4.1 Bortezomib
Bortezomib (PS-341, NSC 681239, Velcade®; Millennium Pharmaceuticals, Inc.) was the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsed MM [
44] and MCL [
45]. Bortezomib was initially used in the clinic as single agent for the treatment of MM patients who relapsed after standard dexamethasone therapy, where it resulted in an improved overall survival rate. Subsequent studies have shown that bortezomib treatment for newly diagnosed MM significantly increased patient survival rates [
46].
Bortezomib is a synthetic dipeptide boronic acid, which slowly and reversibly inhibits the chymotrypsin-like and to a lesser extent the trypsin-like and caspase-like activities of the 20S CP. Inhibition is achieved through an initial nucleophilic attack on the active N-terminus threonine residues located on the β5, β2, and β1 subunits, leading to loss of proteolytic activity [
47]. It has been reported that bortezomib inhibits the activation of NF-κB pathway in squamous cell carcinoma [
48], MM [
49]and in MCL [
50]. However, other studies have reported that canonical NF-κB activation is induced by bortezomib in MM cells suggesting that its cytotoxic effects cannot be entirely attributed to inhibition of NF-κb [
28]. For this, the anti-cancer and pro-apoptotic activities of bortezomib may cell type-dependent and driven by specific signaling pathways. It has been reported that bortezomib induces both transcriptional and post-translational increases in cyclin-dependent kinases, p21 and p27 in hepatocellular carcinoma cells, and NSCLC [
51,
52]. Bortezomib-mediated proteasome inhibition is p53-dependent in renal and NSCLC cell lines [
52,
53]. Bortezomib activates the stress kinase JNK and the c-Jun/AP-1 signal pathway, thus inducing the expression of the cyclin-CDK inhibitor p21, leading to growth arrest and apoptosis of NSCLC cells [
52]. Finally, the induction of pro-apoptotic protein NOXA by bortezomib is directly dependent on the oncogene MYC in melanoma cell lines [
54].
Despite promising results in MM patients, several limitations of bortezomib treatment have emerged. The lack of response observed in some patients as a first-line therapy and relapses and/or resistance after initial favorable responses in MM patients have been described as the main limitations of the drug [
55]. Moreover, bortezomib treatment has been correlated to the onset of adverse effects such as thrombocytopenia, fatigue, GI symptoms, and asthenia. Bortezomib-induced peripheral neuropathy (BIPN) was identified as the main significant dose-limiting toxicity potentially leading to permanent nerve damage to extremities probably due to an off-target effect of the drug [
56] . The incidence of BIPN was reduced by changing the dose frequency from twice weekly to once weekly and choosing subcutaneous administration over intravenous injection [
57‐
61]. In addition, bortezomib has shown successful results when used in combination with other type of treatments. Combination treatments of bortezomib with doxorubicin, dexamethasone, panobinostat, and daratumumab showed improved clinical outcomes in patients with relapsed and refractory MM, suggesting its ability to sensitize malignant cells to conventional chemotherapy [
61‐
63]. However, less promising results were obtained in patients with solid tumor compare to those with hematologic malignancies. The combinational treatment of bortezomib with docetaxel in prostate tumor did not reveal any significant anti-cancer effect [
64].
Several mechanisms of resistance have been reported and investigated in pre-clinical and clinical settings. Alterations at the proteasome level include mutations and overexpression of the catalytic subunits. For instance, alteration of the gene
PSMB5 encoding for the proteasome subunit β5 was found in tumors resistant to bortezomib
in vitro and in pre-clinical studies [
65‐
67]. However, no such mutations were found in MM patients who developed resistance to bortezomib [
68]. Resistance to bortezomib downstream of the proteasome includes enhanced activation of the aggresome-autophagy pathway, alterations in apoptotic signaling, increased expression of anti-oxidants, and dampening of the ER stress response. The role of the aggresome-autophagy pathway has been investigated as a resistance mechanism exploited by cells to overcome proteotoxicity induced by proteasome inhibition. It has been shown that pancreatic cancer cells treated with bortezomib form aggresomes, which are large aggregates of Ub-conjugated proteins that are subsequently cleared by autophagy. Studies revealed that inhibition of aggresome formation with histone deacetylase inhibitors such as panobinostat increases bortezomib anti-cancer activity, suggesting that aggresome formation may be a potential resistance mechanism [
69,
70]. Anti-apoptotic Bcl-2 mediators have also found to be overexpressed in bortezomib-resistant cells, also implicating a reduction in apoptotic capacity as a potential resistance mechanism [
71,
72]. Consistent with this, it has been shown that combination treatments with inhibitors of Bcl-2 and bortezomib showed a synergistic effect [
71,
73]. Overexpression of the chaperone BIP/Grp78 protein involved in the ER stress response to proteotoxicity has also been associated with a reduced sensitivity to bortezomib, which is not surprising considering the role of ER stress in triggering apoptosis. Pretreatment of bortezomib-resistant cells with inhibitors of ER-mediated protein folding restored cell sensitivity to bortezomib, implying that enhanced chaperone activity could also attribute to bortezomib resistance [
74].
4.2 Carfilzomib
Carfilzomib (PR-171; Kyprolis; Onyx Pharmaceutical) is a tetrapeptide epoxyketone, selective and irreversible inhibitor of the chymotrypsin-like activity of the proteasome. Carfilzomib was approved by the FDA in 2012 for the treatment of relapsed MM patients who had previously received at least two therapies, including bortezomib and immunomodulatory drugs, and displayed disease progression within 60 days after the first cycle of therapy [
75]. It is used as single agent in third-line treatments and in combination with lenalidomide and dexamethasone as second-line therapy [
76].
Carfilzomib covalently irreversibly inhibits the β5 subunit, responsible for the chymotrypsin-like activity of the 20S proteasome, resulting in a similar stress response to that observed following bortezomib [
77,
78].
Carfilzomib displayed higher cytotoxicity than bortezomib in several cell lines derived from hematologic tumors as well as solid cancer [
76]. The explanation is found in the higher selectivity of the epoxyketone for the N-terminal threonine active site of the proteasome compared to the boronic acids of bortezomib and β-lactone of salinosporamide A [
78,
79]. Carfilzomib is considered the most specific and potent proteasome inhibitor with good cytotoxic activity in bortezomib-resistant MM cell lines and in samples from patients with bortezomib-resistant MM [
80].
To date, most of the clinical trials with carfilzomib have been performed in patients with relapsed MM. However, many other studies are ongoing and/or recruiting patients with other hematological diseases, such as Hodgkin lymphoma or solid tumors like ovarian and kidney cancer. The treatment design consists of the use of carfilzomib as a single agent or in combination with conventional treatments, such as dexamethasone, melphalan, panobinostat, and irinotecan. The new phase III CLARION study is recruiting patients with newly diagnosed MM to treat with carfilzomib or bortezomib in combination with conventional drugs to evaluate if carfilzomib can replace bortezomib as front line treatment for MM [
81].
Carfilzomib is associated with several side effects mainly correlated not only to the cardiovascular system, such as hypertension, but also to the urinary tract. Moreover, carfilzomib can lead to the onset of fever, anemia, diarrhea, fatigue, and nausea [
82,
83]. The main adverse effects of carfilzomib differ from bortezomib suggesting a potential difference in downstream effects of the two drugs [
84].
4.3 Oprozomib
Oprozomib (ONX0912; PR-047), a truncated derivate of carfilzomib, is a tripeptide epoxyketone that functions as an irreversible and selective inhibitor of the chymotrypsin-like activity 20S CP. The need to find better proteasome inhibitor with better dose flexibility and convenience for patients led to the design and synthesis of oprozomib as a new generation of proteasome inhibitor with better oral bioavailability compared to the intravenously administrated carfilzomib [
85].
Oprozomib has shown similar anti-tumor activity, potency, and selectivity as carfilzomib on MM cell derived from relapsed patients after treatment with conventional anti-MM drugs and therefore can be used to treat patience with resistance to bortezomib, dexamethasone, or lenalidomide.
In vitro studies showed that oprozomib has synergistic/additive anti-MM activity when combined with bortezomib, lenalidomide, and dexamethasone and inhibits the migration of MM cells and angiogenesis. Moreover, oprozomib inhibits the tumor growth in human MM xenograft models reducing tumor progression and increasing the survival [
86]. The inhibitory activity of oprozomib in pre-clinical models of solid tumors had also been investigated. Results showed that oprozomib induced apoptotic pathway through Bik upregulation and activation of caspase-8, caspase-9, caspase-3, and PARP cleavage in head and neck squamous cell carcinoma (HNSCC) cells, leading to cell death. This effect is antagonized by upregulation of Mcl-1 and anti-apoptotic Bcl2 family member that inhibits cytochrome C release form the mitochondria. In response to proteasome inhibition, HNSCC cells upregulate autophagy and ATF4 resulting in increased cell survival. Oprozomib has shown promising inhibition of tumor growth in HNSCC xenograft mice models providing the bases for a further use of this drug in the clinic [
87]. Oprozomib also inhibits NF-κB and activates the JNK pathways [
88]. Oprozomib, similar to carfilzomib, not only targets myeloma growth directly but also decreases myeloma-associated bone disease by inhibition of osteoclast differentiation and reabsorption while enhancing osteoblast formation and function [
89]. Oprozomib is in phase I/II clinical trial for treatment of newly diagnosed, relapsed, and refractory MM as a single agent or in combination with other anti-MM drugs such as dexamethasone. A phase I study in solid tumors has also been performed; however, gastrointestinal toxicities and minimal anti-tumor activity in patients with advanced solid tumors were reported [
90].
4.4 Ixazomib
Ixazomib citrate (MLN9708, Ninlaro®, Takeda Pharmaceutical, Cambridge, MA, USA) and its biologically active form ixazomib (MLN2238) are the first orally administrated proteasome inhibitors tested in the clinic for the treatment of relapsed and refractory MM [
91,
92].
Ixazomib, like bortezomib, is a dipeptide boronate that reversibly and selectively targets the β5 proteasome subunit inhibiting the chymotrypsin-like proteasome activity with IC
50 value of 3.4 nmol/L. Moreover, at higher doses, it also inhibits the β1 caspase-like and β2 trypsin-like proteolytic subunits with an IC
50 of 31 and 3500 nmol/L, respectively [
93].
Ixazomib differs from bortezomib in its physiochemical properties leading to improved pharmacokinetics and pharmacodynamics. In fact, although both drugs have a similar potency and selectivity, ixazomib has a shorter proteasome dissociation half-life (18 min
vs. 110 min). This correlated to an improved blood and tissue distribution, which make administration of higher doses possible. Moreover, increased expression of biomarkers of proteasome inhibition, such as ER stress markers in xenograft tumor tissue, suggests improved pharmacodynamic properties. Improved pharmacokinetic and pharmacodynamic profiles have been correlated to the increased anti-cancer activity of ixazomib compared with bortezomib. In fact, ixazomib showed a better anti-cancer activity in pre-clinical studies performed on solid tumor and hematologic xenograft mice models [
93,
94].
Ixazomib also showed synergistic anti-MM activity when used in combination with dexamethasone or lenalidomide [
93,
95].
A study on microRNA profiling of MM cells treated with ixazomib showed upregulation of the small long-coding RNA miR33b, which seems to be constitutively under-expressed in MM patients. Overexpression of miR33b led to tumor growth inhibition and increased survival in human MM xenograft mice model, elucidating its tumor suppressor role during apoptosis induced by ixazomib treatment [
96].
Ixazomib was approved in 2015 in the USA by FDA and in 2016 in EU by EMA. Ongoing clinical trials are investigating its activity, as single agent or in combination with other standard anti-MM drugs, like melphalan, prednisone, lenalinomide, and dexamethasone, in hematological and solid tumors. Results have shown an evident anti-MM activity and good tolerability and safety [
97]. Like bortezomib, ixazomib’s most common side effects are thrombocytopenia, gastrointestinal symptoms, neutropenia, and fatigue but with a lower incidence of neuropathy, probably due to its higher selectivity [
37].