Trial Watch: Proteasomal inhibitors for anticancer therapy

Abstract

The so-called “ubiquitin-proteasome system” (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients.

Keywords:

• dexamethasone
• immunomodulatory drugs
• ixazomib
• marizomib
• oprozomib
• rituximab

Abbreviations

ALL	=	acute lymphoblastic leukemia
AML	=	acute myeloid leukemia
CLL	=	chronic lymphocytic leukemia
DUB	=	deubiquitinase
DLBCL	=	diffuse large B-cell lymphoma
ER	=	endoplasmic reticulum
FBW7	=	F-box and WD repeat domain containing 7
E3	=	ubiquitin protein ligase
FDA	=	Food and Drug Administration
HDAC	=	histone deacetylase
HCC	=	hepatocellular carcinoma
MCL	=	mantle cell lymphoma
MDS	=	myelodysplastic syndrome
MM	=	multiple myeloma
NHL	=	non-Hodgkin's lymphoma
NSCLC	=	non-small cell lung carcinoma
RNF	=	ring finger protein
TCL	=	T-cell lymphoma
UBE2	=	ubiquitin-conjugating enzyme E2
UPS	=	ubiquitin proteasome system
USP	=	ubiquitin specific peptidase
WM	=	Waldenström's macroglobulinemia

Introduction

The term “ubiquitin–proteasome system” (UPS) is generally used to refer to a multienzymatic machinery that mediates the physiological turnover of short-lived proteins, as well as the removal of misfolded, and hence potentially toxic, polypeptides.^1-3 This process is generally initiated by polyubiquitination, a reversible post-translational modification whereby several copies of ubiquitin, a small (76 residues, 8.5 kDa) highly-conserved polypeptide present in all eukaryotic cells, are covalently conjugated to target proteins.⁴ In general terms, ubiquitination relies on 3 distinct classes of enzymes: (1) ubiquitin-activating E1 enzymes, which catalyze an ATP-dependent reaction that generates a high-energy ubiquitin–adenylate intermediate;⁵ (2) ubiquitin-conjugating E2 enzymes, to which activated ubiquitin is attached to form an E2–ubiquitin thioester intermediate;⁶ and (3) E3 ligases, which transfer ubiquitin from E2 intermediates to specific lysine residues on target proteins.⁷ The human genome appears to encode 1-2 E1, approximately 40 E2, and more than 500 putative E3 enzymes.⁸ When ubiquitination involves previously attached ubiquitin molecules (which contain several lysine residues), target proteins are tagged with multimeric ubiquitin chains, often (but not always) acting as a recognition signal for proteolytic degradation by the 26S proteasome.⁹

The 26S proteasome is a multicomponent enzymatic complex composed of 1 or 2 19S regulatory cap subunits and a central 20S catalytic core.^1,10-14 The 19S subunit is a ring-shaped particle that recognizes polyubiquitinated proteins and promotes either their ATP-dependent unfolding^3,15 or the dismantling of ubiquitin chains, a reaction catalyzed by proteasome-associated deubiquitinases (DUBs).^16-18

The 20S subunit is a cylindrical pore consisting of 4 (2 α and 2 β) stacked rings composed of 7 subunits, 3 of which—β1, β3, and β5—are endowed with caspase-, trypsin-, and chymotrypsin-like enzymatic activities respectively. The 20S catalytic core hence mediates the nonspecific cleavage of polyubiquitinated proteins that have been unfolded by the 19S regulatory caps into small peptides and amino acids.^19-23 A detailed description of the regulation of the UPS, the pathophysiological relevance of alternative ubiquitin linkages (e.g., monoubiquitination, linear polyubiquitination), and deubiquitination reactions goes beyond the scope of this Trial Watch and can be found in several recent reviews.^1,2,6,24-36

Besides playing a critical role in protein quality control, the UPS also regulates the abundance, enzymatic activity, and intracellular localization of several proteins involved in cellular processes as diverse as gene expression, cell cycle progression, differentiation, cell death, macroautophagy (hereafter referred to as autophagy), endocytosis, metabolic adaptation, antigen presentation, and inflammatory signaling.^24,32,37-51 Thus, the UPS resembles autophagy in that it is essential not only for the maintenance of cellular homeostasis in physiological settings, but also for adaptive responses to exogenous alterations of the intra- or extracellular microenvironment.^52-57 Underscoring the importance of the UPS for the preservation of normal cellular functions, defects in the 26S proteasome and defects in E1, E2, or E3 ligases have been associated with several human disorders, including metabolic, cardiac, autoimmune, neurodegenerative, and neoplastic processes.^27,58-62 The survival and proliferation of transformed cells, however, critically rely on an intact UPS,^63-69 possibly reflecting the phenomenon known as “non-oncogene addiction."^70,71 Indeed, the activation of oncogenic pathways and the adverse microenvironmental conditions frequently encountered by growing neoplasms render malignant cells “addicted” to gene products and molecular systems that are not tumorigenic per se, such as members of the heat-shock protein (HSP) family, the autophagic machinery, and the UPS.^63,67,70-74 Targeting non-oncogene addiction represents a novel therapeutic paradigm with potentially high selectivity for cancer cells, stemming from the fact that normal tissues generally do not face adverse conditions, and hence do not rely on the continued activation of adaptive stress responses.^{63,67,70,71,75-77}

Today, 2 inhibitors of the 26S proteasome are approved by the US Food and Drug Administration (FDA) for use in humans: bortezomib and carfilzomib (source: http://www.fda.gov/). Bortezomib was approved for the treatment of relapsed multiple myeloma (MM) as early as in 2003^78-82 and its indications have now been extended to MM patients in general as well as to individuals with mantle cell lymphoma (MCL) who have received at least one prior therapy.^80,83,84 Carfilzomib is currently licensed for use in subjects with MM who have received at least 2 prior therapies, including bortezomib and an immunomodulatory agent, and have demonstrated disease progression on or within 60 d of completion of the last therapy.^80,85-88

Milestone discoveries that have provided more precise insights into the UPS and fostered its exploitation as a target for anticancer therapy include: (1) the original description of a non-lysosomal pathway responsible for the clearance of intracellular misfolded proteins;⁸⁹ (2) the molecular characterization of the UPS as an ATP-consuming machinery that catalyzes the covalent ligation of ubiquitin to intracellular proteins for rapid proteolysis,^90-92 a discovery that earned the 2004 Nobel Prize for Chemistry to the Israeli biologist Aaron Ciechanover, the Israeli biochemist Avram Hershko, and the American biologist Irwin A. Rose;⁹³ (3) identification of the 26S proteasome as the multisubunit component of the UPS that recognizes and degrades polyubiquitinated proteins;^94-96 and (4) the clinical development of bortezomib.^{78,79,81,97-100}

As part of our monthly Trial Watch series,^101-106 in this article we describe the impact of the UPS on oncogenesis and tumor progression, followed by a critical discussion of recent clinical trials investigating the use of proteasome inhibitors in cancer patients. Of note, the robust clinical activity of thalidomide, lenalidomide, and pomalidomide, which are collectively referred to as immunomodulatory drugs, also relies (at least in part) on inhibition of the UPS.^107,108 The clinical development of these agents has been summarized in a recent Trial Watch,^109,110 and will not be discussed further here.

Alterations of the UPS in Cancer

Accumulating evidence links alterations in the UPS to oncogenesis and tumor progression. Several E2 ligases are expressed at abnormal levels in human neoplastic tissues, including (but presumably not limited to) ubiquitin-conjugating enzyme E2Q family member 2 (UBE2Q2);^111,112 UBE2T;¹¹³ UBE2B (also known as HR6B);^114,115 and UBE2C, an enzyme that is involved in the regulation of the metaphase–anaphase transition (also known as UBCH10).^116-119 Of note, high expression levels of UBE2C have been associated with aneuploidy and chromosome instability,¹²⁰ 2 major features of premalignant cells and malignant cells.^121-123

Along similar lines, multiple E3 ligases are frequently overexpressed by transformed cells, including MDM2, the enzyme that targets the tumor suppressor tumor protein 53 (TP53, best known as p53) for proteasomal degradation;^124-131 HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase (HUWE1, also known as HECTH9), which catalyzes the ubiquitination of both v-myc avian myelocytomatosis viral oncogene homolog (MYC) and p53;^132-135 WW domain containing E3 ubiquitin protein ligase 1 (WWP1);¹³⁶ ring finger protein 126 (RNF126);¹³⁷ S-phase kinase-associated protein 2, E3 ubiquitin protein ligase (SKP2);^138-143 seven in absentia homologues 2 (SIAH2);¹⁴⁴ RNF115 (also known as BCA2);¹⁴⁵ and E6, a viral E3 ligase expressed by variants of the human papillomavirus that is associated with nasopharyngeal and cervical carcinomas^146-148 and exerts tumorigenic effects by promoting the degradation of p53.^149-151 In addition, several E3 ligases are lost or affected by loss-of-function mutations in the course of tumorigenesis and tumor progression, including speckle-type POZ protein (SPOP);¹⁵² breast cancer 1, early onset (BRCA1), which is critically involved in transcription and DNA repair;^153-156 von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase (VHL);¹⁵⁷ and F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase (FBW7), which is involved in the degradation of substrates relevant for cell growth, proliferation, and apoptosis.^158-161 Similar to the overexpression of UBE2C, loss-of-function FBW7 mutations have been associated with an oncogenic phenotype characterized by high degrees of chromosomal instability.^159,160

In addition, proteasomal subunits and DUBs can exhibit quantitative or functional alterations in cancer cells. This is the case for proteasome (prosome, macropain) 26S subunit, ATPase, 2 (PSMC2);^69,162 cylindromatosis (CYLD), a tumor suppressor protein involved in NF-κB signaling and regulated variants of necrosis;^163-167 ubiquitin specific peptidase 1 (USP1);¹⁶⁸ USP2A, the DUB that operates on MDM2 and cyclin D1;^169-172 USP9X, whose upregulation correlates with increased levels of the antiapoptotic Bcl−2 family member myeloid cell leukemia 1 (MCL1);^39,173,174 and USP28.¹⁷⁵

In these settings, defects in the UPS appear to contribute to oncogenesis and tumor progression by altering the proper turnover of oncoproteins and tumor suppressor proteins, hence (1) affecting key cellular processes including (but not limited to) cell cycle progression,^137-143 differentiation,¹⁵⁹ and regulated variants of cell death;^{158,163,173,176} (2) favoring genomic instability and/or aneuploidy;^120,159,160 and (3) increasing the resistance of cancer cells to antineoplastic agents.^136,177

Targeting the 26S proteasome as an anticancer intervention

Throughout the past 3 decades the effect of chemical UPS inhibitors on the survival and proliferation of cancer cells has been the subject of an intense wave of investigation, resulting in an abundant scientific literature. Most of these studies originated from the hypothesis that neoplastic cells have an increased demand for protein degradation and therefore rely on proteasomal functions to a greater extent than their non-transformed counterparts.^63-66 This is presumably a consequence of the malignant phenotype itself, which is associated with severe proteotoxic stress,^66,178–180 and the adverse microenvironmental conditions frequently encountered by cancer cells.^66,178-183 In this context, three categories of compounds that have been shown to block the proteolytic activity of the 26S proteasome at the level of the 20S subunit have been, or are being, developed in the clinic: (1) boronate-based agents, encompassing bortezomib, delanzomib, and ixazomib; (2) peptide epoxyketone-based agents, such as carfilzomib and oprozomib; and (3) non-peptide β-lactone-based chemicals, including marizomib.^80,184

The antineoplastic activity of proteasome inhibitors is multifactorial and exhibits at least some degree of context dependency. Thus, the blockade of proteasomal protein degradation may exert cytostatic^185-189 or cytotoxic^185,190-192 effects upon inhibition of the NF-κB signaling pathway,^193-196 overproduction of reactive oxygen species (ROS),^186,197-199 and activation of the mitogen-activated protein kinase 8 (MAPK8, best known as JNK1) and p53 signaling.²⁰⁰ Proteasome inhibitors have also been shown to provoke endoplasmic reticulum (ER) stress by abrogating ER-associated protein degradation,^201-204 de facto favoring the accumulation of misfolded or polyubiquitinated (and potentially toxic) proteins and impairing mitochondrial functions.^202,205 In line with this notion, bortezomib efficiently triggers an immunogenic variant of apoptosis that critically relies on the establishment of ER stress.^206-209 At least in part, the ability of bortezomib to kill cancer cells while promoting the establishment of a tumor-specific immune response may explain its clinical success in MM patients.^51,210-212

Bortezomib

As mentioned above, bortezomib (also known as PS341 or Velcade®) is a reversible inhibitor of the chymotrypsin-like activity of the 26S proteasome.^213-216 This boronate-based agent has been reported to mediate robust antineoplastic effects against a variety of human cancer cell lines in vitro and in vivo.^217-220 This said, the sensitivity of malignant cells to bortezomib varies considerably among cancer cells of distinct histologic origin or that exhibit different oncogenic alterations.^221,222 However, a large amount of preclinical data that accumulated in the early 2000s indicates that bortezomib is particularly efficient against otherwise chemoresistant hematologic malignancies, including MM.^220,223-226

Completed clinical studies

In the past decade, dozens of studies have investigated the therapeutic profile of bortezomib in individuals with relapsed refractory MM (Table 1). The safety and antineoplastic activity demonstrated by bortezomib in initial Phase I-II trials^{78,79,97,227-229} drove the primary approval of this drug by the US FDA for use in MM patients who failed to benefit from at least 2 lines of previous therapy. Indeed, MM patients receiving bortezomib exhibited a response rate of approximately 37% (∼27% partial responses, ∼10% complete or near-to-complete responses) and a median overall survival of 16 mo.⁷⁹ Subsequent clinical trials demonstrated the superior anticancer activity of (1) bortezomib monotherapy compared to high-dose dexamethasone (an FDA-approved glucocorticosteroid that exerts cytotoxic effects against several hematologic malignancies)^230-233 in subjects with relapsed or refractory MM;^98,234 and (2) bortezomib combined with prednisone (another glucocorticosteroid licensed for use in cancer patients)^81,235 and melphalan (an alkylating agent currently employed for the treatment of MM, ovarian carcinoma, and melanoma)²³⁶ compared to melphalan plus prednisone in patients with newly diagnosed MM who were ineligible for high-dose chemotherapy.^237-246 Bortezomib employed as a stand-alone therapeutic intervention has also been associated with a good clinical profile (toxicity, response rate, and duration of response) in subjects with relapsed or refractory MCL,^83,84 and in patients with recurrent or refractory follicular lymphoma.^247,248 These data underpinned the approval of bortezomib for use in MCL patients who have received at least one prior therapy.^80,213 Common toxicities associated with the use of bortezomib as a single agent in individuals affected by MM and MCL include gastrointestinal disorders, anemia, thrombocytopenia, fatigue, and peripheral neuropathy.^{80,229,249-251}

Table 1. Completed clinical trials testing the therapeutic profile of bortezomib in cancer patients.

Indication(s)	Phase	Notes	Ref.
Biliary tract cancers	II	As single agent	326
Breast cancer	II	Combined with doxorubicine	331
Follicular lymphoma	II	As single agent	247
		Combined with bendamustine and rituximab	307
Gastric or gastroesophageal carcinoma	I	Combined with epirubicin, carboplatin and capecitabine	315
	II	As single agent	324
Glioblastoma multiforme	II	Combined with vorinostat	329
Hepatocellular carcinoma	II	As single agent	325
		Combined with doxorubicine	333
Head and neck cancer	I	Combined with cisplatin-based chemoradiotherapy	318
	II	As single agent or combined with irinotecan	328
		Combined with doxorubicine	327
Hematologic neoplasms	I	Combined with doxorubicin	264
	I/II	Combined with chemotherapy and HSCT	311
		Combined with gemcitabine	312
		Combined with R-CHOP	308
Melanoma and soft tissue sarcoma	I	Combined with dacarbazine	321
Mesothelioma	II	Combined with cisplatin	330
Mantle cell lymphoma	II	As single agent	83,84
		Combined with gemcitabine	297
Myelodysplastic syndrome	I/II	Combined with cytarabine	300
	I	As single agent	227–229
	Ib	Combined with panobinostat and dexamethasone	269
	I/II	Combined with bendamustine, rituximab and dexamethasone	286
		Combined with doxorubicin and dexamethasone	266
		Combined with fotemustine and dexamethasone	292
		Combined with melphalan and prednisone	241,243
	II	As single agent	78,79,97,251
		Combined with bendamustine and rituximab	284,285
		Combined with bevacizumab	287
		Combined with dexamethasone	252,254
		Combined with dexamethasone and DLIs	255
		Combined with doxorubicin	263
		Combined with doxorubicin and dexamethasone	265,267
		Combined with fludarabine and melphalan prior to HSCT	295
		Combined with lenalidomide and dexamethasone	278
		Combined with melphalan, lenalidomide, and HSCT	296.
		Combined with melphalan, prednisone, and siltuximab	239
		Combined with panobinostat and dexamethasone	270
		Combined with thalidomide, dexamethasone, and cyclophosphamide	277
	III	As single agent	98,234
		Combined with melphalan and prednisone	237,238,240,242
		Combined with melphalan- and prednisone-based chemotherapy	240,245
		Combined with melphalan and prednisone or thalidomide and prednisone	240,244,246
		Combined with thalidomide and dexamethasone	276
		Combined with vorinostat	268
	IIIb	As single agent or combined with dexamethasone	253
Non-Hodgkin's lymphoma	I	Combined with ^90Y-ibritumomab tiuxetan	303
	II	Combined with rituximab and dexamethasone	306
Non-small cell lung carcinoma	I	Combined with vorinostat and consolidative surgery	322
	II	As single agent	323
		Combined with vorinostat	332
Ovarian carcinoma	II	Combined with doxorubicine	337
Prostate carcinoma	II	Combined with prednisone	334
		Combined with docetaxel	336
Advanced solid tumors	I	Combined with oxaliplatin	320
		Combined with sorafenib	314
		Combined with sunitinib	317
		Combined with tanespimycin	316
		Combined with vorinostat	313,319

Abbreviations: DLI, donor lymphocyte infusion; HSCT, hematopoietic stem cell transplantation; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone.

Bortezomib has also been demonstrated to boost the therapeutic activity of multiple chemotherapeutic agents in MM patients (Table 1), including (1) dexamethasone, employed as a stand-alone therapeutic intervention or followed by donor lymphocyte infusions upon allogeneic stem cell transplantation;^252-259 (2) doxorubicin (an immunogenic anthracycline approved by the FDA for the treatment of various hematologic and solid malignancies),^103,260-262 alone or combined with dexamethasone;^263-267 (3) histone deacetylase (HDAC) inhibitors;^268-272 (4) thalidomide (an immunomodulatory drug licensed for use in MM patients),¹¹⁰ combined with cyclophosphamide (an immunogenic alkylating agent currently approved for the treatment of multiple neoplasms)^273-275 and/or dexamethasone;^276,277 (5) lenalidomide plus dexamethasone (an immunomodulatory chemotherapeutic regimen approved for the treatment of MM, MCL, and myelodysplastic syndromes [MDSs]);^278,279 (6) bendamustine (an alkylating agent currently employed for chronic lymphocytic leukemia [CLL] and non-Hodgkin's lymphoma [NHL]),^280-282 in combination with rituximab (a monoclonal antibody targeting CD20 licensed for the treatment of CLL and NHL)^104,283 and/or dexamethasone;^284-286 (7) bevacizumab (a monoclonal antibody targeting the vascular endothelial growth factor [VEGF] that is currently approved for the treatment of several neoplasms);^287-289 (8) fotemustine (another alkylating agent currently approved for use in melanoma patients)^290,291 and dexamethasone;²⁹² (9) fludarabine (a nucleoside analog used for the treatment of CLL)^293,294 plus melphalan, used as a conditioning regimen before allogeneic stem cell transplantation;²⁹⁵ and (10) intermediate-dose melphalan and autologous stem cell transplantation, followed by lenalidomide-based consolidation.²⁹⁶

Of note, bortezomib-based chemotherapeutic cocktails exert anticancer effects not only in MM patients, but also in subjects bearing other hematologic neoplasms. These include: (1) MCL patients receiving bortezomib plus gemcitabine (an immunostimulatory nucleoside analog used for the treatment of distinct solid malignancies);^297-299 (2) individuals with high-risk MDS treated with bortezomib combined with low-dose cytarabine (a nucleoside analog used for the treatment of different types of leukemia);^300-302 (3) NHL patients receiving bortezomib in combination with the FDA-approved CD20-targeting monoclonal antibody ⁹⁰Y-ibritumomab tiuxetan^303-305 or with rituximab plus low-dose dexamethasone;³⁰⁶ (4) subjects with relapsed or refractory follicular lymphoma treated with bortezomib plus bendamustine and rituximab;³⁰⁷ (5) diffuse large B-cell lymphoma (DLBCL) and MCL patients receiving bortezomib in the context of a rituximab- cyclophosphamide-, doxorubicin-, vincristine-, and prednisone-based chemotherapeutic combination commonly known as R-CHOP;³⁰⁸ (6) NHL and MCL patients treated with bortezomib plus etoposide (an FDA-approved inhibitor of topoisomerase II commonly used for the treatment of several neoplasms),^309,310 cytarabine, melphalan, and autologous hematopoietic stem cell transplantation;³¹¹ (7) subjects with refractory DLBCL or peripheral T-cell lymphoma (TCL) receiving bortezomib plus gemcitabine.³¹²

The results of some Phase I clinical trials (mainly investigating safety, tolerability, and dosing schedules) supported the development of bortezomib in combination with other therapeutic interventions for the treatment of some solid malignancies.^313-322 Nonetheless, the findings of Phase II trials performed so far are quite disappointing. Although well tolerated, bortezomib monotherapy displays limited, if any, clinical activity against chemotherapy-naïve, metastatic non-small cell lung carcinoma (NSCLC),³²³ advanced gastric or gastroesophageal junction adenocarcinoma,³²⁴ unresectable hepatocellular carcinoma (HCC),³²⁵ and advanced tumors of the biliary tract.³²⁶ Along similar lines, various bortezomib-based chemotherapeutic cocktails have been shown to mediate negligible antineoplastic effects in patients with head and neck tumors,^327,328 recurrent glioblastoma,³²⁹ malignant pleural mesothelioma,³³⁰ metastatic breast carcinoma,³³¹ advanced NSCLC,³³² HCC,³³³ castration-resistant metastatic prostate cancer,^334-336 and ovarian carcinoma.³³⁷

Together, these observations suggest that bortezomib, alone or combined with other chemotherapeutic interventions, mediates significant therapeutic benefits exclusively in patients affected by hematologic malignancies. Interestingly, the elevated sensitivity of MM to bortezomib has been ascribed to the fact that MM cells (de facto originating from plasma cells) produce high titers of abnormal immunoglobulins and hence critically rely on the activity of the so-called immunoproteasome, a bortezomib-sensitive variant of the 26S proteasome that is upregulated in response to inflammatory cytokines.^338-341 In this setting, the relatively low efficiency of bortezomib at the molecular level (bortezomib is estimated to reduce the global proteolytic activity of the 26S proteasome by 20–30%)³⁴² may be sufficient to efficiently kill transformed cells.

Ongoing clinical trials

Official sources list 15 ongoing (not terminated, withdrawn, suspended, or completed) clinical trials launched after January 1 2012 that are aimed at assessing the safety and antineoplastic activity of bortezomib as an off-label therapeutic intervention, i.e., in patients affected by malignancies other than MM and MCL (http://www.clinicaltrials.gov/) (Table 2). In particular, bortezomib is being tested in individuals with: (1) relapsed or refractory acute lymphoblastic leukemia (ALL), in combination with doxorubicin, dexamethasone, vincristine (a microtubular poison currently licensed for the treatment of several malignancies),^343-345 and pegylated asparaginase (a recombinant enzyme commonly employed for this oncologic indication) (NCT01769209); (2) acute myeloid leukemia (AML), in combination with arsenic trioxide (NCT01950611), sorafenib (a FDA-approved multikinase inhibitor)^346-348 and decitabine (a nucleoside analog employed for the treatment of AML and MDS)^301,349 (NCT01861314), liposomal doxorubicin (NCT01736943), or sorafenib plus the HDAC inhibitor vorinostat³⁵⁰ (NCT01534260); (3) DLBCL, either as a stand-alone maintenance therapy (NCT01902862), or as an induction therapy in combination with rituximab, dexamethasone, cytarabine, and cisplatin (a platinum derivative commonly employed against several solid neoplasms)^351-354 prior to high-dose chemotherapy and autologous stem cell transplantation (NCT01805557), or combined with rituximab, cyclophosphamide, doxorubicin, and prednisone (NCT01848132); (4) low- or intermediate-risk MDS, as a single agent (NCT01891968); (5) Waldenström's macroglobulinemia (WM), combined with cyclophosphamide, rituximab and dexamethasone (NCT01788020) or with cyclophosphamide, rituximab, and fludarabine (NCT01592981); and (6) various hematologic malignancies, in combination with the experimental inhibitor of aurora kinase A (AURKA) alisertib and rituximab (NCT01695941) or a multicomponent chemotherapeutic cocktail (NCT02112916). Moreover, the therapeutic potential of bortezomib is being investigated in subjects with relapsed or refractory neuroblastoma, who receive it in combination with the ornithine decarboxylase inhibitor eflornithine (which is currently approved as a topical intervention against facial hirsutism and as a systemic treatment for sleeping sickness)^355-358 (NCT02139397), and in NSCLC patients bearing KRAS mutations or with a limited smoking history, who are treated with bortezomib plus acyclovir (a guanosine analog currently approved for the treatment of herpes simplex virus infection)^359-362 (NCT01833143).

Table 2. Ongoing clinical trials recently launched to evaluate the safety and efficacy of off-label bortezomib in cancer patients.*

Indication(s)	Phase	Status	Notes	Ref.
Acute lymphoblastic leukemia	II	Recruiting	Combined with doxorubicin-based radiotherapy	NCT01769209
Acute myeloid leukemia	I	Recruiting	Combined with decitabine and sorafenib	NCT01861314
	I/II	Recruiting	Combined with sorafenib and vorinostat	NCT01534260
	II	Recruiting	Combined with arsenic trioxide	NCT01950611
			Combined with liposomal doxorubicin	NCT01736943
Diffuse large B-cell lymphoma	II	Recruiting	As single agent	NCT01965977
			Combined with rituximab, cyclophosphamide, doxorubicin, and prednisone	NCT01848132
	II/III	Recruiting	Combined with rituximab-based chemotherapy prior to HSCT	NCT01805557
Hematologic neoplasms	I	Recruiting	Combined with alisertib and rituximab	NCT01695941
	III	Not yet recruiting	Combined with cyclophosphamide- and doxorubicin-based chemoradiotherapy	NCT02112916
Myelodysplastic syndrome	II	Recruiting	As single agent	NCT01891968
Neuroblastoma	I/II	Recruiting	Combined with eflornithine	NCT02139397
Non-small cell lung carcinoma	II	Recruiting	Combined with acyclovir	NCT01833143
Waldenström's macroglobulinemia	II	Recruiting	Combined with cyclophosphamide, dexamethasone, and rituximab	NCT01788020
	III	Recruiting	Combined with cyclophosphamide, fludarabine, and rituximab	NCT01592981

Abbreviation: HSCT, hematopoietic stem cell transplantation.

*initiated after January 1 2012 and not terminated, suspended, withdrawn, or completed at the date of submission.

Carfilzomib

Several MM patients either do not respond or become refractory to bortezomib monotherapy.^363-365 A variety of molecular alterations have been proved to contribute to such innate or acquired resistance, including overexpression of wild-type or mutant proteasome components;^366-370 constitutive activation of NF-κB^371,372 or insulin-like growth factor 1 receptor (IGF1R)^373,374 signaling; a block in mitochondrial apoptosis;³⁷⁵ upregulation of the chaperones involved in the ER unfolded protein response;³⁷⁶ increased expression levels of multidrug transporters;^377-379 and the elicitation of nuclear factor, erythroid 2-like 2 (NFE2L2)-dependent responses to oxidative stress.³⁸⁰ This has driven the development of carfilzomib (also known as PR-171), a second-generation, epoxyketone-based, irreversible inhibitor of the chymotrypsin-like activity of the 26S proteasome.^381-383 Carfilzomib rapidly turned out to mediate robust antineoplastic effects against several hematologic malignancies (including MM) in vitro and in vivo.^381-383 Similar to that of bortezomib, the pronounced antimyeloma activity of carfilzomib has been attributed to its ability to inhibit the immunoproteasome.³⁸³

Completed clinical studies

The safety profile and efficacy of carfilzomib monotherapy in MM patients have been demonstrated by several clinical studies, including 2 Phase I^384,385 and 4 open-label, single-arm Phase 2^85-88 trials (Table 3). In one of these studies, carfilzomib was associated with durable clinical responses (overall response rate 23.7%, median duration of response 7.8 mo, median overall survival 15.6 mo) and an acceptable toxicity profile,⁸⁷ supporting approval of this agent by the FDA for the treatment of relapsed and refractory MM patients who have received at least 2 prior therapies, including bortezomib.³⁸⁶ Importantly, a prospective analysis performed on this patient cohort revealed that single-agent carfilzomib has the potential to at least partially overcome the impact of high-risk cytogenetics in heavily pretreated MM patients.³⁸⁷ Moreover, carfilzomib appears to be associated with a reduced incidence of peripheral neuropathy (13.9%).³⁸⁸ The combination of carfilzomib with lenalidomide and dexamethasone also seems to be well tolerated and to promote robust, rapid, and durable responses in patients with both relapsed/progressive^389,390 and newly diagnosed³⁹¹ MM. In particular, 62% of individuals with newly diagnosed MM achieved at least a near-complete clinical response in response to this chemotherapeutic cocktail, with a 2-y progression-free survival estimate of 92%.³⁹¹ Recently, an open-label, intra-patient Phase I/II clinical trial demonstrated that replacing bortezomib with carfilzomib is safe and can provide therapeutic benefits to MM patients who are progressing on bortezomib-based combinatorial chemotherapy.³⁹² Moreover, the results of a Phase II study indicate that combining carfilzomib with cyclophosphamide and dexamethasone is associated with a good safety profile and high rates of complete response among patients with newly diagnosed MM.³⁹³ A randomized, open-label Phase III study is currently ongoing to compare the overall survival of carfilzomib monotherapy to best supportive care in relapsed or refractory MM patients.³⁹⁴ Of note, similar to bortezomib, carfilzomib is well tolerated by patients with advanced solid tumors but exerts limited, if any, antineoplastic activity.³⁹⁵

Table 3. Completed clinical trials testing the therapeutic profile of carfilzomib in cancer patients.

Indication(s)	Phase	Notes	Ref.
Multiple myeloma	I	As single agent	384,385
	Ib	Combined with dexamethasone and lenalidomide	389
	I/II	Combined with a panel of chemotherapeutics	392
		Combined with dexamethasone and lenalidomide	391
	II	As single agent	85–88,387
		Combined with dexamethasone and lenalidomide	390
		Combined with dexamethasone and cyclophosphamide	393
	III	As single agent	394
Advanced solid tumors	I/II	As single agent	395

Ongoing clinical trials

Official sources list 14 ongoing (not terminated, withdrawn, suspended, or completed) clinical trials launched after January 1 2012 to investigate the therapeutic profile of carfilzomib as an off-label therapeutic intervention, i.e., in patients affected by neoplasms other than MM (http://www.clinicaltrials.gov/) (Table 4). Carfilzomib is being evaluated as a stand-alone therapeutic intervention in patients with (1) relapsed or refractory MCL (NCT02042950); (2) refractory renal cell carcinoma (NCT01775930); or (3) advanced malignancies (NCT01949545). Moreover, the safety and efficacy of carfilzomib are being tested in (1) patients with relapsed or refractory DLBCL, receiving carfilzomib in the context of rituximab-based chemotherapy (NCT01959698; NCT02073097); (2) subjects with relapsed or refractory MCL, who are treated with carfilzomib plus lenalidomide and rituximab (NCT01729104); (3) patients with relapsed or refractory NHL, receiving carfilzomib in combination with an FDA-approved histone deacetylase inhibitor (belinostat)^271,396-398 (NCT02142530) or bendamustine and rituximab (NCT02187133); (4) patients with relapsed WM, with carfilzomib, rituximab, and dexamethasone (NCT01813227); (5) subjects with cutaneous TCL, receiving carfilzomib plus the FDA-approved HDAC inhibitor romidepsin^397-400 (NCT01738594); and (6) MCL, TCL, and DLBCL patients, who are treated with carfilzomib and dexamethasone as consolidation therapy after autologous stem cell transplantation (NCT01926665). Finally, the clinical profile of carfilzomib is being assessed in (1) previously untreated subjects affected by extensive stage small-cell lung cancer, who are treated with carfilzomib plus carboplatin (a platinum derivative employed for the treatment of multiple solid tumors, including ovarian carcinoma)^401-403 and etoposide (NCT01987232); (2) subjects with relapsed lung cancer, receiving carfilzomib in combination with irinotecan (a topoisomerase I inhibitor mainly used for the treatment of colorectal carcinoma)^404,405 (NCT01941316); and (3) patients with metastatic castration-resistant prostate cancer,³³⁵ who are treated with carfilzomib, dexamethasone, and acyclovir (NCT02047253).

Table 4. Ongoing clinical trials recently launched to evaluate the safety and efficacy of off-label carfilzomib in cancer patients.*

Indication(s)	Phase	Status	Notes	Ref.
Diffuse large B-cell lymphoma	I/II	Not yet recruiting	Combined with rituximab and CHOP	NCT02073097
		Recruiting	Combined with rituximab, ifosfamide, carboplatin, and etoposide	NCT01959698
Hematological neoplasms	I	Recruiting	Combined with dexamethasone and HSCT	NCT01926665
Mantle cell lymphoma	I/II	Recruiting	Combined with lenalidomide and rituximab	NCT01729104
	II	Not yet recruiting	As single agent	NCT02042950
Non-Hodgkin's lymphoma	I	Not yet recruiting	Combined with belinostat	NCT02142530
			Combined with bendamustine and rituximab	NCT02187133
Prostate carcinoma	II	Recruiting	Combined with dexamethasone and acyclovir	NCT02047253
Renal cell carcinoma	II	Active, not recruiting	As single agent	NCT01775930
Small cell lung carcinoma	I/II	Recruiting	Combined with carboplatin and etoposide	NCT01987232
T-cell lymphoma	I	Recruiting	Combined with romidepsin	NCT01738594
Waldenström's macroglobulinemia	II	Recruiting	Combined with dexamethasone and rituximab	NCT01813227
Advanced tumors	I	Recruiting	As single agent	NCT01949545
	I/II	Recruiting	Combined with irinotecan	NCT01941316

Abbreviations: CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; HSCT, hematopoietic stem cell transplantation.

*initiated after January 1 2012 and not terminated, suspended, withdrawn, or completed at the date of submission.

Additional proteasome inhibitors

Other clinically relevant inhibitors of the 26S proteasome include: (1) marizomib (also known as NPI-0052), an irreversible inhibitor of both the chymotrypsin- and trypsin-like enzymatic activities of the 20S subunit^406-410 that exhibits improved bioavailability compared to bortezomib and carfilzomib, perhaps because of its non-peptidic nature,⁴¹¹ and robust antineoplastic activity in preclinical models;^190,412-415 ixazomib (also known as MLN9708), a boronate-based agent characterized by increased oral availability and antitumor activity compared to bortezomib;^184,416 (3) oprozomib (also known as ONX-0912), a carfilzomib-like orally bioavailable inhibitor of the chymotrypsin-like activity of the 20S subunit,^417,418 which is active against MM and head and neck cancers;^419,420 and (4) delanzomib (also known as CEP-18770), a potent, reversible, and orally bioavailable agent^421-424 exhibiting high antineoplastic activity in preclinical models of MM both as monotherapy⁴²⁵ and in combination with other chemotherapeutic agents.^425,426 Intriguingly, some of these chemicals, including marizomib, have been reported to synergize with bortezomib in the killing of MM cells,^409,427 suggesting that the mechanisms of action of distinct proteasome inhibitors may not be completely overlapping.

Clinical studies

Marizomib-based monotherapy has been associated with a promising safety profile (no evidence of thrombocytopenia and peripheral neuropathy) and clinical efficacy in Phase I trials enrolling relapsed and refractory MM patients.^{80,407,411,428} In addition, the combination of marizomib and vorinostat was well tolerated by patients with advanced solid tumors.⁴²⁹ According to official sources (http://www.clinicaltrials.gov/, ongoing trials initiated after January 1 2012), the safety and antineoplastic activity of marizomib are currently being assessed in relapsed or refractory MM patients, receiving marizomib either as a stand-alone therapeutic intervention (NCT00461045) or in combination with pomalidomide (an immunomodulatory agent approved by the US FDA for the treatment of MM)^110,430-433 and low-dose dexamethasone (NCT02103335) (Table 5).

Table 5. Clinical trials recently launched to evaluate the safety and efficacy of third-generation proteasomal inhibitors in cancer patients.*

Inhibitor	Indication(s)	Phase	Status	Notes	Ref.
Ixazomib	Acute myeloid leukemia	I	Not yet recruiting	Combined with cytarabine, etoposide, and mitoxantrone	NCT02070458
		II	Recruiting	As single agent	NCT02030405
	Follicular lymphoma	II	Recruiting	As single agent	NCT01939899
	Hematologic neoplasms	I	Recruiting	Combined with doxorubicin-based chemotherapy	NCT01887587
		II	Not yet recruiting	As single agent after HSCT	NCT02169791
	Multiple myeloma	I	Recruiting	Combined with dexamethasone and lenalidomide	NCT01645930
				Combined with dexamethasone	NCT01830816
				Combined with dexamethasone and panobinostat	NCT02057640
		I/II	Not yet recruiting	Combined with dexamethasone and pomalidomide	NCT02119468
			Recruiting	Combined with dexamethasone and cyclophosphamide	NCT01864018
				Combined with dexamethasone and pomalidomide	NCT02004275
		II	Not yet recruiting	As single agent after HSCT	NCT02168101
				Combined with dexamethasone and lenalidomide	NCT01936532
			Recruiting	Combined with dexamethasone	NCT01415882
				Combined with lenalidomide after HSCT	NCT01718743
				Combined with dexamethasone and cyclophosphamide	NCT02046070
		III	Not yet recruiting	As single agent after HSCT	NCT02181413
		III	Recruiting	Combined with dexamethasone and lenalidomide	NCT01564537
					NCT01850524
	T-cell lymphoma	II	Not yet recruiting	As single agent	NCT02158975
	Advanced tumors	I	Not yet recruiting	Combined with vorinostat	NCT02042989
			Recruiting	As single agent	NCT01912222
					NCT01953783
Marizomib	Multiple myeloma	I	Recruiting	Combined with dexamethasone and pomalidomide	NCT02103335
		I/II	Recruiting	As single agent	NCT00461045
Oprozomib	Multiple myeloma	I/II	Recruiting	Combined with dexamethasone and lenalidomide or cyclophosphamide	NCT01881789
				Combined with dexamethasone	NCT01832727
				Combined with dexamethasone and pomalidomide	NCT01999335
				Combined with melphalan and prednisone	NCT02072863

Abbreviations: HSCT, hematopoietic stem cell transplantation.

*initiated after January 1 2012 and not terminated, suspended, withdrawn, or completed at the date of submission.

The safety and tolerability of ixazomib have been evaluated in several Phase I clinical trials enrolling subjects with relapsed/refractory MM.^434-440 In 2 of these studies, 15–18% of patients were reported to achieve at least a partial response to therapy,^434,435 supporting further clinical development. Along similar lines, ixazomib (co-administered with dexamethasone and lenalidomide) was well tolerated by individuals with previously untreated MM and exerted some degree of clinical activity.^441,442 According to official sources, no fewer than 14 clinical trials have been initiated after January 1 2012 to evaluate the therapeutic potential of ixazomib in subjects with hematologic malignancies (http://www.clinicaltrials.gov/). In these studies, ixazomib is being tested (1) as a single agent for post-transplantation maintenance in patients with MM (NCT02168101; NCT02181413) or various hematologic tumors including MM (NCT02169791); (2) in combination with dexamethasone for relapsed and refractory (NCT01830816) or relapsed but not refractory (NCT01415882) MM; (3) in combination with dexamethasone and cyclophosphamide in newly-diagnosed MM patients (NCT01864018, NCT02046070); (4) in combination with dexamethasone and panobinostat (an experimental non-selective HDAC inhibitor)^397,398 in subjects with relapsed and/or refractory MM (NCT02057640); (5) in combination with dexamethasone and pomalidomide in refractory (NCT02004275) or relapsed/refractory (NCT02119468) MM patients; (6) with lenalidomide only, as a maintenance regimen upon autologous stem cell transplantation in MM patients (NCT01718743); (7) with lenalidomide and dexamethasone, in patients with either newly diagnosed (NCT01850524, NCT01936532) or relapsed/refractory (NCT01564537, NCT01645930) MM. Moreover, ixazomib monotherapy is being evaluated in non-MM patients, including (1) subjects with relapsed or refractory AML (NCT02030405); (2) individuals with relapsed/refractory cutaneous and peripheral TCL (NCT02158975); (3) patients with relapsed/refractory follicular lymphoma (NCT01939899); (4) subjects with hematologic malignancies or advanced solid tumors (NCT01912222); and (5) individuals with lymphomas or advanced solid tumors (NCT01953783). Finally, ixazomib is being assessed in combination with (1) mitoxantrone (an FDA-approved immunogenic anthracycline used for the therapy of NHL, AML, and breast carcinoma),^443-446 etoposide, and intermediate-dose cytarabine in relapsed/refractory AML patients (NCT02070458); (2) vincristine, doxorubicin, pegylated-asparaginase, and dexamethasone in subjects with relapsed/refractory ALL or lymphoma (NCT01887587); and (3) vorinostat, in individuals with advanced solid tumors (NCT02042989) (Table 5).

The safety and tolerability of oprozomib have been evaluated in Phase I studies performed on patients with hematologic malignancies and advanced solid tumors.^447,448 According to official sources (http://www.clinicaltrials.gov/, ongoing trials initiated after January 1 2012), the clinical profile of oprozomib is being investigated in (1) transplant-ineligible patients with newly diagnosed MM, receiving oprozomib plus dexamethasone and lenalidomide (NCT01881789), or dexamethasone and oral cyclophosphamide (NCT01881789), or prednisone and melphalan (NCT02072863), (2) relapsed and/or refractory MM patients, treated with oprozomib plus dexamethasone (NCT01832727); and (3) subjects with primary refractory or relapsed/refractory MM, receiving oprozomib with pomalidomide and dexamethasone (NCT01999335) (Table 5).

Finally, results from a relatively recent Phase I clinical trial enrolling patients with advanced solid tumors and MM demonstrated a favorable safety profile for delanzomib, which in this setting was not associated with significant neurotoxicity and skin toxicity.⁴⁴⁹ The clinical development of this proteasome inhibitor has nonetheless been discontinued due to a lack of efficacy documented in a Phase I/II trial conducted on relapsed refractory MM patients.^411,450

Concluding Remarks

The clinical advantages provided by proteasome inhibitors to MM patients have been demonstrated by a large number of clinical studies. However, bortezomib, carfilzomib, and similar agents generally lack therapeutic activity against solid tumors. In addition, the clinical activity of proteasome inhibitors in MM patients can be limited by (1) side effects, including thrombocytopenia and peripheral neuropathy,^251,451-456 that call for reductions in dosage or the discontinuation of therapy²⁵¹ and often result in poor therapeutic effects;^{78,97,98,254,457,458} (2) innate or acquired resistance;^363-365 and (3) the absence of validated predictive biomarkers that allow preselection of patients who have a high chance of truly benefitting from therapy.^64,459,460 Modifications in drug administration protocols (e.g., alternative routes),⁴⁶¹, the implementation of novel combinatorial chemotherapeutic regimens, and the development of third-generation proteasome inhibitors with improved bioavailability and reduced toxicity may broaden the therapeutic utility of these compounds against hematologic malignancies and solid tumors. As an alternative, therapeutic strategies targeting other components of the UPS and DUBs have been proposed. Although such an approach holds promise, only a few compounds such as MDM2 antagonists (e.g., nutlin-3, serdemetan) and NEDD8-activating enzyme (NAE) inhibitors (e.g., MLN4924) have entered clinical development to date.^462,463

Accumulating evidence indicates that regulation of the UPS in both physiologic and pathologic settings is more complex than originally thought, which complicates the development of clinically useful proteasome targeting agents^64,464 Moreover, limiting proteasomal protein degradation in healthy tissues may favor tumorigenesis (by stabilizing oncoproteins or inhibiting tumor suppressors) and/or neurodegenerative disorders (by promoting the accumulation of potentially neurotoxic misfolded proteins).^64,464 Along similar lines, the UPS plays a critical role in the processing of intracellular proteins for antigen presentation,^{32,50,465-468} implying that proteasome inhibitors may negatively affect the elicitation of therapeutically relevant anticancer immune responses.

An improved understanding of the composition, function, and regulation of the UPS, as well as the molecular mechanisms underlying the intrinsic or acquired resistance of some neoplasms to proteasome-targeting agents, may pave the way to the design of novel effective anticancer chemotherapies based on proteasome inhibition and to their successful translation from the bench to the bedside.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

Authors are supported by the European Commission (ArtForce); Agence National de la Recherche (ANR); Associazione Italiana per la Ricerca sul Cancro (AIRC: MFAG 2013 #14641 and triennial tellowship “Antonietta Latronico”, 2014); Italian Ministry of Health (GR-2011-02351355); Programma per i Giovani Ricercatori “Rita Levi Montalcini” 2011; Ligue Nationale contre le Cancer; Fondation pour la Recherche Médicale (FRM); Institut National du Cancer (INCa); Association pour la Recherche sur le Cancer (ARC), LabEx Immuno-Oncologie; Fondation de France; Fondation Bettencourt-Schueller; AXA Chair for Longevity Research; Cancéropôle Ile-de-France, Paris Alliance of Cancer Research Institutes (PACRI) and Cancer Research for Personalized Medicine (CARPEM).