro and in patients with one of several types of

treatment of lymphoma, detailing current preclinical and clin- lymphoma [26–28]. Geminin inhibits DNA replication dur-

ical developments and describing apoptotic mechanisms that ing S, G2, and M phases by preventing the incorporation of

may underlie the antitumor activities of the inhibitors. MCM proteins into the prereplication complex, thus playing

an important role in DNA replication licensing. Geminin ubi-

quitination by the anaphase-promoting complex/cyclosome

2. Signaling pathways regulated by the UPS (APC/C) ubiquitin-protein ligase and subsequent degradation

by the 26S proteasome permits DNA replication to proceed

Numerous regulatory protein participants in cellular [29]. Unbalanced expression of geminin is associated with

processes undergo proteolytic degradation. The UPS has B-cell lymphomagenesis [30] and genomic instability, espe-

been targeted as part of the chemotherapeutic regimen cially in MCL with p53/ARF inactivation [31]. In addition,

for solid tumors and hematological malignancies, such as the overexpression of geminin has been proposed to play a

non-Hodgkin’s lymphoma (NHL). The roles of proteins or role in the pathology of acute leukemia [32].

molecular targets that are proteolytically degraded by the 26S Cyclins have been directly implicated in the pathogenesis

proteasome of the UPS in basic cellular activities (Fig. 1), in of lymphoma. Cyclin A is required for entry into mitosis and

lymphomagenesis, and in tumor progression are described S-phase progression, and is ubiquitinated and targeted for

below. proteasomal degradation by APC/C during late M to early

Cell cycle progression requires programmed and peri- G1 phase [33]. Bcl-1 (CCND1 and PRAD1) has been impli-

odic expression and degradation of speciﬁc proteins [15]. cated in the pathogenesis of multiple types of NHL, and its

Cyclins, cyclin-dependent kinases, and their inhibitors are protein product, cyclin D1, plays an important role in the

target proteins degraded by the 26S proteasome in an orderly transition from G1 to S phase in response to mitogens. Over-

and sequential manner to regulate the cell cycle [16]. G1/S expression of cyclin D1 shortens the G1 phase and reduces

phase progression requires coordination of positive regula- the dependency of the cell on extrinsic mitogenic signals.

tors (cyclins, cyclin-dependent kinases [CDKs], CDK-cyclin Defects in the proteasomal degradation of cyclin D lead to

complexes, E2F, and Cdc6) and negative regulators (CDK its accumulation within the cell and contributes to prolif-

inhibitors (CKIs) of the Cip/Kip and INK4 families). Dys- eration; the prototypical NHL with dysregulation of cyclin

regulation of these proteins may lead to tumor progression. D is MCL [34]. Cyclin E is also required for the onset of

Proteasomal degradation of p27 (Cip/Kip inhibitor) promotes DNA replication during late G1 phase and early S phase,

cell cycle progression from G0 to G1 [17], consequently ini- and is targeted for ubiquitination and subsequent proteaso-

tiating the early onset of S phase [18]. For example, high mal degradation [35,36]. Cyclin E overexpression has been

levels of cyclin E and low levels of p27 (Kip1) expres- observed in lymphoma [37], particularly DLBCL, and its

sion have been associated with malignant lymphomas in overexpression has been associated with poor response to

humans [19], and the loss of p27 (Kip1) decreases sur- standard treatment and inferior outcomes [38]. Of the non-

vival in murine Myc transgenic lymphoma models [20]. cyclin factors, E2F1 is required for the completion of the

An increase in proteasomal degradation of p27 was also G1-to-S-phase transition, and is degraded by the 26S protea-

associated with decreased overall survival in mantle cell some at the S-to-G2-phase transition after dissociating from

lymphoma (MCL) in a study of 157 NHL patients with lym- the Rb protein [39]. It has been proposed that dysregula-

phoproliferative disorders [21]. A more recent study of 671 tion of E2F plays an oncogenic role in sporadic Burkitt’s

diffuse large B cell lymphoma (DLBCL) patients treated lymphoma [40] and in lymphomagenesis in murine models

with rituximab and CHOP (cyclophosphamide, doxoru- [41]. The Cdc6 protein is a component of the prereplication

bicin/hydroxydaunomycin, vincristine/oncovin, prednisone) complex required to initiate DNA unwinding and replication

(R-CHOP) or without rituximab (CHOP) showed that low before S phase [42]. During completion of DNA replication

expression of p27 (Kip1) and increased Skp correlated in S phase, Cdc6 dissociates from DNA-bound replication

with poor overall and progression-free survival rates [22]. complexes and is ubiquitinated and targeted for proteasomal

Similarly, p19INK4d is governed by ubiquitination and degradation by the APC/C [43]. Cdc6 is also overexpressed

subsequent proteasomal degradation during cell cycle pro- in a subset of MCL, in which it is associated with poor clin-

gression [23]. Genetic defects in the tumor suppressor ical outcome [31], and is differentially expressed in primary

INK4a/ARF locus accelerate lymphomagenesis in an Emu- cutaneous large B-cell lymphoma [44]. In addition, a Cdc6

myc transgenic murine model, and cytogenetic alterations of G1321A polymorphism is associated with decreased risk of

the INK4a/ARF locus are directly related to drug responses NHL [45]. These observations suggest that a failure of the

in primary tumors [24]. The prognostic signiﬁcance of these UPS to effectively maintain optimal levels of intracellular

functional deletions is made evident by the poor progno- cyclins can result in hematological malignancies.

sis of subsets of MCL patients with higher levels of cyclin Terminal differentiation of eukaryotic cells requires

D1, which has been attributed to deletion of the INK4a/ARF withdrawal from the cell cycle, which is accomplished

locus (CDKN2A) [25]. Dysregulated expression or inacti- by the down-regulation of CDK activities during the G1

vation of p21 promotes cell cycle progression in lymphoma phase [46]. This mainly occurs in mammalian cells through

K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322 309

Ink4a

binding of CDKs to the CKIs of the INK4 family (p16 , blocking proteasomal degradation may enhance the activities

Ink4b Ink4c INK4d

p15 , p18 , and p19 ) and the Cip/Kip family of pro-apoptotic proteins rather than those of anti-apoptotic

WAF1/Cip1 Kip1 Kip2

(p21 , p27 , and p57 ) [47]. More recent stud- proteins. Emerging evidence regarding the proteins that con-

WAF1/Cip1

ies suggest that proteasomal degradation of p21 and stitute the apoptotic core machinery and upstream signaling

other cell cycle regulators, such as cyclin D1 and Cdc25A molecules has revealed multiple new opportunities for ther-

phosphatase, can promote differentiation of certain mouse apeutic intervention, some of which are already under

and human cells [48,49]. In aggregate, the data indicate investigation in clinical trials. With respect to core apopto-

that the 26S proteasome plays a complex role in cellular sis targets, for example, nuclease-resistant (phosphothioate)

differentiation [50]. antisense oligonucleotides directed against BCL-2 mRNA

Heat shock proteins (HSP110, HSP90, HSP70, and are currently in phase III trials for patients with multiple

HSP27) perform a crucial role as cytosolic molecular chap- myeloma (MM), and are in phase II trials for patients with

erones in regulating the balance between protein synthesis NHL, B-cell chronic lymphocytic leukemia (B-CLL), and

and degradation, stabilizing regulation of signal transduction acute myeloid leukemia. Small-molecule compounds that

pathways, and protecting cells from apoptosis in response mimic the BH3 domains of pro-apoptotic proteins have also

to cellular stress [51]. The role of HSP90 in tumorigenesis been described [60–62].

has been studied in vitro in lymphoma and leukemia models. The stability and activation of transcription factors are

HSP90 is commonly upregulated in high-grade or large-cell also regulated by proteasomal degradation. Dysregulated

NHL types of B- and T-cell lymphomas [52,53]. Immuno- expression of c-Myc is associated with many human can-

histochemical data also show that among B-cell lymphomas, cers including Burkitt’s lymphoma, and proteolysis of c-Myc

HSP90 is moderately to strongly expressed in Burkitt’s lym- is mediated by the UPS. Protein levels of c-Myc are over-

phoma (5/5, 100%) and in subsets of follicular lymphoma expressed up to 6-fold in Burkitt’s lymphoma-derived cell

(17/28, 61%), DLBCL (27/46, 59%), nodal marginal zone B- lines, and blocking of proteasomal degradation may enhance

␬

cell lymphoma (6/16, 38%), plasma cell neoplasms (14/39, c-Myc-mediated apoptosis. Aberrant, dysregulated NF- B

36%), small lymphocytic lymphoma/chronic lymphocytic activation is directly associated with several lymphoid malig-

leukemia (3/9, 33%), MCL (12/38, 32%), and lymphoplas- nancies, and blockage of proteasomal degradation of its

␬

macytic lymphoma/Waldenstrom macroglobulinemia (3/10, inhibitory I B proteins ablates the survival signals medi-

␬

30%) [54]. These observations identify a role of HSP90 in ated by NF- B and substantially shifts the balance to

the pathology of lymphomas, and suggest that HSP90 is a pro-apoptotic proteins [63,64].

viable therapeutic target of HSP inhibitors in lymphomas. In addition to the UPS, autophagy machinery is also

The proteasomal degradation pathway plays a role in the important for protein degradation in cells via a lysosome-

induction and suppression of apoptosis in eukaryotic cells dependent degradative pathway [65]. When proteasomes are

[55]. Among molecules that are regulated by proteasomal inhibited, autophagy is triggered as a compensatory mech-

degradation are members of the Bcl-2 family of anti-apoptotic anism of protein degradation through activation of the ER

and pro-apoptotic proteins. The anti-apoptotic Bcl-2 and stress-induced IRE1 pathway; thus, both systems are func-

Bcl-XL proteins are integral mitochondrial proteins that tionally coupled in protein degradation. Recent research

share sequence homology in all four Bcl-2 homology (BH1 investigating the role of autophagy in myc-induced lym-

TAM

to BH4) domains but vary in both structure and function. phoma derived from p53ER cells demonstrated the

Upon apoptotic stimuli, these proteins block apoptosis and signiﬁcance of autophagy inhibitors (chloroquine) in com-

preserve mitochondrial integrity [34]. Proteasomal degrada- bination with other therapies for successfully inducing

tion of Bcl-2 results in release of proapoptotic signals and apoptosis in malignant cells [66]. Similarly, in leukemia,

consequent promotion of apoptosis [56]. Another class of deregulation of autophagy points to the fact that autophagy

anti-apoptotic proteins includes the inhibitor of apoptosis inhibitors could be included in combination therapies for

(IAP) family (XIAP and c-IAP2), which inhibits the activa- suppressing cancer progression [65].

tion and enzymatic activity of caspases by targeting them for

ubiquitination [56,57]. IAPs auto-ubiquitinate via the RING 3. Current therapeutic options for lymphoma

ﬁnger domain, and subsequent 26S proteasomal degrada-

tion initiates IAP binding to ubiquitin-conjugating enzymes To treat NHL, the combination chemotherapy regi-

(UbCs, E2) [58]. In these cases, inhibition of proteasomal men CHOP is most commonly used [67]. Other treatment

degradation of IAP family members enhances proliferative combinations for NHL include common chemotherapy

potential of the treated cells. However, the mechanism under- agents such as chlorambucil, methotrexate, vinblastine,

lying this effect has not been well demonstrated. Similarly, etoposide, cytarabine, ﬂudarabine, and cladribine, and

pro-apoptotic members of the Bcl family (Bax, Bak, Bad, radioimmunotherapeutic agents including tositumomab and

Bim, Bik, and Bid) also undergo proteasomal degradation ibritumomab. For Hodgkin’s lymphoma (HL), which can be

[59], and inhibition of proteasomal degradation signiﬁ- cured in about 75% of cases overall and 90% of cases in

cantly increases apoptotic potential in treated cells. Thus, younger patients [68], a combination chemotherapy regimen

310 K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322

of ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) non-small cell lung carcinoma [80]. More recently, accumu-

is most commonly used. Radiation therapy may be used alone lating evidence has shown that the efﬁcacy of bortezomib

for treating HL, or may be combined with chemotherapy and can be enhanced through combination with chemother-

with stem cell transplantation after failure of primary therapy apy. When combined with doxorubicin-based chemotherapy,

[69]. For MCL, which is clinically aggressive with poor prog- bortezomib resulted in an 83% higher response rate in

nosis, rituximab and ASCT (Allogeneic/Autologous Stem relapsed/refractory activated B-cell-like DLBCL, and in a

Cell Transplant) prolongs progression-free survival. How- 13% higher response rate in relapsed/refractory germinal cen-

ever, for patients who are not ﬁt for intensive treatment, ter B-cell-like DLBCL [81]. In addition, a combination of

less intensive treatment with various combinations of ritux- bortezomib with irinotecan in a phase II clinical trial pro-

imab and other compounds, such as rituximab with CHOP or duced improvements in the ORR from 9% (bortezomib alone)

with bendamustine is recommended, and for relapsed MCL, to 44% (bortezomib/irinotecan) in advanced gastric cancer

ASCT can be effective if a compatible donor is available (n = 28) [82]. More recently, in a phase II trial of 63 refractory

(reviewed in Cortelazzo et al. [70]). In addition to con- and relapsing FL patients, bortezomib combined with ben-

ventional combination chemotherapy, proteasome inhibitors damustine and rituximab resulted in an 88% ORR (including

have been used to treat lymphomas. This class of compounds a 53% CR), with toxicity within manageable limits [83]. In

consists of synthetic and natural products and can be catego- a separate phase II clinical trial of 94 untreated advanced

rized into ﬁve different classes (A to E) based on chemical stage (III/IV) FL patients, bortezomib added to rituximab,

structure. cyclophosphamide, vincristine, and prednisone resulted in

an intent-to-treat outcome of 46 of 94 patients (49%; 95%

3.1. Class A compounds conﬁdence interval [CI] of 38.8–59.0%) who achieved a

CR or unconﬁrmed CR, and 32 of 94 patients (34%) who

Class A compounds are peptide aldehydes and include achieved a partial response, for an overall response rate of

MG 132 and MG 115. These compounds primarily inhibit 83% (95% CI of 75.4–90.6%) [84]. In this study (Table 1),

chymotrypsin-like activity through the formation of a cova- no grade 4 neurotoxicity was observed, although 5 patients

lent bond between the aldehyde and an N-terminal threonine developed largely reversible grade 3 neurotoxicity. Other

moiety in a reversible manner. The speciﬁcity of class A combinations that have shown encouraging results in hemato-

compounds is relatively low. Well-known peptide aldehyde logical malignancies include thalidomide and its derivatives,

inhibitors have been further refined for specificity by using flavopiridol (cyclin inhibitor), rapamycin (mTOR inhibitor),

peptide vinyl sulfones and peptide-boronic acid derivatives. and dexamethasone, doxorubicin, melphalan, and prednisone

These efforts have led to the development of highly potent [85,86]. In fact, a multicenter phase II trial of 29 evaluable

and speciﬁc drugs like PS-341 (bortezomib) [71] and CEP- relapsed/refractory indolent and mantle cell NHL patients

18770, which is currently being clinically evaluated (Table 1) treated with 90 mg/m of bendamustine on days 1 and 4;

2 2

(Fig. 2). 375 mg/m rituximab on day 1, and 1.3 mg/m bortezomib

on days 1, 4, 8, and 11, on six 28-day cycles resulted in

3.2. Class B compounds a 47% 2-year progression-free survival rate (15/29 patients

achieved complete response) [87]. In another phase II clin-

Class B compounds are synthetic peptide boronates, ical trial of 16 relapsed and chemotherapy-refractory MCL

and including benzamide, alpha-ketoamide, bortezomib, and patients treated with bortezomib combined with rituximab

Cbz-Leu-Leu-Leu-boronic acid. Bortezomib binds with high and dexamethasone (treatment cycle consisted of bortezomib

afﬁnity and speciﬁcity (yet reversibly) to the threonine [1.3 mg/m on days 1, 4, 8, and 11; six 21-day cycles], rit-

hydroxyl groups in the chymotrypsin-like active site of the uximab [375 mg/m on day 1], and dexamethasone [40 mg

proteasome ␤5 subunit and inhibits the peptidyl-glutamyl orally on days 1–4]) resulted in 13 patients (81.3%) achiev-

peptide-hydrolyzing activity of the ␤1 subunit. Bortezomib ing responses and 7 patients achieving complete responses

has shown promising activity and has led to durable responses (43.8%) [88]. In addition, in chemotherapy-naïve low-grade

as a single agent in relapsed or refractory MCL patients and NHL, bortezomib combined with rituximab, dexamethasone,

MM patients [72,73]. In a phase II trial (n = 202), the over- and cyclophosphamide (bortezomib given at 1.6 mg/m , on

all response rate (ORR) and complete response rate (CR) days 1, 8, 15, and 22 of every 35-day cycle; rituximab given

of patients with MM treated with bortezomib were 35% at 375 mg/m on the same days as bortezomib during cycle

and 4%, respectively, with a median overall survival of 16 1 and then only on day 1 in subsequent cycles; dexametha-

months [74]. Similarly, heavily pre-treated MCL patients sone given orally at 40 mg on days 1, 2, 8, 9, 15, 16, 22, and

(n = 36) responded to bortezomib favorably with an ORR of 23; and cyclophosphamide administered orally at 400 mg/m

46.5% and a CR of 16% [75]. While somewhat less success- on days 1–4) resulted in an ORR of 90% (CR of 54%),

ful, encouraging responses have also been seen in patients with a 75% (n = 9) survival rate at a 22-month follow-up

with DLBCL, follicular lymphoma (FL) [76], Waldenstrom [89]. In this study, there were no grade 3 or 4 periph-

macroglobulinemia [77], and in patients with a few solid eral neuropathies. In addition, results from in vitro studies

tumors including sarcoma [78], renal cell carcinoma [79], and with cells derived from primary effusion lymphomas showed

K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322 311 Status Completed Completed Completed Completed Completed Completed Completed Completed Recruiting

NCT NCT00030875 [154] NA NCT00038571 NCT00038571 NCT00636792 NCT00374699 [155] NCT00295932

ORR 46% 31% 58% 41% 19% 88% NA 75% NA

months

months months months months

years

TTP 10 15 11–24+ 24 24 NA NA 4 NA

PR NA NA 37% 18% 5% 35% NA 5% NA

CR NA 8% 7% 18% 15% 53% NA 95% NA

2 1 2 2 2 1–2 1 2

Phase Phase Phase Phase Phase Phase2 Phase Phase Phase Phase

(patients)

155 20 n 55 115 30 26 73 33/60 27/60 toxicity,

hematologic constipation,

neuropathy neuropathy peripheral

myalgia/edema

neutropenia, neutropenia, anorexia

nausea, peripheral peripheral toxicity Neuropathy, dyspnea, Neurological Toxicity Lymphopenia, thrombocytopenia NA NA Thrombocytopenia, fatigue, Thrombocytopenia, fatigue, neuropathy, Asthenia, Pancytopenia, thrombocytopenia, neutropenia 2 2 8, 8, 4, 1,

a a

21 of 8, and

4, 4, IV oral

of and 1,

the

1 each

4, 6 2

for for

22 5

mg/m

1, 1, mg/m Total 1, days

phase

days

IV 2–6, and

of (at 1,

is dose

5. every

for in

days 21 day1 therapy. 3-week 2

3 and

375 day 8. Days 15 days days

was

was courses

mg/m

days days

cycles cycles

days on 50–90 8 of and administered days

IV IV on 6 6 and

21 21 15,

cycle; on on

each 1 and 1.6 cycle

to 4,

every 1 1, on ) )

given i.v. on

8, repeats of of 2 and IV 2 2

duration

was cycle.

2 2 2 3,

bortezomib up

11 1

with every every 2,

rituximab lymphoma days days of

determined

days for 35-day

21-day

each

and mg/m mg/m 11 11 11 bortezomib Bortezomib Bortezomib

mg/m mg/m mg/m

on on days a

cycle

8 and

[B] administered each of prednisone levels MTD cycles bendamustine and administered rituximab 4 (1.5 (1.5 Dose maximum maximum on on cycle; and Treatment 1.3 1.3 Bortezomib IV IV R-CHOP Cyclophosphamide 8, treatment [A] 4, days I) NA 1.5 cycles, and and cycle cycles. administered

inhibitors lymphoma and cell

NHL

for

NHL

NK/T

DLBCL

indolent

B-cell proteasome

treatment

RR MCL, R/R-FL Condition MCL MCL Indolent-NHL T-cellor R/R Lymphomas MCL in

major

the

of CHOP R- R-CP

Inhibitors + + +

trials

Bendamustine Rituximab

+ CHOP +

Bortezomib Bortezomib Bortezomib Bortezomib Bortezomib Bortezomib Bortezomib Table Proteasome PI Bortezomib Clinical

312 K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322 Recruiting Completed Recruiting Recruiting Completed Recruiting Recruiting Completed Status

NCT00572637 NCT00893464 NCT00667082 NCT01111188 [109] NCT01276717 [156] NCT00396864 NCT

NA 3.5% NA NA NA ORR NA NA NA

NA NA NA NA NA NA NA TTP NA

NA NA 3.5% NA NA NA PR NA 3.5% http://clinicaltrials.gov/ (

trial

NA NA NA 3.5% NA None NA CR NA

clinical 1 1 1 1 1 1 1 1

Phase Phase Phase Phase Phase Phase Phase Phase Phase

number

NCT,

(patients)

55 34 29 50 40 30 24 30 n rate;

response

and

status balance

overall

fatigue

mental loss

ORR,

nausea NA NA NA NA NA Diarrhea, NA Toxicity change, Fatigue, progression; 8, 9

at a and 15,

9, to

2 will cycle

dose

8, 9, 2.4,

to 8, by

days

cycles. 2

infusion 1, 8,

dose 6 3,

time

while up

of on

be each

1.2, mg/m

days

to by 2,

and

min daily

15 at 17. of

days cycles. Starting mouth 1, cycles. 28 0.1 weekly

continuously days, up TTP,

and will

30 15 days bolus

followed 18

without

with on 13 by 13 8, and Bortezomib and

oral IV cohorts

days 2 28 days

2 2 2 days 11,

toxicities dose

4, 8,

every

cycle, cycle and 16

days 14 , mg/m

1, 1, on for

× 4.4, period 0332991 15, 15, Every

mg/m 12 response; 3

every

administered mg/m mg/m mg/m

Maximum 28-day limiting Starting be day Vorinostat vorinostat 20 20 21-day 4.6, for 10, Days Days daily 11, daily IV NPI-0052 16, treatment. PD Every Carﬁlzomib 0.7 15 0.125 intravenous Dose 15 maximum administered partial

PR,

lymphoma

for

MCL

response;

treatment Relapsed Lymphoma R/Lymphoma NHL Lymphoma MCL/MM R/R-Lymphoma Lymphoma Condition

complete )

CR,

Vorinostat PD0332991 :

+ Inhibitors +

Vorinostat

Continued ( +

18770

0052

CEP MLN9708 NPI-0052 NPI-0052 NPI Carﬁlzomib Carﬁlzomib Proteasome PI Bortezomib Abbreviations Table

K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322 313

Fig. 2. Chemical structures of major proteasome inhibitors in the treatment of lymphoma bortezomib, CEP 18770, marizomib, ixizomib and carﬁlzomib.

that treatment with bortezomib in combination with doxoru- assessed. Bortezomib was well tolerated with no grade 4

bicin and Taxol was schedule-dependent, and was additive toxicity. The most common grade 3 hematologic toxicities

with tumor necrosis factor-related apoptosis-inducing ligand were neutropenia (2 patients, 17%) and thrombocytopenia

(TRAIL) and synergistic with dexamethasone [90]. A recent (2 patients, 17%). However, no grade 3 or higher anemia

report demonstrated that cytarabine interferes with DNA was recorded. The most common non-hematologic toxic-

polymerases and incorporates into the genome during DNA ity was sensory neuropathy, which was observed in 50% of

replication, consequently arresting cell cycle progression patients. One patient (8%) reported grade 3 sensory neuropa-

[91]. Furthermore, cytarabine shows sequence-dependent thy but had complete recovery within six weeks. There was no

synergism with bortezomib in MCL [92]. A combination observation of GI toxicity or fatigue, perhaps due to inherent

of rituximab and bortezomib for treatment of MCL, FL, differences between diseases or differences in prior therapy.

and marginal zone lymphoma is under clinical development The safety proﬁle of bortezomib for use in lymphoma

[93]. treatment appears to be similar to previous experiences in

The safety of bortezomib has been demonstrated by clini- MM. During the ﬁrst phase II clinical trial, the Study of

cal trials (Table 4). One such study was a phase II, multicenter Uncontrolled Multiple Myeloma Managed with Proteasome

clinical trial of patients with relapsed or refractory MCL Inhibition Therapy (SUMMIT), bortezomib was adminis-

(PINNACLE study). In total, 152 patients were treated with tered at 1.3 mg/m IV on days 1, 4, 8, and 11 of a 21-day

bortezomib administered at 1.3 mg/m intravenously (IV) on cycle for a maximum of eight cycles [96]. Grade 3 toxicities

days 1, 4, 8, and 11 of a 21-day cycle for up to 17 cycles included thrombocytopenia (28%), peripheral neuropathy

[94]. The most common non-hematologic adverse effects of (12%), fatigue (12%), and neutropenia (11%). A second

grade 3 or higher were diarrhea (11 patients, 7%), fatigue (19 phase II trial, the Clinical Response and Efﬁcacy Study of

patients, 12%), and peripheral neuropathy (20 patients, 13%). Bortezomib in the Treatment of Relapsing Multiple Myeloma

The median onset of neuropathy was estimated at 12 weeks. (CREST) (Table 2), was an open-label, randomized trial

However, data on reversibility was not collected. The most of 54 patients who were previously treated for MM [97].

common hematologic toxicity was thrombocytopenia, which Patients were randomized to receive bortezomib at a dose of

2 2

occurred in 17 (11%) patients. Five (3%) patients died from 1.3 mg/m or 1 mg/m IV using the same dosing schedule as

bortezomib-associated causes, including non-neutropenic the SUMMIT trial (Table 2). The incidence of drug-related

sepsis and respiratory failure. The most common adverse adverse effects was considerably lower for patients receiv-

2 2

effects leading to treatment discontinuation were peripheral ing 1 mg/m versus 1.3 mg/m . The most common adverse

neuropathy (10%) and fatigue (6%). The safety of bortezomib effects were peripheral neuropathy (58% vs. 19%), diarrhea

was evaluated in another phase II clinical trial in patients (65% vs. 25%), nausea (62% vs. 46%), and vomiting (38%

with relapsed or refractory cutaneous T-cell lymphoma vs. 14%). The relatively common occurrence of peripheral

[95]. Patients were treated with bortezomib administered at sensory neuropathy associated with bortezomib necessitates

1.3 mg/m IV on days 1, 4, 8, and 11 of a 21-day cycle for up early detection, and recommendations for dose modiﬁcations

to six cycles. Fifteen patients were registered and 12 were are detailed in Table 3 [98].

314 K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322

Table 2

Combined safety data for pivotal phase II clinical trial results for patients with multiple myeloma at standard doses.

Study Dose Patients Toxicity ≥ 20% Overall (%)/Grade 3 (%)

1.3 mg/m 202 Nausea and vomiting 55%/6% and 27%/8%

Diarrhea 44%/7%

Fatigue 41%/12%

SUMMIT trial [96]

Thrombocytopenia 40%/28%

Peripheral neuropathy 31%/12%

Anemia 21%/8%

1.3 mg/m 28 Nausea 41%/0%

1 mg/m 26 Diarrhea 39%/4%

CREST trial [97] Fatigue 56%/7%

Thrombocytopenia 28%/22%

Peripheral neuropathy 35%/9%

Table 3

Prescribing information: recommended dose modiﬁcations for bortezomib-related neuropathic pain and/or peripheral sensory or motor neuropathy [97].

Severity of peripheral neuropathy Modiﬁcation of dose and regimen

Grade 1 (asymptomatic; deep tendon reﬂex loss or No action needed

paresthesia) without pain or loss of function

Grade 1 with pain or Grade 2 (moderate symptoms; limiting Reduce bortezomib dose to 1 mg/m

instrumental activities of daily living)

Grade 2 with pain or Grade 3 (severe symptoms; self-care Withhold bortezomib therapy until symptoms of toxicity resolve; after symptoms resolve,

limiting activities of daily living) reinitiate bortezomib at 0.7 mg/m and change treatment schedule to once weekly

Grade 4 (life-threatening consequences with urgent Discontinue bortezomib treatment

intervention indicated; permanent sensory loss)

A number of conclusions and recommendations can be during cycles 1–2. Nausea and vomiting may require the use

drawn from these trials. Both of these myeloma trials con- of anti-emetics; diarrhea may be controlled with antidiar-

ﬁrm that thrombocytopenia is not associated with serious rheal medications, and constipation can be managed with

bleeding events (Table 2), although one patient developed stool softeners and laxatives (Table 4).

an episode of gastrointestinal bleeding with grade 3 throm- There have been a number of attempts to identify biomark-

−1

bocytopenia (platelet count < 50,000 mL ). Anemia and ers to predict responses to bortezomib to spare a population

neutropenia associated with bortezomib are usually not prob- of patients that is unlikely to respond favorably to the drug.

lematic. Fatigue usually occurs during cycles 1–2 and may A few studies showed that antitumor activity of bortezomib

persist for several cycles before subsiding. Treatment should is greater against tumors with an NF-␬B mutation and cyclin

be withheld at the onset of any treatment-associated grade D overexpression (JNCI). Such pre-selection may potentially

3 fatigue. Once symptoms resolve, bortezomib therapy may improve ORR while sparing unnecessary exposure to therapy.

be reinitiated with a 25% dose reduction. Bortezomib does

not appear to be directly cytotoxic to most normal bone 3.3. Class C compounds

marrow cells or to destroy progenitor cells [99]. Complete

blood counts should be performed prior to bortezomib dose Class C compounds are the ﬁrst natural proteaso-

administration. Dose adjustments for bortezomib-associated mal inhibitors discovered and contain ␤-lactone structures.

hematologic toxicity are detailed in Table 4. Gastrointestinal More recent experiments have demonstrated that lacta-

adverse events occur frequently, with the most common being cystin blocks cell-cycle progression, inhibits degradation

nausea, diarrhea, constipation, and vomiting. These gastroin- of KIP1, and induces apoptosis of activated B-CLL cells

testinal disturbances can occur at any time during treatment, with little effect on resting B-CLL cells [3]. NPI-0052

but in clinical trials they have occurred most frequently (marizomib; Fig. 2) was originally isolated from marine

Table 4

Prescribing information: recommended dose modiﬁcations for bortezomib-related hematologic toxicities [97].

Hematologic toxicity Modiﬁcation of dose

−3

Platelets ≤30,000 mm or absolute neutrophil count Withhold bortezomib; if several bortezomib doses in consecutive cycles are withheld, reduce

−3 2 2 2

≤750 mm on bortezomib treatment day(s) (except day 1) dose 1 level (1.3 mg/m /dose reduced to 1 mg/m /dose; 1 mg/m /dose reduced to 2 0.7 mg/m /dose)

−3 2

Grade 4 hematological toxicity: platelets ≤25,000 mm or Withhold until toxicity resolved; may reinitiate with a 25% dose reduction (1.3 mg/m /dose

−3 2 2 2

absolute neutrophil count ≤500 mm on bortezomib reduced to 1 mg/m /dose; 1 mg/m /dose reduced to 0.7 mg/m /dose)

treatment day(s)

K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322 315

bacteria. It resembles lactacystin, and is used largely for exhibited grade 3 thrombocytopenia, thus establishing

cancer treatment. However, unlike bortezomib, NPI-0052 15 mg/m as the maximum tolerated dose (Table 3). Anti-

irreversibly inhibits the three major enzymatic activities tumor responses were observed at doses ≥11 mg/m . More

(chymotrypsin-, trypsin-, and caspase-like) of the 20S pro- than one-third of patients experienced grade 1 or 2 non-

teasome [71,100]. When compared to bortezomib, NPI-0052 hematologic toxicities including diarrhea, fatigue, and nausea

exhibits an improved therapeutic ratio and signiﬁcant activity in the absence of grade 3–4 peripheral neuropathy, whereas

in hematologic malignancies and solid tumors, includ- 48% (n = 14) of patients experienced grade 3–4 toxicity, thus

ing bortezomib-resistant MM. To overcome inherent and establishing the reduced incidence of neuropathy associ-

acquired bortezomib resistance and for broader anti-cancer ated with carﬁlzomib [109]. Co-administration of carﬁlzomib

effects, NPI-0052, with its structural and pharmacological (subtoxic or minimally toxic levels) in combination with

uniqueness, is being tested both as a single agent and as com- the histone deacetylase inhibitor vorinostat potentiates the

bined with biologics and targeted therapeutic agents (Table 4) activities of carﬁlzomib including mitochondrial injury, cell

[101]. This marine-derived, orally active, broad-spectrum cycle arrest, and caspase activation, leading to apoptosis in

proteasome inhibitor has shown potent antitumor activity in both germinal-center B cell-like DLBCL and activated B

leukemia in vitro and in a leukemic mouse model in vivo cell-like DLBCL [110]. Given the promising results of com-

[102,103]. Interestingly, combining NPI-0052 with either binatorial drug administration with proteasome inhibitors and

MS-275 or valproic acid (VPA) induced greater levels of cell histone deacetylase inhibitors, a phase I clinical trial has been

death than the combination of bortezomib with these histone established for carﬁlzomib (PR-171) in combination with

deacetylase (HDAC) inhibitors [102]. A phase II clinical trial vorinostat (SAHA) in patients with relapsed/refractory B cell

(n = 30) demonstrated that NPI-0052 was well tolerated and lymphomas [111] (Table 1).

inhibited caspase- and trypsin-like activities with 51% and

72% efﬁcacy, respectively, which led to stable disease in 31%

of cases in MCL, HL, FL, and other solid tumors [104]. Simi- 3.5. Class E compounds

lar to bortezomib, NPI-0052 increased cytotoxic effects when

used in combination with 5-ﬂuorouracil, CTP-11, Avastin Class E compounds are macrocyclic vinyl ketones

(bevacizumab), leucovorin, or oxaliplatin in tumor xenograft that irreversibly inhibit proteasomal activity. While small-

models (Fig. 2) [105]. Due to the greater potency of NPI-0052 molecule inhibitors in general suffer from poor pharma-

compared to bortezomib in increasing apoptotic potential in cokinetics due to rapid renal clearance, more potent and

CLL and in preclinical settings [106], further clinical devel- sustained effects can be expected from these newly developed

opments are expected. irreversible proteasomal inhibitors.

As a part of developing combination therapy for hemato-

3.4. Class D compounds logical malignancies, the BH3- mimetic ABT-737 was found

to exhibit synergistic potential in combination with borte-

Class D proteasomal inhibitors have epoxyketone struc- zomib against CLL, MCL, and DLBCL. Among these, MCL

tures. The efﬁcacy of the class D compound carﬁlzomib is showed the highest sensitivity toward ABT-737 and DLBCL

under investigation in clinical trials (Table 1). Carﬁlzomib showed the least [112]. Currently ABT-737 is in a clinical trial

(Fig. 2) is an irreversible proteasomal inhibitor that primarily in oral form as ABT-263, which exhibits high-afﬁnity binding

inhibits chymotrypsin-, trypsin-, and caspase-like activities to Bcl-2 and Bcl-xL, thus inducing apoptosis by preventing

of the proteasome. It has equal potency but greater selectiv- the sequestration of proapoptotic molecules in hematological

ity for the chymotrypsin-like activity of the proteasome than malignancies, including MM [113,114]. Similarly, the anti-

bortezomib, causing stronger cytotoxic effects and enhanc- tumor effects and sensitivity of tumor cells to ABT-737 have

ing cell cycle arrest and apoptosis in vitro. In animal studies, been assessed in six MCL cell lines and in primary MCL cells

carﬁlzomib-mediated inhibition of proteasome activity was (n = 13); the results showed that ABT-737 induces apoptosis

high low

found to occur in a dose-dependent manner in all tissues in MCL cells that express a Bcl-2 /Mcl-1 proﬁle [115].

examined, with the exception of the brain [71]. Epoxomicin This study shows that down-regulation of Mcl-1 enhances

is a selective, irreversible inhibitor of the chymotrypsin-like the effects of ABT-737, and indicates that the Bcl-2/Mcl-1

activity of the proteasome, which promotes proapoptotic proﬁle of the tumor should guide the use of ABT-737 in the

activity in anaplastic large-cell lymphoma cell lines [107] treatment of MCL.

and in Bcl-2-overexpressing recombinant Jurkat cells in vitro In addition to the use of drugs that inhibit proteasomal

[108]. Carﬁlzomib is being evaluated in early clinical trials activity, other enzymes such as E3 ligase or de-ubiquitylating

and in a phase I trial in patients with refractory and relapsed enzymes may be additional, alternative molecular targets

hematologic malignancies, including MM and MCL. In the for effectively inhibiting the proteasomal degradation path-

latter trial (n = 29), carﬁlzomib was administered every day way [116]. The ubiquitin-like molecules represent another

for 5 consecutive days every 14 days at 1.2, 2.4, 4.6, 4.4, family of enzymes of interest for drug development. Numer-

11, 15, and 20 mg/m . No dose-limiting toxicities were ous substrates for SUMO and NEDD8 have been identiﬁed

2 2

observed up to 15 mg/m . At 20 mg/m , two of ﬁve patients as drugable targets by proteomics approaches [71]. Major

316 K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322

proteasomal inhibitors currently being evaluated in clinical a recent study analyzing 18 MCL samples including cell

trials are listed in Table 1. lines, Roué et al. demonstrated that loss of sensitivity of

Similarly, HDAC inhibitors have been reported to have a MCL cells to proteasome inhibitors is related to the up

synergistic effect in MCL therapy when combined with pro- regulation of prosurvival chaperone BiP/Grp78 and to the

teasomal inhibitors, where the combination induces higher stabilization of this protein by increased chaperoning activ-

cytotoxicity and cell death. Studies on interactions of SAHA ity of Hsp90 (Heat shock protein 90 kDa). Knocking down

(HDAC inhibitor) with bortezomib in MCL reveal that the of BiP/Grp78 coupled with Hsp90 inhibitor IPI-504 (class:

combination of both drugs synergistically induces apoptosis ansamycin) in combination with bortezomib resulted in apo-

in MCL cells [117]. ptosis of MCL cells. Results of this study further indicate that

in MCL cultures and tumors the combination of bortezomib

and IPI-504 can overcome both intrinsic and acquired resis-

4. Unfolded protein response (UPR) tance via dissociation of Hsp90/BiP complexes, BiP/Grp78

depletion, inhibition of UPR, and thus, enhanced cell death,

Cellular stress can cause disruption in peptide processing suggesting the effectiveness of this combination in treat-

in endoplasmic reticulum either by altering Calcium levels ment of patients with MCL refractory to bortezomib therapy

or by interfering with glycosylation and disulﬁde bond for- [124].

mation, leading to the induction of a cascade of intracellular

stress signaling—the UPR (unfolded protein response)—due

to the accumulation of the unfolded proteins. Induction of 5. Apoptosis

the UPR results in a reduction in general protein synthesis,

an increase in clearing misfolded proteins, and a restoration Proteasome inhibitors mediate cell cycle arrest and apo-

of peptide processing in the ER lumen. IRE-1 and ATF6 ptosis by causing dysregulation of a number of proapoptotic

can induce UPR since they are ER proximal stress sensing proteins in lymphoma cells [125]. Earlier studies reported

molecules [118,119]. In stressed normal cells, UPR ame- the accumulation of p53 in cells treated with proteasome

liorates protein misfolding and helps the cells to survive inhibitors, causing up regulation of the p53-inducible gene

the stress. However, in tumorigenesis, UPR plays a signif- products p21 and Mdm-2[126]. In normal cells, the RING

icant role in maintaining malignancy and chemoresistance domain of Mdm2 or Hdm2 (the human counterpart of Mdm2)

of malignant cells by upregulating the expression of XBP1, binds to the tumor suppressor p53, and an E3 ubiquitin ligase

inducing ATF4 and CHOP (C/EBP homologous protein), and then targets p53 to enhance its rapid degradation [127]. Upon

activating ATF6 [120]. proteasome inhibitor treatment, p53-Mdm2 binding is inter-

Recent studies have revealed that proteasomal inhibitors rupted; p53 becomes phosphorylated, and initiates proapop-

(e.g., bortezomib) induce endoplasmic reticular stress and totic signaling [128]. Disruption of the p53–Mdm2 interac-

UPR in MM cells. The intensity of the response to borte- tion leads to defective apoptosis [129], and overexpression of

zomib depends on levels of XBP1 [121]. Also, proteasomal wild type p53 increases sensitivity to proteasome inhibitors

inhibitors cause accumulation of unfolded proteins in tumor in Burkitt’s lymphoma, suggesting that effective apoptosis

cells, and trigger proapoptotic signaling leading to cell death mediated by proteasome inhibitors requires an intact p53

[120]. In MM cells, bortezomib and tunicamycin activate pathway [130]. In contrast, recent reports indicate that borte-

ER stress speciﬁc PERK (PKR-like ER kinase) in less than zomib induces apoptosis by mitotic catastrophe independent

an hour. Similarly, constitutive expression of physiologic of p53 activity in B-cell lymphoma cells in vitro [72].

UPR genes is necessary in MM cells since they produce Proteasome inhibitors activate apoptosis in Lymphoma

signiﬁcant amounts of immunoglobulin which causes them cells. Two major pathways are involved in apoptosis-

to be inherently sensitive to proteasome inhibitors, espe- associated caspase activation in mammalian cells: the

cially bortezomib, which selectively induces apoptosis in extrinsic death receptor pathway, which is regulated by mem-

MM cells. However; MM cells constantly express ER stress bers of the death receptor superfamily, and the intrinsic

survival proteins necessary to support their role as secre- mitochondrial pathway, which is associated with extracel-

tory cells; hence, they have a lower threshold for proteasome lular cues and internal insults [131]. The active form of

inhibitor-induced UPR induction and ER stress-induced apo- caspase-3 is necessary to cleave the other initiator caspases,

ptosis [122]. Moreover, as detailed analysis of the role of which ultimately leads to the cleavage of many cellular pro-

UPR in balancing apoptosis of cancer cells, dormancy, and tein substrates and apoptosis. It has recently been shown that

invasive growth and increasing susceptibility of tumors to caspase-3 is degraded by the 26S proteasome. Proteasome

chemotherapeutic agents has shown (reviewed earlier by Ma inhibitors induce the accumulation of caspase-3 subunits and

and Hendershot [123]), prolonged activation of UPR could enhance apoptosis induced by overexpression of pro-caspase-

result in apoptosis. 3 [132]. Another indication that caspase-3 is targeted by

Similarly, inhibition of UPR plays a signiﬁcant role ubiquitination for degradation is that monoubiquitination or

in reducing the resistance of MCL cells to proteasome diubiquitination of caspase-3 subunits was seen after protea-

inhibitors, thereby increasing the efﬁcacy of treatment. In some inhibitor treatment of cells [133,134].

K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322 317

␬

TRAIL has the ability to induce apoptosis in cancer cells pathways in the cell including (1) the inhibition of NF- B,

with little or no toxicity to normal cells. Death receptor 5 which contributes to many features of the malignant phe-

(DR5; also called TRAIL-R2, Apo2, TRICK2, or KILLER) notype, (2) maintenance of p27 and p21 levels, which are

is a receptor for TRAIL. In fact, preclinical studies in mice cell cycle-dependent kinase inhibitors, (3) accumulation of

provided the ﬁrst evidence that the soluble form of recom- proapoptotic proteins in the mitochondria, and (4) effects on

binant TRAIL would suppress the growth of human tumor cytokines and cell adhesion molecules [147]. The UPS tar-

xenografts with no apparent systemic toxicity [135]. DR5 gets the p27 protein in MCL, whereas, in FL and aggressive

mediates TRAIL-induced apoptosis through the interaction lymphoma, NF-␬B is targeted.

with adapter proteins, such as FADD, and through caspase Proteins like p21 and p27 are members of the Cip/Kip

activation [136]. Ubiquitin ligases bind to caspases, resulting family of cyclin-dependent kinase inhibitors that promote

in the degradation of these caspases and possibly tumori- cell cycle progression at the G1-S phase junction by inac-

genesis. However, proteasome inhibitors can induce DR5 tivating cyclin/cdk complexes [147,148]. These proteins are

expression and sensitize tumor cells to TRAIL-induced apo- substrates for the UPS, and drugs that affect this pathway

ptosis [137–139]. could lead to the accumulation of regulatory proteins that

Proteasome inhibitors also induce the activation of Bax, a could cause cell cycle arrest. Most cases of MCL (91/112)

member of the proapoptotic Bcl-2 family, which is required and DLBCL (12/19) have been shown to have lost expres-

for apoptosis [140]. As a result of apoptotic signaling and sion of p27 [21,149], but other subtypes of NHL such as

caspase activation, the BH3 domain of Bax dimerizes and small lymphocytic lymphoma and extranodal marginal zone

becomes resistant to proteasomal degradation. Bax then lymphomas have not been found to have any loss of p27

translocates into the mitochondria, which leads to the release expression [149].

of proapoptotic mitochondrial factors such as cytochrome c Molecular and in vivo protein degradation assays showed

resulting in apoptosis [139]. that MCL exhibited normal p27 mRNA expression but

Proteins known as ␤-transducin repeat-containing pro- increased p27 protein loss. Correlation of the p53 and p27

teins (␤-TrCPs) play a role as substrates of E3 ubiquitin defects with clinical data from patients with MCL indicates

ligases that control stability of important regulators of the cell that patients with both defects exhibit a signiﬁcant reduc-

cycle and signal transduction [141]. One of the major mech- tion in overall survival. The explanations for the loss of p27

anisms of the anticancer effects of proteasomal inhibitors is might be (1) the possible sequestration of p27 by cyclins

thought to be the suppression of prosurvival NF-␬B due to sta- D1 and D3, which are overexpressed in MCL and (2) an

bilization of its inhibitor I␬B [142]. Proteasomal degradation over-accumulation of Skp2, which is a part of the p27Kip1

of I␬B requires phosphorylation-dependent ubiquitination, ubiquitin ligase (E3) combination that plays a signiﬁcant role

which is mediated by ␤-TrCPs [143,144]. Thus, ␤-TrCP func- in the degradation of the CDK inhibitor [147,149]. High

tion is essential for the induction of NF-␬B transcriptional levels of Skp2 correlate with greater E3 enzyme activity,

activities, which play a key role in proliferation and survival which ultimately leads to proteasome-mediated degradation

of cancer cells and are often augmented in human cancers of the target protein p27. In large B-cell lymphomas and

[141]. blastic MCL, elevated Skp2 levels were signiﬁcantly asso-

␬

NF- B activation contributes to many aspects of tumor ciated with a low p27Kip1 level, indicating that increased

development, such as accelerated cell cycle progression, cell proteasome-mediated degradation of p27Kip1 contributes

proliferation, tumor initiation, and metastasis. As a major to the malignant phenotype [149], an observation in MCL

antiapoptotic factor, NF-␬B is involved signiﬁcantly in the that provides a powerful rationale for the use of proteasome

resistance of tumors to chemotherapy and radiation. Constitu- inhibitors in lymphoma [147].

tive activation of NF-␬B is seen in many human malignancies, FL is characterized by the translocation of the Bcl-2 proto-

including breast cancer [145]. Furthermore, ␤-TrCP1 and oncogene from chromosome 18q21 to the immunoglobulin

␤-TrCP2 proteins seem to play a redundant role in ubiquitin- heavy chain locus at chromosome 14q32 [150]. This

ation and degradation of I␬B. Also, expression of ␤-TrCP2 is translocation t(14:18) leads to the overexpression of the anti-

induced in human breast cancer cell lines and primary tumor apoptotic Bcl-2 protein, which is driven by NF-␬B, protecting

samples [143,146]. cells from apoptosis. Furthermore, NF-␬B overexpression

Targeting ␤-TrCP for inhibition with an RNAi approach is seen in lymphoma cells carrying t(14:18), and it has

or forced expression of a dominant-negative ␤-TrCP mutant been demonstrated that cell lines expressing an I␬B␣ super-

suppresses growth and survival of human breast cancer cells. repressor exhibit decreased levels of Bcl-2 protein, indicating

In addition, inhibiting ␤-TrCP augments the antiproliferative a role for NF-␬B in cells harboring this translocation. Since

effects of anticancer drugs such as doxorubicin, tamoxifen, proteasome inhibitors are employed in FL, a relationship

and paclitaxel on human mammary tumor cells. These data between NF-␬B and the apoptotic pathways is suggested in

indicate that targeting ␤-TrCP is a likely beneﬁcial approach FL [147,151]. Also, limited effects of proteasome inhibitors

for anticancer therapies [141]. have been seen in DLBCL, whereas some evidence suggests

Bortezomib is capable of inhibiting the 26S protea- that NF-␬B helps to deﬁne the chemotherapy-resistant post-

some, and has the ability to target a number of different GC B-cell lymphomas [152].

318 K.S. Suh et al. / Critical Reviews in Oncology/Hematology 87 (2013) 306–322

In hematological malignancies, increased levels of TNF-␣ generation proteasome inhibitors such as carﬁlzomib, mari-

result in poor prognosis, and this in turn heightens the risk zomib, MLN9708, CEP-18770, and ONYX-0912 along with

of MM and other cancers such as hepatocellular carcinoma, targeted agents such as heat shock protein inhibitors, histone

gastric carcinoma, and breast and bladder cancer. Promyelo- deacetylase inhibitors, and AKT inhibitors may also drive

cytic leukemia protein (PML) is a known molecular marker lymphoma therapy in a promising direction. Bortezomib

for cancer, and since PML regulates cell growth, DNA repair, in combination with widely used chemotherapeutic drugs

and cell death, patients with normal expression of PML and such as dexamethasone, rituximab, ifosfamide, carboplatin,

P53 have a better survival chance compared to patients with melphalan, doxorubicin, thalidomide, lenalidomide, and

poor expression of PML and P53 [153]. etoposide is being evaluated under clinical trials for MCL,

MM, WM (Waldenstrom macroglobulinemia), and other

hematological malignancies (http://www.clinicaltrials.gov/).

6. Conclusion However, there are challenges and limitations regarding

toxicity proﬁles of proteasome inhibitors when used in com-

Multiple therapeutic targets based on proteasomal inhibi- bination therapy such as peripheral neuropathy, fatigue,

tion have been identiﬁed in hematological malignancies such nausea, anxiety, insomnia, and transient cognitive changes.

as MCL, NHL, Hodgkin’s lymphoma, MM, CLL, FL, ALL, The future of proteasome inhibition in lymphoma therapy

and DLBCL. As alternatives to conventional combination resides in successfully managing these challenges, improv-

chemotherapy, proteasome inhibitors have been used to treat ing safety and tolerability, and decreasing chemotherapy

lymphomas as single agents or in combination with HDAC resistance for the betterment and prolonged survival of

inhibitors or BH3 family inhibitors. Proteasome inhibitors patients.

consist of synthetic and natural products, and belong to ﬁve

different classes based on chemical structure (A to E). It has

been shown that proteasome inhibitors enhance antiprolif- Reviewers

erative, proapoptotic, antitumor, and antiangiogenic effects

of conventional therapeutic agents in many hematologi- Samir Parekh, Albert Einstein Cancer Center, Jack adn

cal malignancies. Among proteasome inhibitors, bortezomib Pearl Resnick Campus, 1300 Morris Park Avenue, Bronx,

(a class B drug) was the ﬁrst to receive FDA approval NY 10461, United States.

– against MCL (for patients who had received one prior Andrés J. Ferreri, M.D., Fondazione IRCCS, START

therapy) and MM (as an initial treatment). Substantial clin- Project, Via Venezian 1, IT-20133 Milan, Italy.

ical beneﬁts of bortezomib treatment have been observed

in patients with MCL (relapsed or refractory). Further-

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University Medical Center in Hackensack, New Jersey. He

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Andrew Pecora, MD, is a vice president of cancer services

events that predicts survival in mantle cell lymphoma. Cancer Cell

and Innovation ofﬁcer of the John Theurer Cancer Center of

2003;3:185–97.

the Hackensack University Medical Center. He is a certiﬁed

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Translational Cancer Research in the John Theurer Cancer

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Center of the Hackensack University Medical Center. He

zomib plus R-CHOP in previously untreated patients with

is the chief of lymphoma division and an internationally

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