Multiple myeloma (MM) is a cancer of plasma cells characterized by increased survival and proliferation of terminally differentiated plasma cells in the bone marrow.1 Clinically, MM diagnosis is prompted by the detection of monoclonal intact immunoglobulin (M spike), also known as free light chains (not in association with heavy chains), in the serum and urine of patients presenting with 1 or more of hypercalcemia, renal failure, anemia, and bone disease (the CRAB criteria). Major progress in our understanding of MM biology over the past 4 decades has led to significant improvements in how we treat MM, reflected by a 3- to 4-fold increase in patient median survival. Although MM is now better controlled over longer periods for many patients, it remains incurable and resistance to novel agents represents a major clinical problem. This review will focus on the molecular mechanisms underlying protein handling in MM and on bench-to-bedside translation of therapies targeting protein synthesis, folding, and degradation in MM. Rational combination of these agents holds promise to help overcome proteasome inhibitor (PI) resistance in MM, with the goal of achieving prolonged remission, if not cure, for most patients with multiple myeloma.
The process of protein synthesis and folding is intrinsically prone to errors, and eukaryotic cells are equipped with quality control mechanisms to ensure that native proteins adopt proper tertiary and quaternary conformations. The cytotoxic accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR), which, together with autophagy, aggresome, and the ubiquitin-proteasome system (UPS), has the goal of maintaining protein homeostasis.2,3 It is estimated that one-third of newly synthesized proteins are de- graded via the proteasome within minutes of their synthesis dueto an intrinsic inability to achieve stably folded conformations.4 These rapidly degraded proteins are termed “defective ribosomal products” (DRiPs). Due to high protein turnover, cancer cells typically produce an even higher percentage of DRiPs, making them reliant on an intact UPS for survival.5-8 This is especially true for MM, a cancer characterized by a high synthesis rate of immunoglobulins. In fact, MM cells exhibit stigmata of ongoing proteotoxic stress with baseline induction of UPR and accumulation of polyubiquitinated proteins, providing a substrate for proteasome-mediated degradation.9-11
Studies have shown that an imbalance between the cargo for proteasomal degradation (polyubiquitnated proteins) and the activity of the proteasome is a key determinant of PI sensitivity in MM.12 Drugs that increase proteasome workload (eg, heat shock protein [HSP] inhibitors, ER stressors) synergize with drugs that decrease proteasome activity (eg, PIs) in MM. The results of in vitro studies have shown that proteasome inhibition, perhaps even UPR induction, results in the compensatory activation of aggresome, autophagy, and heat shock response pathways in an effort to protect MM cells from proteotoxicity (Figure 1).2,13-15 Further work assessing the combinatorial effects of blocking 2 or more of these pathways in MM are currently ongoing, some of which are highlighted below.
At the core of protein homeostasis in eukaryotes is the UPS (Figure 1A). Proteins targeted for proteasome degradation are polyubiquitinated via a 3-enzyme cascade involving E1 (activating), E2 (conjugating), and E3 (ligase) enzymes, while deubiquitinating enzymes (DUBs) act in opposition to E3 ligases to remove ubiquitin.16-19 The 26S proteasome is an ATP-dependent, multicatalytic complex comprising a 20S catalytic core flanked on either side by 19S regulatory caps.20 Polyubiquitinated substrates are recognized by the 19S regulatory subunit that, in concert with DUBs, remove ubiquitin and facilitate engagement with the 20S core that contains the catalytically active β1 (caspase-like activity), β2 (trypsin-like activity), and β5 (chymotrypsin-like activity) subunits.19,21,22 The PIs bortezomib, carfilzomib, ixazomib, and oprozomib primarily target the β5 subunit, while marizomib appears to have activity against all 3 β-subunits.23-26
Prior to degradation, proteasome-associated DUBs (eg, RPH11, UCH37, and USP14) remove ubiquitin chains, which would otherwise sterically hinder the translocation of target proteins to the 20S core.27 Similar to PIs, DUB inhibitors trigger poly- ubiquitinated protein accumulation and apoptosis in MM, but without inhibiting the catalytic subunits of the proteasome.28-30 Thus, DUB inhibitors could theoretically overcome resistance to proteasome inhibition when this is mediated by mutations in the catalytic subunits of the proteasome. Furthermore, DUB inhibition offers the opportunity to promote the degradation of proteins that are preferential clients of specific DUBs.
Autophagy, a conserved process of autoproteolysis that plays a key role in maintaining protein homeostasis (Figure 1B), participates in the quality control of protein synthesis/degradation by sequestering misfolded/aggregated proteins in autophagosomal vesicles for subsequent lysosome degradation.31 Studies have shown that crosstalk exists between UPS, ER stress, and autophagy.32-34 Although elevated basal autophagic activity in primary MM cells is associated with shorter overall survival (OS) and progression-free survival (PFS), autophagy’s role in MM is controversial given that it can be pro-survival and pro-apoptotic depending on factors we have yet to fully understand.32 The current consensus is that a basal level of autophagy is essential for MM survival as an alternative proteolytic pathway in the face of decreased proteasome activity/increased proteotoxic stress, thus providing a rationale for the combination of autophagy inhibitors with PI in MM. However, persistent, sustained, and uncontrolled autophagy is likely to result in cell death, outlining the difficulties in therapeutically targeting autophagy.35
In vitro, the aggresome pathway is activated when proteasomes are blocked. Polyubiquitinated protein aggregates are transported along the microtubule to the microtubule-organizing center in a histone deacetylase 6 (HDAC6)-dependent manner to form aggresomes that target proteins for refolding or degradation by autophagy (Figure 1C).36-39 The results of in vitro studies show that combined inhibition of the proteasome and aggresome leads to synergistic cell death in MM, providing strong rationale for combining PI with HDAC6 selective inhibitors.40
Heat Shock Chaperone Proteins
Heat shock chaperone proteins (HSPs) are a class of enzymes that chaperone the proper folding and function of proteins, and that direct misfolded proteins to degradation; therefore, they participate in protein quality control (Figure 1D).41-44 In MM, HSPs support proliferation and survival by 1) facilitating proper folding of newly synthesized proteins to prevent proteotoxic stress and 2) preferentially supporting the folding and expression of several oncogenes.45 Two main families of HSPs are being targeted therapeutically in MM: HSP90 and HSP70. Interestingly, HSP70 overexpression in neurons results in inhibition of caspase-dependent and -independent apoptosis, suggesting a third pro-survival function.46,47 Inhibition of HSP90 or the proteasome results in compensatory upregulation of HSP70, thereby making the latter an attractive target in combinatory anti-MM therapy. Recently, there has been growing interest in developing inhibitors against heat shock factor 1 (HSF1), the “master regulator” of heat shock response, in an attempt to avoid compensatory upregulation of individual chaperones.45
Endoplasmic Reticulum Stress and Unfolded Protein Response
The UPR is a tripartite response triggered by the accumulation of unfolded/misfolded proteins in the ER (Figure 1E).48,49 The UPR functions to restore equilibrium in the ER; however, prolonged/ persistent activation of UPR results in apoptosis.50 The 3 distinct UPR branches are regulated by 3 kinases: IRE1, PERK, and ATF6. Activation of IRE1 results in the splicing of XBP1 mRNA which, together with activated ATF6, regulates ER expansion, increases expression of chaperone proteins, and initiates ER-associated de-gradation to reduce ER stress.51 Depending on the magnitude and duration of stress, IRE1 can either activate antiapoptotic signaling through protein kinase B or trigger apoptosis through c-Jun N-terminal kinases (JNK) activation.52-54 Further- more, JNK activation can initiate autophagy, thereby serving as a link between ER stress and autophagy.54 Finally, protein kinase R-like ER kinase (PERK) activation inhibits eIF2α, leading to a repression of global protein synthesis while selectively inducing the translation of ATF4.55 ATF4 activates cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), and together they upregulate protective autophagy in the face of transient proteotoxic stress.56 However, if the stress is prolonged, CHOP can trigger apoptosis, outlining the double-edged nature of this stress response pathway.57
Forced expression of spliced X-box binding protein 1 (XBP1) in B cells induces an MM-like phenotype in mice, and high XBP1 expression in primary MM cells correlates with poor OS, suggesting that chronic IRE1-XBP1 activation may be important for MM survival.58 However, it was recently demonstrated that decreased XBP1 splicing confers bortezomib resistance in MM.59 By suppressing XBP1s, MM cells decommit to plasma cell maturation and decrease immunoglobulin production, proteasome load, and ER stress, resulting in acquired resistance to PI.12,59
Drugs Targeting the Ubiquitin-Proteasome Pathway
Apart from FDA-approved bortezomib, carfilzomib, and ixazomib, there are 2 novel PIs in advanced clinical development, oprozomib and marizomib. Oprozomib (ONX 0912), an oral analogue of carfilzomib, is an irreversible epoxyketone PI. In preclinical studies, oprozomib demonstrated cytotoxicity in MM in combination with lenalidomide and/or HDAC inhibitor molecules, as well as bone anabolic effects.60,61 A phase Ib/II trial of single-agent oprozomib showed an overall response rate (ORR) of 22% to 34% in relapsed/refractory MM (R/R MM), including bortezomib- and carfilzomib-refractory MM (NCT01416428).62 In the R/R MM setting, the combination of oprozomib with dexamethasone (NCT01832727) or with pomalidomide and dexamethasone (NCT01999335) has resulted in ORRs of 42% and 50% to 59%, respectively.63,64
Attempts at overcoming PI resistance by blocking all proteasome catalytic subunits prompted the development of marizomib, a pan-proteasome inhibitor.65,66 A phase I study of marizomib (NCT00461045) reported an ORR of 7.4% in bortezomib-, lenalidomide-, and/or thalidomide-refractory patients.67 Based on encouraging preclinical data supporting the combination of marizomib and pomalidomide/dexamethasone, clinical trials evaluating this combination are now underway (NCT02103335).68Deubiquitinating Enzyme Inhibitors
The small-molecule DUB inhibitors RA190, P5091, and B-AP15 target RPN13, USP7, and USP14/UCHL5, respectively. In vitro, they induce proteotoxicity and apoptosis in MM without direct inhibition of catalytic subunits of the proteasome.69-71 The USP14 inhibitor VLX1570 also demonstrated promising preclinical activity and is currently being evaluated in an early-phase clinical trial in MM.28
Heat Shock Chaperone Protein Inhibitors
HSP90 is the most well-studied chaperone protein in MM. Several HSP90 inhibitors have completed phase I clinical studies (Table). Notably, among patients who were evaluable (n = 67), the combination of tanespimycin with bortezomib in R/R MM was associated with an ORR of 15%.72 However, modest activity and/ or significant toxicity hampered clinical development of next-generation HSP90 inhibitors.73
Histone Deacetylase Inhibitors
Panobinostat, in combination with bortezomib and dexamethasone, was recently approved as a third-line therapy in patients with MM with prior bortezomib and immunomodulatory drug (IMiD) exposure. Vorinostat is a class I and II HDAC inhibitor currently undergoing clinical trials. The phase III Vantage 008 trial reported improvements in ORR (54% vs 41%; P <.0001) and PFS (7.6 vs 6.8 months; P = .10) when vorinostat was added to bortezomib.74 Although nonselective HDAC inhibitors show promising clinical activity, they are associated with significant adverse effects (particularly of a gastrointestinal and hematologic nature) due to the indiscriminate targeting of multiple HDACs, leading to extensive modulation of downstream histone and nonhistone protein functions.75 Isoform-specific HDAC inhibitors, focusing on inhibition of HDAC6 and the aggresome pathway, have been developed in an effort to maintain efficacy and limit toxicities. Two promising HDAC6-selective inhibitors (ACY-241 and ACY-1215) are currently undergoing clinical trials. A phase Ib study of ricolinostat (ACY- 1215) in combination with bortezomib/dexamethasone reported 45% and 25% ORRs in R/R MM and bortezomib-refractory MM, respectively, while ricolinostat in combination with lenalidomide/ dexamethasone had an ORR of 55% in R/R MM.76,77 BG45, an HDAC3-selective inhibitor, has also shown promising preclinical results, demonstrating anti-myeloma activity alone and in combination with bortezomib; translation to an early phase clinical trial is anticipated soon.78
Unfolded Protein Response Modulators
Pharmacological induction of UPR via ER stressors, such as tunicamycin, thapsigargin, and brefeldin A, has proven to potently synergize with a PI in vitro; however, clinical translation is limited by anticipated toxicities based on animal models.11,79-81 A more elegant way to exploit the UPR against MM hinges on identifying which of the tripartite signaling pathways and their downstream effectors are necessary for MM survival and proliferation. Such identification efforts were recently undertaken, revealing that single knockdown of each branch of the UPR has modest effects on MM viability at baseline; however, an intact IRE1-XBP1 pathway is required for bortezomib-mediated cytotoxicity.59 Inhibition of either the endoribonuclease or kinase domains of IRE1 was found to have an anti-MM effect, especially combined with PI.82,83
PERK inhibition has also recently emerged as a potential therapeutic option in MM. In preclinical studies, GSK2606414, a selective PERK inhibitor, was shown to synergistically enhance the apoptotic effect of bortezomib in MM.84 Nelfinavir is an HIV-protease inhibitor that has demonstrated anti-MM activity through UPR induction, CHOP upregulation, poly (ADP-ribose) polymerase cleavage, and proteasome inhibition in preclinical studies.85 Nelfinavir could also re-sensitize bortezomib-refractory primary MM cells towards bortezomib treatment.85 A phase II trial of nelfinavir and bortezomib reported an ORR of 30% in the dose escalation cohort and an ORR of 50% in an exploratory extension cohort comprising patients with both bortezomib-refractory and lenalidomide-resistant MM.86 No clinical-grade inhibitors of ATF6 have been reported at the time of writing.
The clinical synergism between proteasome inhibitors and the IMiDs thalidomide, lenalidomide, and pomalidomide is well established.87,88 The molecular mechanisms of activity of these compounds have long been obscure, and the anti-angiogenic effect was initially thought to be primarily responsible for their anti-MM activity.89 However, the degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) was recently shown to be the base of the anti-MM effect of lenalidomide.90,91 In an unexpected twist, lenalidomide was shown to bind to the E3 ubiquitin-ligase complex made up of the damage-specific DNA-binding protein 1 (DDB1) and cereblon, enhancing its activity and facilitating ubiquitination and proteasome-mediated degradation of IKZF1 and IKZF3. IMiD-mediated stimulation of thymus and natural killer (NK) immunity similarly depends on the degradation of IKZF1 and IKZF3, resulting in IL-2 production in T lymphocytes. Based on these findings, the clinical synergism between bortezomib, a PI, and lenalidomide, a facilitator of proteasome-mediated IKZF1 and IKZF3 degradation, appears paradoxical and remains to be clarified at the cellular and molecular levels.
Although disrupting proteostasis via a PI has been successful in MM, innate or acquired resistance remains a major clinical challenge. Combination treatments have only partially overcome these issues, and progressive acquisition of resistance to multiple agents with each disease relapse is a well-known phenomenon in MM. Recent research efforts have focused on modulating other facets of protein homeostasis pathways (ie, aggresome, autophagy, UPR, DUB, HSP), with the goal of exacerbating proteotoxcity and overcoming MM drug resistance (Figure 1).
Recent insight into the mechanism of IMiDs has led to a novel therapeutic strategy (degronomid) that exploits the ability of IMiDs to redirect the cereblon E3 ubiquitin ligase complex toward specific proteins, thus targeting them for degradation.92,93 As a proof-of-concept, the phthalimide conjugate d-bromodomain and extra-terminal 1 was able to selectively induce cereblon-dependent BET protein degradation both in vitro and in mice (Figure 1F).94 This ability to hijack the UPS to selectively degrade proteins that are otherwise considered undruggable (eg, MYC, β-catenin, and myeloid cell leukemia 1) could be a powerful tool in the treatment of MM and other malignancies.
In conclusion, the understanding of MM reliance on protein- handling pathways paved the way to therapeutically target this Achilles’ heel by exacerbating baseline proteotoxic stress. In combination with IMiDs and immunotherapies, drugs targeting the protein synthesis/degradation machinery hold the key to achieving sustained remission, if not cure, in most MM patients.
Acknowledgements: We would like to thank Steven C. Smith, MD, PhD, Department of Pathology, Virginia Commonwealth University Medical Center, Richmond, Virginia, for his assistance with this report.
Financial disclosures: The authors report no relationship or financial interest with any entity that would pose a conflict of interest with the subject matter of this article
Author affiliations: Both Matthew Ho Zhi Guang and Giada Bianchi are with the department of Medical Oncology, Jerome Lipper Multiple Myeloma Center and LeBow Institute for Myeloma Therapeutics, Dana-Farber Cancer Institute, and Harvard Medical School, Boston, Massachusetts.
Address correspondence to: Giada Bianchi, MD, Dana-Farber Cancer Institute, 450 Brookline Ave, Boston, MA 02215. E-mail: [email protected].
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