Cancer Chemotherapy & Biotherapy: Principles & Practices, 4th Edition

Proteasome Inhibitors

Owen A. O'Connor

In 2004, Ciechanover, Hershko, and Rose were awarded the Nobel Prize in Chemistry for their seminal work regarding the discovery of an ATP-dependent ubiquitin-mediated protein degradation pathway. 1, 2, 3, 4, 5, 6, 7 Only a year before, in May of 2003, bortezomib, the very first drug capable of inhibiting the ubiquitin-proteasome pathway (UPP), was approved by the US Food and Drug Administration (FDA) for the treatment of a disease universally characterized by an overaccumulation of protein in the body. The approval of bortezomib in multiple myeloma has now underscored the therapeutic merits of targeting this novel pathway in the treatment of cancer. It has become evident that targeting the ubiquitin-proteasome pathway is associated with a seemingly infinite array of different biological effects on the cell. These effects are not only providing new treatment options but are teaching us much about how and why cancer cells behave the way they do.

Intracellular proteolytic mechanisms may not seem like the most obvious target for anticancer drug development. Such processes are ubiquitous in all cells in the body. Drugs that affect such a “common” target might not have a therapeutic index or sufficient tumor specificity. While lysosomal pathways of protein degradation by acid hydrolases have been well clarified in eukaryotes for decades, it was a focus on protein dynamics in denervated muscle that provided some of the first clues to a nonlysosomal pathway for intracellular protein degradation.8 The observation that the rate of protein degradation, and not protein synthesis, was the basis for muscle wasting and cachexia spawned major research initiatives on how cells maintain protein homeostasis.9 In the early 1970s, work by Goldberg and others10, 11 revealed the presence of a soluble ATP-dependent proteolytic system that mediated the breakdown of intracellular proteins in Escherichia coli. While the basis for the energy requirements remained elusive for many years, Ciechanover, Hershko, and Rose made the critical observation that ATP was an absolute necessity for the covalent linkage of ubiquitin (a small 76 amino acid [~8,000 MW] heat-stable protein) to the protein substrate targeted for degradation.

Ubiquitin-conjugating reactions are a simple cellular strategy for “tagging” or earmarking specific protein substrates for proteolytic degradation at the proteasome. The structure and function of the 26S proteasome (a grouping of proteolytic enzymes into a cell organelle) had been alluded to over the years in the literature by a variety of different groups in a variety of ways. 12, 13, 14, 15, 16 In about 1990, the three essential elements of the pathway came together when it was demonstrated that, in the presence of ATP, the 20S proteasome was a component of the larger 26S proteasome, which was responsible for the proteolysis of the ubiquitin-conjugated proteins. Eighty percent to 90% of all intracellular proteins are degraded by the ubiquitin-proteasome pathway, including both short-lived and long-lived proteins.

The generation of early inhibitors of the proteasome proteases, mostly aldehyde-based peptides, soon led to the identification of a panoply of different unexpected biological effects. For example, Palombella et al.17 showed that the proteasome was important in the activation of NF-κB, which plays a major role in inflammation and malignant cell growth. The proteasome also plays a major role in regulating at least five major categories of proteins. These include (1) proteins involved in cell cycle regulation; (2) proteins involved in regulating apoptosis; (3) IκB, an inhibitor of the transcription factor NF-κB; and (4) the processing of proteins in antigen-presenting cells (APCs), peptides of which are loaded into MHC molecules for presentation to T cells; and(s) proteins involved in cell adhesion. Thus, targeting this pathway could alter a broad spectrum of cellular functions.

CHEMISTRY

The very first inhibitors of the proteasome were potent inhibitors of other proteases, some of which possess enzymatic functions similar to that of the proteasome. Four broad classes of compounds that inhibit the proteasome have been identified (Fig. 29.1 and Table 29.1), including (1) natural products (e.g., lactacystin, eponemycin, gliotoxin, epoxomicin); (2) synthetic peptide-based aldehyde inhibitors (e.g., MG132, MG 115; calpain inhibitors I and II, PSI, dipeptide aldehyde, glyoxal, CEP 1612, and bifunctional aldehydes); (3) synthetic reversible inhibitors of peptide amides and boronates (e.g., benzamide, dipeptidyl boronic esters, α-ketoamide, and bortezomib); and (4) synthetic irreversible inhibitors that are derivatives of vinyl sulfones and epoxyketones.

Figure 29.1 A–F: Representative classes of select proteasome inhibitors.

While several natural product inhibitors have been used and studied in the laboratory, none of these compounds has emerged to become a commercially viable therapeutic. One of these compounds, known as lactacystin, is a metabolite produced by the microorganism Streptomyces lactacystinaeus. Lactacystin and its derivative clasto-lactacystin β-lactone are structurally complex compounds unrelated to any of the peptide-based inhibitors (Fig. 29.1), which makes its total synthesis and clinical application more difficult and expensive.18 The β-lactone derivative of lactacystin covalently modifies the amino-terminal threonine residues of the β-subunits in the 20S proteasome, interfering with the primary mechanism of catalysis for both the tryptic-like and chymotryptic-like proteases.19 Unfortunately, like many of the compounds known as “proteasome inhibitors,” lactacystin derivatives inhibit a host of other proteases, limiting their specificity and therapeutic potential. These compounds have been shown to have significant antitumor effects and are capable of inhibiting cell cycle progression in sarcoma cell lines.20, 21 Isolation of an unidentified strain of actinomycete (Q996-17) in the early 1990s by Hanada et al.22 led to the isolation and characterization of a family of compounds known as “peptidyl epoxyketones,” including one derivative called “epoxomicin.” The subsequent total synthesis of epoxomicin23 eventually led to the demonstration that this compound is a highly specific inhibitor of the 20S proteasome. While these two molecules are among the best-known natural product inhibitors of the proteasome, most other natural product compounds have been shown to be less specific inhibitors, including some cyclic hexapeptides produced from other strains of Streptomyces.

TABLE 29.1 SELECT PROTEASOME INHIBITORS AND THEIR KI FOR INHIBITING THE 26S PROTEASOME.

Compound

CT-L (k obs / [I] (m/s))

T-L (k obs / [I] (m/s))

PGPH (k obs / [I] (m/s))

In Vivo Activity

Cross-reactivity

Reference

A. Synthetic Peptide Aldehyde Inhibitors

MG132

Ki = 4.0 nM

Ki = 2,760 nM

Ki = 900 nM

0.4 µMa

Calpain, cathepsins

Kisselev et al. (38)
Fevig et al. (131)
Loidl et al. (132)

Bifunctional aldehyde

IC50 > 100 µM

IC50 = 0.5 µM

IC50 > 100 µM

ND

ND

Myung et al. (133)

B. Synthetic Reversible Inhibitors: Peptide Amides and Boronates

Benzamide

IC50 = 0.14 µM

IC50 > 20 µM

IC50 > 20 µM

IC50 = 8 µMa

No calpain I

Lum et al. (134)

Dipeptidyl boronic ester

IC50 = 8 nM

No inhibition up to 1 µM

ND

ND

100-fold selective vs. chymotrypsin

Iqbal (135)

Bortezomib

53,000 IC50 = 0.62 nM

150

3,200

0.02 µMb

None found

Adams et al. (30)
Harding et al. (136)

Cbz-LLL-boronic acid

Ki = 0.03 nM

ND

ND

0.04 µMb

ND

Adams et al. (31)
Harding et al. (136)

C. Synthetic Irreversible Inhibitors: Vinyl Sulfones and Epoxyketones

NLVS

5000

3.4

4.0

8 µMa

Cathepsin S and B

Bogyo et al. (137)

D. Natural Product Inhibitors

Lactacystin

1,500

110

17

4 µMa

Cathepsin A, TPPII

Bogyo et al. (138)

Clasto-lactacystin β-lactone

7,400

68

47

ND

Cathepsin A, TPPII

Elofsson et al. (139)

Epoxomicin

20,000

300

40

0.03 µMa

None found

Myung et al. (133)

Dihydroeponemycin

114

17

217

2 µMb

Cathepsin (weak)

Meng et al. (140)
Myung et al. (133)

CT-L, chymotrypsin-like; T-L, trypsin-like; PGPH, post-glutamyl peptide hydrolase; ND, not determined;
Ki, inhibition constant.
aEighty-percent growth inhibition of EL4 cells
bFor intracellular proteolysis of proteasome substrate and MHC1 processing.
Adapted from Kim KB, Crews CM. Natural product synthetic proteasome inhibitors. In: Adams J, ed. Proteasome Inhibitors in Cancer Therapy. Totowa, NJ: Humana Press, 2004:47–63.

Figure 29.2 Chemical structure of bortezomib.

 

Like lactacystin, peptides containing a C-terminal vinyl sulfone moiety have been shown to covalently modify the N-terminal active site on the threonine of the β-subunits. These compounds are typically irreversible inhibitors and were originally introduced as cysteine proteases. Subsequent efforts to refine their structure to generate analogs with increased selectivity and potency succeeded only in generating compounds with dual inhibitor properties (i.e., they inhibited both threonine proteases and cysteine proteases).24, 25 As a result, while these compounds still serve as valuable tools in the laboratory, their lack of specificity has relegated them to experimental use only.

Several classes of reversible and irreversible synthetic inhibitors of the proteasome have been described. The synthetic peptide-based aldehydes were among the first potent reversible inhibitors described. Their structures have provided a model upon which many new analogs have been synthesized with even better and more selective inhibitory effects against the 20S proteasome. For example, leupeptin is a well-known inhibitor of serine proteases (e.g., trypsin and plasmin) and cysteine proteases (papain and cathepsin B) and is also a relatively potent inhibitor of the trypsin-like activity in the 20S protesasome.26 In contrast, the calpain inhibitors have been shown to be potent inhibitors of the chymotrypsin-like activity.12, 27 Many of these compounds, including MG115 (acetyl- Leu-leu-norleucinal, also known as Calpain Inhibitor I or ALLN) and MG132 (Cbz-leu-leu-leucinal), are tripeptide aldehydes with potent inhibitory activity against the chymotryptic-like component of the 20S proteasome.28 MG132 exhibits a Ki against the chymotrypsin-like, trypsin-like, and peptidyl glutamyl hydrolase enzymes, of 4, 2,760, and 900 nM, respectively (Table 29.1).

Most aldehyde-based proteasome inhibitors were found to be relatively nonspecific.29 Chemical synthetic efforts to develop novel compounds with more selective properties eventually led to the identification of a new class of chemicals that were synthetic reversible peptide amide and boronic acid derivatives. Early efforts focused on altering the chemically interactive moiety of the inhibitor (or “warhead”) that interfaces with the active site of the proteasome. A series of peptidyl aldehyde compounds that were derivatives of boronic acid were found to display dramatically enhanced potency inhibiting the chymotryptic-like enzymatic activities.30 To date, a variety of boronic acid peptide inhibitors have been developed and studied,30, 31 of which bortezomib (formerly known as PS-341) was selected as a lead compound (Fig. 29.2). Independently, Lowe et al.32 determined the x-ray crystallographic structure of the 20S proteasome from an archaebacterial species. These pivotal studies revealed a unique mode of catalysis in the proteasome, a mechanism that centered around a threonine residue.32 Targeting the unique threonine residue within the proteasome proteases provided the selectivity of the aldehyde boronic acid derivatives. N-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid (Fig. 29.2; formerly known as PS-341, now known as bortezomib and Velcade) selectively inhibited the threonine residue on the chymotryptic-like enzyme (Ki ~0.6 nM) specifically through the boronic acid moiety (Table 29.2) and did not inhibit other important proteases in the cell, including chymotrypsin (Ki ~320 nM), thrombin (Ki ~13,000 nM), and trypsin.

MECHANISM OF ACTION

How inhibiting the proteasome leads to cell death is still an area of intensive research. What is clear is that there is a remarkable diversity of biological processes that are affected by proteasome inhibition. The sentinel event leading to cell death could be distinct in different diseases.

TABLE 29.2 THE Ki (nM) FOR BORTEZOMIB AGAINST SELECT INTRACELLULAR PROTEASES

Specific Protease

Bortezomib (Ki app) [nM]

20S proteasome

0.62

Chymotrypsin

970

Trypsin

>10,000

Cathepsin B

>10,000

Elastase

5,700

Calpain I

>10,000

Calpain II

>10,000

Angiotensin-converting enzyme (ACE)

490

Thrombin

>10,000

Factor XIIa

>10,000

Tissue plasminogen activator (TPA)

>10,000

Papain

>10,000

The Ubiquitin-Proteasome Pathway (UPP)

The ubiquitin-proteasome pathway (UPP) consists of two major components: the ubiquitinating enzyme complex and the proteasome. Three enzymes make up the ubiquitinating enzyme complex, the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin protein ligase (E3). E1 is a generic enzyme used by the pathway regardless of the protein substrate targeted. In contrast, there are 20 to 30 different ubiquitin-conjugating enzymes (E2) and likely hundreds of ubiquitin protein ligases (E3). The ligase step is the point where the specificity of the ubiquitylation process is controlled, with most protein substrates having their own distinct ubiquitin protein ligase. 33, 34, 35 Each of these enzymes represents a potential therapeutic target, offering unique ways to selectively inhibit the degradation of very specific proteins or groups of proteins. The initial step of this process involves the binding of this three-enzyme complex, known also as the ubiquitinating enzyme complex (UEC), to the N-terminus of the target protein (Fig. 29.3). This enzyme complex catalyzes the covalent linkage of ubiquitin molecules to the ε-amino moieties of internal lysine residues in a processive manner. These ATP-dependent catalytic reactions eventually lead to the generation of a branched polyubiquitylated protein. This ubiquitylation process is the fundamental means by which the cell “tags” or “earmarks” specific proteins for proteolytic degradation at the 26S proteasome.

The second major component of the pathway includes the proteasome proper (Fig. 29.4). The proteasome is composed of two components, the 20S proteasome (720 kd) and the 19S regulatory subunit (890 kd). Collectively, they form a complex called the 26S proteasome.36 The isolated 20S proteasome exhibits no proteolytic activity in the absence of the 19S regulatory subunit. The 19S subunit mediates the ATP-dependent process of denaturing and unfolding the ubiquitin conjugates. The 26S proteasome is a very large (2.5 MDa) structure composed of 44 distinct polypeptides. The proteases within the proteasome possess different mechanisms of catalysis relative to other proteases. Specifically, they are threonine proteases, not the usual serine and cysteine proteases. In addition, the proteasome proteases are known to be highly processive. Normally, most proteases cleave their substrate once, generating two new fragments. In contrast, the proteasome, by virtue of the fact that its is a redundant multicatalytic enzyme, cleaves polypeptides at multiple sites, releasing very small peptides ranging in length from 2 to 24 amino acids, with a median size of six to seven residues each. 37, 38, 39

Figure 29.3 Schematic of the ubiquitin-proteasome pathway.

Figure 29.4 Structure of the 26S proteasome and its assembly.

The 19S regulatory subunit consists of at least nine polypeptides, including multiple isopeptidases that disassemble and unfold the polyubiquitin conjugates. It also plays a major role in facilitating and regulating the many ATP-dependent processes used by the proteasome. In fact, this energy-consuming process requires ATP for a number of functions including complex formation resulting from the 20S and 19S assembly; protein unfolding; ubiquitylation; the actual translocation of ubiquitin conjugates into the 20S lumen; the opening of the regulatory “gate” that allows the protein to enter the 26S lumen; and the action of the isopeptidases, which result in the recycling of the ubiquitin molecules.40

The 20S proteasome (720 kd) consists of four rings, each containing seven individual globular proteins. As can be seen from Figure 29.4, the assembly of the four rings forms a central lumen through which proteins are funneled. The two outer or flanking layers of the 20S proteasome are referred to as the α-layers and are basically a structural feature that allows anchorage of the 19S regulatory subunit. The two inner rings are referred to as the β-layers and contain the distinct proteases that account for the proteolytic activity of the proteasome. The 19S regulatory subunit sits on the top and bottom of the 20S subunit, controlling the entry of ubiquitylated proteins into the core of the proteasome.40 Once the protein enters the lumen of the proteasome, the proteases digest the protein into smaller peptide fragments. The activity of these proteases can be quantitatively assessed by using specific fluorogenic peptides that are substrates for only one of these proteases. These assays have provided a valuable tool by which biochemists can decipher which of the three different proteolytic functions are being affected by test inhibitors. To date, at least three different enzymatic activities have been ascribed to the β-layers, including (1) a peptidylglutamyl activity (β1) that cleaves proteins near glutamate residues; (2) a tryptic-like function (β2) that cleaves proteins next to the basic amino acids lysine and arginine; and (3) a chymotryptic-like function (β5) that cleaves proteins near the aromatic amino acids phenylalanine, tyrosine, and tryptophan. 36, 41, 42, 43, 44 Bortezomib is a reversible and selective inhibitor of the chymotryptic-like protease found in the 26S proteasome (Fig. 29.5).

Influencing the NF-κB Pathway

The early discovery by Palombella et al.17 that proteasome inhibition could lead to inactivation of NF-κB helped formulate the hypothesis that inhibitors of this pathway could have effects on a vast array of biological processes. NF-κB is the prototype for a family of dimeric transcription factors involved in the inflammatory and immune responses, promotion of cell growth, and blocking of apoptosis. Constitutive activation of NF-κB has been described in a number of solid tumors, including pancreatic and colorectal cancer, as well as several hematological malignancies. Its role in drug resistance and oncogenesis has now been firmly established.

Figure 29.5 Cross-sectional view of the B-ring and the binding of bortezomib (Bort) to the chymotryptic-like protease.

Five members of the NF-κB family—p65/RelA, c-Rel, Rel B, p52/100, and p50/105—have been defined, all capable of forming a number of different homodimeric or heterodimeric complexes.45, 46 The activation of NF-κB is tightly regulated through its inhibitor IκB. IκB, in fact, belongs to a family of proteins that includes IκBα, IκBβ, IκBε, and Bcl-3. The interaction of IκB with the dimeric transcription factors blocks the nuclear translocation of NF-κB, preventing access of the transcription factor to DNA. Activation of NF-κB typically involves the induction of IκB kinase (IKK), leading to the phosphorylation of IκB, followed by its ubiquitylation and eventual degradation by the 26S proteasome. Interestingly, a number of different stimuli have been shown to induce IκB, including tumor necrosis factor-α (TNF-α), lipopolysaccharide, various interleukins (ILs) and interferons, hypoxia, and even cytotoxic drug exposure. 45,47, 48, 49, 50 Liberation of free NF-κB leads to its translocation into the nucleus, where it binds to the promoter region of several genes, resulting in transcriptional activation. If serine residues on IκB are mutated to alanine, which cannot be phosphorylated, IκB cannot be degraded, and it permanently sequesters NF-κB in the cytoplasm. This mutated form of IκB has proven to be a model for mimicking the effect of proteasome inhibition.51

One of the first clues that NF-κB was important in cancer biology came in the mid-1980s, when Sen and Baltimore52 first described the protein as a B-cell transcription factor that bound a site in the immunoglobulin enhancer. Subsequently, these same authors demonstrated that NF-κB could be activated in a variety of other cells following exposure to assorted stimuli like phorbol esters and that the activation of NF-κB was independent of protein synthesis.53 The range of NF-κB inducers has continued to grow beyond those typically associated with immune function. The variety of stimuli known to induce NF-κB47 are enormous and are known to include (a) a number of immune effector proteins, such as tumor necrosis factor (TNF-α), lymphotoxin, IL-1 and -2, granulocyte/macrophage colony-stimulating factor (GM-CSF), allogeneic stimulation, lectins, phorbol ester, diacylgycerol, CD40 ligand, leukotriene B4, and prostaglandin E2; (b) physical stresses, such as UV irradiation, ionizing radiation, and hypoxia; oxidative stresses like hydrogen peroxide, oxidized lipids, and butyl peroxide; (c) many different chemical agents, such as conventional chemotherapy drugs and protein synthesis inhibitors; and (d) a host of infectious agents, including bacterial products, viruses, and some parasites. These stimuli have been shown to activate NF-κB primarily through the induced degradation of IκB, allowing for transcriptional activation within minutes of exposure.

Perhaps even more daunting are the number of NF-κB responsive genes,47 which include (a) a host of cytokines and growth factors, such as IL-1, IL-2, IL-6, IL-8, TNF-α, GM-CSF, G-CSF, and interferon-β; (b) a number of immunoreceptors, such as immunoglobulin κ light chains, the T-cell receptor β-chain, major histocompatibility complex class I and II (MHC-I and II), and β2 microglobulin; (c) several adhesion molecules, such as endothelial-leukocyte adhesion molecule-1 (ELAM-1), vascular endothelial adhesion molecule-1 (VCAM-1), and intracellular cell adhesion molecule-1 (ICAM-1); and (d) a number of transcription factors, such as Rel, p105, IκB-α, c-myc, and interferon regulatory factor-1. These lists suggest the complex biology influenced by NF-κB. While targeting this pathway may be a fruitful strategy in drug development, it is also clear that identifying a singular mechanism of action across different diseases will likely remain elusive. Furthermore, it provides credence to the idea that inhibitors of the proteasome that can influence such complex signaling cascades are likely to be associated with a multitude of biological consequences and that inhibition of NF-κB alone is not likely to be the sole mechanism of action in any disease. The broad range of biological effects of NF-κB and its ubiquitous expression also raise concerns about undesired effects of NF-κB inhibition.

Effects on Cell Cycle Regulation

Control of cell cycle transition points depends heavily on both transcriptional and posttranscriptional mechanisms. The timely degradation of essential regulatory proteins through the UPP allows for the coordinated progression of a cell through the cell cycle. Inhibition of the degradation of any of the host of proteins that play a role in orchestrating this process can have a profound influence on cell cycle kinetics. For example, p21 and p27 are members of the Cip/kip family of cyclin-dependent kinase inhibitors, which halt cell cycle progression at the G1-S junction by inactivating the cyclin/cdk complexes.54 The cdk inhibitor p21WAF/CIP1, a transcriptional target of p53, is thought to be what couples cellular differentiation with cell cycle arrest. Treatment of the human colon cancer cell lines RKO and HCT-116 with the proteasome inhibitor lactacystin results in cell cycle arrest mediated by p21 accumulation.55 Another well-established cell cycle control mechanism revolves around the expression of cyclin B, the synthesis of which begins very early in the S phase, accumulating to its highest levels during G2 and early mitosis (M phase). During these phases of growth, cyclin B is complexed with the CDK/cdc2, with specific phosphorylation and dephosphorylation of cdc2 governing the activity of the complex.56 Progression into and through anaphase is tightly dependent on the degradation of cyclin B, which is tightly controlled through its ubiquitylation and proteolytic degradation by the 26S proteasome.57 Similarly, entrance into the S phase from G1 is controlled by the cyclin E-CDK2 complex. Association of this complex with p27 is responsible for inhibiting the kinase activity, leading to cell cycle arrest.58, 59 Degradation of the cell cycle inhibitory protein p27, allows the cell to pass the G1/S transition, allowing for DNA synthesis within only hours. This critical event is also tightly controlled through the ubiquitylation- and proteasome-mediated degradation of p27.58, 60, 61Hence, one important mechanism of action of proteasome inhibitors is to dysregulate normal cell cycle kinetics, usually by inducing cell cycle arrest.

The significance of these particular changes was underscored in an evaluation of several forms of non-Hodgkin's lymphoma (NHL), in which most mantle cell lymphomas (91 of 112) and diffuse large B-cell lymphomas (12 of 19) were shown to have lost expression of p27.62, 63 In contrast, small lymphocytic lymphoma and extranodal marginal zone lymphomas were found to have ample p27 expression.62 Mantle cell lymphomas actually had normal p27 mRNA expression but increased p27 protein degradation through the ubiquitin-proteasome pathway. Interestingly, overexpression of p53 and/or loss of p27 in patients with MCL correlated with a statistically significant reduction in overall survival.63 While the mechanism of p27 loss is not entirely clear, some have suggested sequestration with cyclins D1 and D3, while others have suggested that the level of Skp2, a component of the p27Kip1 ubiquitin ligase, may be important in select NHLs.62 The basis for the involvement of Skp2 revolves around the observation that high Skp2 levels correlate with greater E3 activity and hence greater proteasome-mediated degradation of the target protein (i.e., p27), establishing an inverse relationship between the two. In aggressive lymphomas and blastic MCL, high Skp2 levels were associated with a low p27Kip1 level, suggesting increased proteasome-mediated degradation of p27Kip1.62Loss of p27 identified a poorer outcome among the p53 negative cases and established this pathway as an important high-risk marker in these patients, potentially providing a therapeutic rationale for proteasome inhibitors in mantle cell lymphoma. Furthermore, MCL cell lines (i.e., Granta 519 and NCEB) treated with lactacystin failed to accumulate cyclin D1 or cdk4 (despite the overexpression mediated by the t[11:14] translocation), though cell cycle arrest and induction of apoptosis was accompanied by accumulation of the cdk inhibitor p21 and p27 in both cell lines.64 These observations have been corroborated in human non–small cell lung cancer (NSCLC) cell lines as well, where a pronounced G2-M phase arrest was associated with the inhibited degradation of p21cip/waf-1.65 There is much to be learned about how proteasome inhibition affects the cell cycle regulation and whether those actions will translate into a specific antitumor effect.

Induction of Apoptosis

Proteasome inhibition can lead to apoptosis through both direct and indirect influences.66 NF-κB mediates an antiapoptotic effect. For example, Beg and Baltimore67 and Van Antwerp et al.68 have shown that activation of NF-κB suppressed TNF-α–induced apoptosis. NF-κB also protects cells exposed to either ionizing radiation or daunorubicin from death. The inhibition of NF-κB nuclear translocation has been found to enhance apoptotic killing by radiation or chemotherapy.69 This represents one mechanism through which proteasome inhibitors can influence cell death signaling.

Other more direct influences of proteasome inhibition on the induction of apoptosis have been shown in leukemic cells following exposure to the proteasome inhibitor lactacystin. Marshansky et al.70 demonstrated in both Jurkat (T-cell tumor) and Namala (B-cell tumor) cell lines that inhibition of the proteasome differentially up-regulated a proapoptotic Bcl-2 family member known as Bik by decreasing its proteolytic degradation. Interestingly, other family members in this model, including Bax, Bak, and Bad, were not similarly affected. Up-regulation and accumulation of Bik was shown to be sufficient for inducing apoptosis in these leukemic cells. Additionally, these authors showed that proper functioning of the electron transport chain is dependent on proteasome activity and that interruption of protein turnover adversely influenced the transmitochondrial membrane potential, leading to the induction of apoptosis. It was shown that Bik and the antiapoptotic member Bcl-XL coprecipitated, leading to the hypothesis that excess Bik could trap and theoretically nullify the influences of Bcl-XL, since the level of Bcl-XL is not changed following proteasome inhibition. This “trapping” of the Bcl-XL then offers a theoretical mechanism for overriding the antiapoptotic effects of Bcl-XL, leading to cell death.

The temporal sequence of events following exposure to the proteasome inhibitor bortezomib on Bcl-2 was shown by Ling et al.71 Treatment of the H460 cell line with bortezomib resulted in a both a time-dependent and concentration-dependent set of effects on Bcl-2 phosphorylation and cleavage. For example, treatment of the cells with bortezomib resulted in cleavage of Bcl-2, with the identification of a unique cleavage product (Mr 25,000). The generation of this cleavage product was not prevented by caspase inhibitors, which was the case with the Mr 23,000 cleavage product typically seen following exposure to conventional cytotoxic therapies, suggesting the possibility of a caspase-independent pathway. The Bcl-2 cleavage products accumulated in the mitochondrial membrane early, usually 12 hours after exposure, while poly(ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation were seen about 36 hours post exposure. The authors concluded that inhibition of the proteasome resulted in a prompt phosphorylation of Bcl-2, leading to the formation of a unique cleavage product that was associated with G2-M arrest and the induction of apoptosis.71

A link between the NF-κB and apoptotic pathways was also established by Heckman et al.,72 and it may provide some insight into one of the possible mechanisms of action in follicular lymphoma. Follicular lymphomas are well characterized by the translocation of the bcl-2 proto-oncogene from chromosome 18q21 to the immunoglobulin heavy chain locus (IgH) at chromosome 14q32. 73, 74, 75 The resulting translocation, t(14:18), is the sine qua non lesion seen in follicular lymphoma and leads to the overexpression of the antiapoptotic bcl-2 protein, protecting cells from apoptosis. In addition, lymphoma cells carrying the t(14:18) also overexpress NF-κB. These authors demonstrated that cell lines expressing an IκBα super-repressor exhibited marked reductions in bcl-2 protein, implying a role for NF-κB in cells carrying this translocation. These observations could provide a rationale for employing proteasome inhibitors in follicular lymphoma.72 While the impact of the NF-κB signaling pathway in these studies was not analyzed, it is reasonable to suggest that the pathways leading to the induction of apoptosis in cancer cells will involve both direct and indirect influences on the mitochondria. The ultimate pathway leading to cell death is likely to depend on the molecular perturbations that drive the behavior of that malignant clone. For example, in mantle cell lymphoma, inhibition of the constitutive activation of NF-κB leads to the induction of both cell cycle arrest and apoptosis through the down-regulation of bcl-2 family members and the activation of caspases.76

The Unfolded Protein Response

One of the lesser recognized mechanisms that may lead to cell death or injury following inhibition of the ubiquitin-proteasome pathway is based on the cell's need to “protect” itself from misfolded or damaged intracellular proteins. Such mechanisms for damaged protein elimination have been established in prokaryotes long before the discovery of the ubiquitin-proteasome pathway was discovered in eukaryotes.11, 77 Misfolded or damaged proteins can arise in any cell through errors in DNA repair or spontaneous mutation. In addition, there are a host of other physical and chemical factors that can lead to damaged intracellular protein, including denaturing conditions at high temperatures; the presence of oxidizing agents or high redox conditions; activated proteases or kinases, high salt concentrations, which may favor disaggregation of large multimeric complexes; or the presence of detergents. Resulting proteins can be highly noxious to the cell and are capable of inducing cell death. This mechanism of cell death, though not well defined, is likely due to the ability of these damaged proteins to form insoluble aggregates. Such protein inclusions have been seen in a variety of inherited and neurodegenerative disease, strongly pointing towards a failure of the cell to break down intracellular protein as the basis for the underlying pathology.

Lee et al.78 have provided some valuable insight into this process, especially in multiple myeloma. Using a variety of myeloma cell lines they demonstrated that the transcription factor XBP-1 is an important inducer of plasma cell differentiation. XBP-1 is a major regulator of this unfolded protein response in plasma cells. Treatment of myeloma cells with proteasome inhibitors resulted in impaired production of XBP-1, leading to the induction of their apoptosis.78When unfolded proteins accumulate inside cells, they tend to saturate the proteolytic capacity of the cell (as might occur following treatment with any proteasome inhibitor), leading to the activation of the heat shock response42, 79 and the induction of more proteasome biosynthesis.80 The continued accumulation of these proteins eventually triggers the activation of Jnk kinases and apoptosis.81, 82 Blocking the cell's mechanisms for protecting itself against damage by misfolded proteins with proteasome inhibitors alone or in combination with agents that inhibit heat shock proteins may provide new therapeutic opportunities for both malignant and nonmalignant diseases.

PRECLINICAL PHARMACOLOGY

Proteasome inhibitors have potent anticancer properties. Imajoh-Ohmi et al.83 demonstrated that the microbial metabolite lactacystin induced cell death in U937 cells at concentrations of about 5 µM. These insights were broadened when several groups reported that tripeptide inhibitors of the proteasome but not lysosomal protease inhibitors induced apoptosis in Rat-1 and PC12 cells in a p53-dependent manner.84 Normal cells were found to be more resistant to proteasome inhibitors than transformed cells. For example, the proteasome inhibitor CEP1612 induced apoptosis selectively in simian virus 40 (SV-40)–transformed cells but not in normal human fibroblasts.85 Orlowski et al.86 demonstrated that fibroblasts transformed with ras and myc, lymphoblasts transformed by c-myc alone, and a Burkitt's lymphoma cell line that overexpresses c-myc were up to 40-fold more susceptible to apoptosis induced by proteasome inhibitors than were primary rodent fibroblasts or immortalized nontransformed human lymphoblasts. The explanation for these observations remains unclear.

Boronate proteasome inhibitors have been shown to kill tumor cells in culture, as demonstrated by their activity in the NCI tumor cell line screen.31 Data from this well-characterized cell line screen established an excellent correlation between the intrinsic potency of different proteasome inhibitors (the Ki [nM]) and their cytotoxicity.31, 87 Using data from the NCI's algorithm COMPARE, the analysis established that the mechanism of cytotoxicity of bortezomib was markedly different from any of the other 60,000 compounds already in the library. The average growth inhibition of (GI50%) for bortezomib was 7 nM across the entire panel of cells. More detailed in vitro and in vivo data were obtained using the prostate cancer cell line PC-3, in which bortezomib was found to result in an accumulation of cells in G2-M, which was attributed to the accumulation of CDK inhibitors p21 and p27. Cell death was noted at 20 and 100 nM for cells incubated for 24 and 48 hours in the presence of bortezomib.31 In a nude mouse model with PC-3, bortezomib was found to kill the tumor in a dose-dependent manner.

Intravenous dosing with radiolabeled [14C]-bortezomib in rats followed by whole-body autoradiography revealed that the highest radioactivity levels 10 minutes after drug administration were in the adrenal glands, renal cortex, liver, prostate, and spleen. Lower levels were found throughout other organs, including skeletal muscle, skin, and blood, while no detectable drug could be found crossing the blood-brain barrier (brain, eye, testes). Sixty-six percent of the radiolabeled drug was recovered from the bile, while the remainder was recovered from the urine.31

Multiple myeloma is one disease where significant activity has been seen in both clinical and preclinical models. 88, 89, 90 In vivo models of drug-resistant human myeloma have shown that twice weekly schedules of bortezomib administration resulted both in tumor shrinkage and prolonged median survival of treated mice. 89, 90, 91 Preclinical models of multiple myeloma have shown that proteasome inhibitors induce the down-regulation of the cytokine-induced expression of E-selectin, vascular cell-adhesion molecule-1 (VCAM-1), and intracellular cell adhesion molecule 1.92 VCAM-1 is essential for facilitating the interactions between the myeloma cells and the bone marrow stromal cells, an interaction that leads to the protection of the myeloma cells from apoptosis.93,94 Second, the inhibition of NF-κB reduces the transcription of the NF-κB–responsive IL-6 gene. IL-6 is a growth and survival factor for multiple myeloma cells lines.89 Most recently, lactacystin, bortezomib, and MG262 have been shown to have antiangiogenic properties and may help to reduce the degree of neovascularization in the bone marrow, adversely affecting the survival of the myeloma cells. 95, 96, 97, 98, 99 Obviously, many of these latter effects would not be appreciated in the standard in vivo or in vitro models of anticancer drug screening.

CLINICAL PHARMACOLOGY

Pharmacokinetic (PK) Profiles and Metabolism

Studies in nonhuman primates have shown that the tissue distribution is extensive, though the drug does not appear to cross the blood-brain or blood-testes barriers. After a single intravenous dose of bortezomib, plasma concentrations decline in a classic biphasic manner, which is characterized by a very rapid distribution period, with a t½α of approximately 10 minutes.100 The terminal elimination phase in humans has been estimated between 5 and 15 hours. Figure 29.6 presents a plot of the mean plasma concentrations of bortezomib following a single and one-repeat dose of bortezomib in patients receiving 1.0 or 1.3 mg/m2. Multiple doses of drug appear to result in some decrease in clearance, with a resulting increase in the terminal elimination half-life and AUC, but they have no effect on the estimated Cmax or distribution half-life. For example, in patients with solid tumor malignancies, the mean terminal half-life increased from 5.45 to 19.7 hours, while the AUC increased from 30.1 after the first dose to 54 hr × ng/mL after the third dose of the first cycle (MPI, on file, 2004). These pharmacokinetic profiles have also been observed in preclinical studies in rodents and cynomolgus monkeys and do not appear to result in increased toxicity from accumulation of the drug with repeat dosing. The overall disposition of bortezomib is most consistent with a two-compartment PK model.

Figure 29.6 Plasma pharmacokinetics of the 1.0 and 1.3 mg/m2 doses.

Figure 29.7 Major metabolites of bortezomib.

The principal pathway of elimination of bortezomib is through oxidative deboronation (Fig. 29.7). Based on in vitro studies, the major phase 1 metabolic reactions are mediated by cytochrome P450 isoforms 3A4 and 2C19, while the major phase 2 conjugation pathways do not appear to play any major role in elimination.101 The inactive deboronated metabolites then undergo a series of hydroxylations, leading to their elimination. Radiolabeled studies using [14C]-bortezomib have confirmed elimination of some metabolites through both renal and hepatic routes (approximately 66% of the initial drug load), though only a small quantity of intact bortezomib is recovered from the urine (MPI, on file, 2004).

Pharmacodynamic Endpoints

The critical pharmacodynamic endpoint for bortezomib is proteasome inhibition in vivo. As shown in Figure 29.8, 20S proteasome inhibition was quantified as a function of both dose and time. 102, 103, 104, 105 Blood samples were collected before therapy and then 1, 6, and 24 hours after each dose of bortezomib in the first cycle for measurement of the 20S proteasome activity. The degree of proteasome inhibition was measured by quantitating the rate of hydrolysis of sample fluorogenic peptide substrates relative to the normalized chymotryptic activity. Bortezomib induced a dose-dependent inhibition of the 20S proteasome compared with pretreatment controls, with the 0.40, 1.04, 1.20, and 1.38 mg/m2 doses producing inhibition levels of 36%, 60%, 65%, and 74%, respectively. Doses of bortezomib less than 0.4 mg/m2 produce marginal inhibition of the proteasome. The majority of the proteasome inhibition is attained at about 1 hour post drug infusion, after which proteasome inhibition decays with a half-life of about 24 hours. The data exhibit a classic sigmoidal maximal effect distribution, where larger increases in dose produce correspondingly smaller interval increases in proteasome inhibition. At 72 hours post bortezomib infusion, there is little to no residual inhibition of the proteasome. Figure 29.9 shows that the plasma levels of bortezomib predict the level of proteasome inhibition. This pharmacodynamic endpoint has helped to establish the clinical basis for the once-every-72-hours dosing strategy, along with the recognition that dosing bortezomib more frequently would lead to a “stacking” of the proteasome inhibition, eventually leading to complete inhibition of the proteasome. Based on several in vivo models, complete inhibition of the proteasome is not compatible with life, while about 60 to 70% inhibition of the proteasome may be required for the bulk of the anticancer activity.

Figure 29.8 Proteasome inhibition as a function of dose.

Single-Agent Phase I Experience

To date, three phase I experiences with bortezomib have been completed (Table 29.3), with one of these devoted to patients with hematologic malignancies. 102, 103, 104 These phase I experiences have explored weekly and twice-weekly intravenous bolus schedules. In general, the pattern of toxicity paralleled what was noted in the preclinical findings. In the phase I study conducted by Papandreou et al.,104 bortezomib was administered weekly × 4 weeks every 6 weeks, establishing 1.6 mg/m2 as the maximum tolerated dose (MTD) on this schedule. The authors noted that hypotension and syncope were dose-limiting toxicities on the weekly schedule up to 2 mg/m2. In what has now emerged as the “standard schedule” for bortezomib in most of the early phase II studies done to date, Aghajanian et al.103 conducted a phase I study of bortezomib in a range from 0.4 to 1.5 mg/m2 twice weekly for 2 weeks every 3 weeks. On this schedule, diarrhea and neuropathy were the dose-limiting toxicities. While there is some debate about the MTD established in this study (1.3 vs. 1.5 mg/m2), most have adopted 1.3 mg/m2 as the MTD on this schedule.

Figure 29.9 Plot of the plasma pharmacokinetic profile and the kinetics of proteasome inhibition.

TABLE 29.3 PHASE I STUDIES OF BORTEZOMIB

Schedule

Length of Cycle (weeks)

Doses of Bortezomib (mg/m2)

No. of Patients

MTD (mg/m2)

DLT

Study

Weekly × 4 weeks

6

0.13–2

53

1.6

Hypotension, syncope

Papandreou et al. (104)

Twice weekly × 2 weeks

3

0.4–1.5

43

1.3a

Diarrhea, neuropathy

Aghajanian et al. (103)

Twice weekly × 4 weeks

6

0.4–1.38

27

1.04

Hyponatremia, hypokalemia, malaise

Orlowski et al. (102)

MTD, maximum tolerated dose; DLT, dose-limiting toxicity.
aMTD in publication noted to be 1.56 mg/m2; generally accepted MTD based on available studies with this schedule is 1.3 mg/m2.

The phase I study reported by Orlowski et al.102 was the only one devoted to patients with hematologic malignancies. This multicenter study conducted by investigators at the University of North Carolina and Memorial Sloan-Kettering Cancer Center included 27 heavily pretreated patients with a variety of hematologic malignancies, including Hodgkin's disease (n = 4), small lymphocytic lymphoma/chronic lymphocytic leukemia (n = 2), diffuse large B-cell lymphoma (n = 2), mantle cell lymphoma (n = 3), Waldenstrom's macroglobulinemia (n = 1), and multiple myeloma (n = 11). The patients had received a median of 3 prior forms of chemotherapy (range, 1 to 12), and over one third (n = 10) had had prior high-dose chemotherapy and peripheral blood stem cell transplantation. The drug was administered twice a week for 4 consecutive weeks every 6 weeks at dose levels of 0.4, 1.04, 1.2, or 1.38 mg/m2. The major dose-limiting toxicities included thrombocytopenia, which was observed at every dose level; hyponatremia; hypokalemia; fatigue; and malaise. This twice-weekly schedule for 4 of 6 weeks established a slightly lower MTD of 1.04 mg/m2. Interestingly, among the nine fully assessable patients with heavily pretreated plasma cell dyscrasias completing at least one cycle of therapy, one achieved a complete remission, and eight other patients demonstrated a major reduction in paraprotein levels and/or bone marrow plasma cells. In addition, one of three patients with mantle cell lymphoma achieved a durable partial remission, as did one patient with follicular lymphoma.

Phase II Experience

A variety of single-agent studies have been conducted in patients with solid tumors and hematologic malignancies. A summary of these experiences is presented in Table 29.4. The promising activity seen in multiple myeloma has led to FDA approval for that disease. Other studies focusing on lymphoma, NSCLC, and renal cell carcinoma have also been reported.

Multiple Myeloma

Based on the responses seen in multiple myeloma in the phase I study, a large multicenter, open-label, nonrandomized phase II trial of bortezomib for patients with relapsed or refractory multiple myeloma was initiated (the Summit trial). Patients were required to have relapsed and progressive disease following conventional chemotherapy and to have disease refractory to salvage chemotherapy, as defined by progression of disease during treatment or within 60 days of completion of their therapy. Two hundred and two patients were registered to the study. Treatment consisted of bortezomib administered at a dose of 1.3 mg/m2 as an intravenous push given on days 1, 4, 8, and 11 every 21 days. The study was conducted in a very heavily pretreated population of patients with a median of 6 lines of prior treatment, which included high-dose chemotherapy and stem cell transplantation in almost two thirds of patients. Based on the report of Richardson et al.106 and the analysis by the FDA,107 the overall response rate according to the stringent Blade criteria was 27.7%, with 2.7% (n = 5) of patients meeting criteria for complete remission (complete disappearance of paraprotein). The median time to response was 38 days (range, 30 to 127 days), and the median duration of response was 365 days (range, 41 to 509 days). Interestingly, these responses were independent of the number and type of previous therapies, performance status, β2 microglobulin level, chromosome 13 deletion status, or type of myeloma. The most common adverse effects (all grades) included nausea (64%), diarrhea (49%), fatigue (49%), thrombocytopenia (44%), constipation (43%), vomiting (36%), anorexia (34%), and sensory neuropathy (34%). Overall, most of these adverse effects were grade 1 or 2. Severe adverse events (>grade 3) included thrombocytopenia (29%), peripheral neuropathy (14%), neutropenia (15%), asthenia (11%), and anemia (9%). The frequency and severity of the diarrhea appeared to be dose-dependent. Based on these data, bortezomib was approved by the FDA for the treatment of relapsed or refractory multiple myeloma in May 2003.

TABLE 29.4 SUMMARY OF SELECT EFFICACY STUDIES OF BORTEZOMIB

Disease1

n

ORR

CR

Duration of Response (months)

Comments

Reference

Multiple myeloma (Summit study)

202

35%

4%

12

Median overall survival was 16 months

Richardson et al. (106)

Multiple myeloma (Crest study)

54

1.3 mg/m2 = 38%
1.1 mg/m2 = 30%

3.7%

ND

Compared 1.3 vs. 1.1 mg/m2

Jagannath et al. (108)

Indolent and mantle cell lymphoma

25
59

61%
54%

4%
10%

6–19 months

Marked differences among the different subtypes of NHL; marked activity in MCL

O'Connor et al. (110)
O'Connor et al. (109)

B-cell lymphoma

60

41% in MCL and 15% in all other B-cell neoplasms

20% in MCL

6 months MCL

Significant activity in MCL, minimal in other subtypes, though heavily treated population of patients

Goy et al. (112)

Mantle cell lymphoma

30

33%

3%

2.5–14.8 months

10 patients received no prior therapy, 14 had prior therapy; minimal difference between the two

Belch et al. (141)

Non–small cell lung cancer

27

4%

0%

NR

12 patients experienced stabilization of their disease

Stevenson (115)

Renal cell carcinoma

21

5%

0%

2 months

Allowed dose escalations to 1.7 mg/m2 with no clear benefit

Davis et al. (113)

Renal cell carcinoma

37

11%

0%

8–20+ months

Median survival was 7.5 months

Kondagunta et al.(112)

ORR, overall response rate; CR, complete remissions.

Following the Summit trial, a second dose-response phase II study of bortezomib in myeloma was launched (the Crest study). This trial was an open-label, randomized phase II dose-response study in 54 patients who received bortezomib at a dose of 1.3 or 1.0 mg/m2 on days 1, 4, 8, and 11 every 21 days.108 The median number of prior therapies was three for both cohorts, with about 48% of all patients having received prior stem cell transplantation. In the Crest study, no grade 4 adverse events were reported, and the side effect profile was similar to what had been seen in the Summit trial. The overall response rate according to the European Group for Blood and Marrow Transplantation criteria (cases of complete remission plus cases of partial remission) was 38% and 30% in the 1.3 mg/m2 (n = 26) and 1.0 mg/m2 (n = 28) cohorts, respectively. One complete remission was noted at each dose level (about a 3.7% complete remission rate). There was no statistically significant difference in response rate between the two cohorts. As expected, the incidence of adverse effects was less at the 1.0 mg/m2 dose level, with less overall peripheral neuropathy, fewer neuropathic pain syndromes, less weakness, and less neutropenia being noted. This study, while not statistically powered to address the issue of equivalency between these two dose cohorts, clearly demonstrated that a lower dose of bortezomib was effective in myeloma and had a more favorable adverse effects profile.

The promising single-agent activity in patients with relapsed or refractory multiple myeloma has prompted a multitude of clinical trials studying the activity of bortezomib as primary treatment as well as in the relapsed setting in combination with other active drugs. For example, the APEX study randomly assigned patients to receive dexamethasone at a dose of 40 mg/day orally on days 1 to 4, 9 to 12, and 17 to 20 every 5 weeks or bortezomib at a dose of 1.3 mg/m2on the typical schedule used in the Summit study. Patients in the bortezomib arm experienced a 58% improvement in median time to progression (5.7 months) compared with patients receiving dexamethasone (3.6 months); the difference was statistically significant. Jagannath et al.108 reported a phase II study in which untreated patients with myeloma received bortezomib as in the Summit study; however, for those patients who did not achieve partial remission or remission, dexamethasone was added. Of 24 evaluable patients, 6 patients attained either a complete remission or near complete remission, while 13 attained a partial remission, for an overall response rate of 79%. Of the 14 patients who received additional dexamethasone, 8 demonstrated an improvement in their response.

Non-Hodgkin's Lymphoma

Based upon the activity seen in the phase I study, a single-agent phase II study of bortezomib in patients with indolent and mantle cell lymphoma was initiated.109, 110 This study was limited to patients with follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma/ chronic lymphocytic leukemia, and marginal zone lymphoma. Unlike in the Summit study, all patients were initially treated with a dose of 1.5 mg/m2 on days 1, 4, 8, and 11 every 21 days, with criteria for dose reduction to 1.3 and 1.1 mg/m2. Patients were required to have three or fewer lines of prior therapy and were allowed to have been treated with prior radioimmunotherapy and/or peripheral stem cell transplantation.109, 110 The median age of the 59 patients was 62, with a median KPS of 90%, with a slight male predominance. Thirty patients (51%) had mantle cell lymphoma, 18 (31%) had follicular lymphoma, 6 had marginal zone lymphoma, and 5 had small lymphocytic lymphoma. Overall the drug was well tolerated, with only one half of all patients requiring a dose reduction to 1.3 mg/m2, mostly for issues related to thrombocytopenia, asthenia, and neuropathy. The median number of prior therapies was three, and virtually all patients had received rituximab, about half having received multiple courses of rituximab. The different forms of lymphoma had quite variable overall response rates and time to response. Overall, the response rate was 54%. Sixty-five percent of patients with follicular lymphoma achieved a response, with 2 patients meeting criteria for complete response or unconfirmed complete response. Among patients with mantle cell lymphoma, 54% of these patients achieved a major response, with 4 meeting criteria for complete response or unconfirmed complete response. Interestingly, patients with follicular lymphoma tended to respond later, on average after cycle 3 or 4, while patients with mantle cell lymphoma typically responded by the second cycle. In all cases, the responses were durable, with most patients having a duration of remission that exceeded that seen with the last treatment before receiving the study drug. Following this study, a second study of bortezomib in B-cell lymphomas was launched, which included patients with any subtype of B-cell non-Hodgkin's lymphoma, with no cap on the number of prior therapies. Adopting virtually the same study design, this study initially established cohorts for patients with mantle cell lymphoma alone and patients with other types of B-cell neoplasms. Overall, the patients reported in the study by Goy et al.111 were more heavily pretreated. Of 29 patients with MCL, 12 met the criteria for a major response, with 6 attaining a complete remission. Among the patients with other B-cell neoplasms, 12 had diffuse large B-cell lymphoma, of which only 1 attained a partial remission. Overall, the experience in NHL is encouraging; however, response rates will need to be defined for each histologic form of lymphoma.

Solid Tumors

Early clues to the activity of bortezomib in solid tumors were seen in the two phase I studies of the drug. In one of these phase I studies, Aghajanian et al.103treated 43 patients on days 1, 4, 8, and 11 every 21 days, with doses ranging from 0.13 to 1.56 mg/m2. In this heavily treated patient population, one patient with NSCLC (bronchioloalveolar type) achieved a partial remission. The remission lasted about three months. In the second single-agent phase I experience, a weekly schedule of bortezomib administration at doses ranging from 0.13 to 2 mg/m2 was studied. Responses were seen in patients with androgen-independent prostate cancer (AIPCa) but not in patients with renal cell carcinoma or transitional cell carcinoma.104 Two (4%) of the 47 patients with AIPCa had a greater than 50% decline in serum prostate-specific antigen (PSA) while 9 (19%) of 47 patients had stable PSA measurements over the study period. Two patients with measurable disease attained partial remission, with shrinkage of their retroperitoneal lymphadenopathy. One of these responses was seen at a dose of 0.4 mg/m2, the other at a dose of 1.6 mg/m2.

Some suggestion of activity has also been reported in patients with metastatic renal cell carcinoma. Thirty-seven patients were treated at a dose of 1.5 mg/m2on days 1, 4, 8, and 11.112 Four of these patients achieved a partial remission, while another 14 were found to have stable disease after having documented disease progression at the time of enrollment. The remissions were durable, lasting 8, 8+, 15+, and 20+ months. With a median follow-up of 11.7 months, the proportion of patients alive at one year was 36%, and the median survival time was 7.5 months. In contrast, a second phase II study in patients with renal cell carcinoma reported on 21 assessable patients, of which only 1 partial remission was reported.113 Interestingly, a large retrospective study of over 251 patients with renal cell carcinoma enrolled in 29 consecutive clinical trials at Memorial Sloan Kettering Cancer Center between 1975 and 2002 demonstrated that patients treated after 1990 showed slightly longer survival compared with patients treated prior to 1990.114 More importantly, the authors established three major prognostic factors for this patient population that adversely affect outcome: low Karnofsky performance status, low hemoglobin, and a high corrected serum calcium level. The median time to death in patients with zero risk factors was 22 months. In comparison, the median time to death for patients with one or two to three risk factors was 11.9 and 5.4 months, respectively. This kind of analysis is essential for deciphering the true benefit of novel drugs in the single-agent phase II setting.

Based on the response seen in the original phase I experience, a single-agent study of bortezomib inpatients with advanced NSCLC was initiated using a dose of 1.5 mg/m2 on days 1, 4, 8, and 11 every 21 days. Among 27 evaluable patients, 1 achieved partial remission, and 12 had stable disease.115 It is not clear what biological features separate responding from nonresponding patients with NSCLC. Additional phase II studies to define activity in other tumor types are underway or planned.

Combination Phase I/II Experiences

The natural evolution of most drug treatment strategies is to explore integration into standard chemotherapy regimens. The rationale for integrating drugs that target the ubiquitin-proteasome pathway, in particular, the NF-κB signaling pathways, is that there is a strong theoretical basis for drug synergy with this class of molecules. Many types of environmental stress, including chemotherapy exposure, hypoxia, and radiation exposure, are known to activate NF-κB, presumably as part of a survival response. For example, irinotecan (CPT-11) has been shown to markedly increase NF-κB levels, which leads to the increased transcription of antiapoptotic factors and cell survival.116, 117 If all the cells within in any tumor population are not completely eradicated with a given cytotoxic drug, then those cells left behind will activate various survival pathways to overcome the antitumor activity of the cytotoxic agent. If one could block the induction of this survival response by inhibiting NF-κB, then one might overcome some of these survival strategies and improve antitumor efficacy.

A number of studies have documented synergistic effects by combining bortezomib with a host of conventional drugs, including irinotecan, paclitaxel, doxorubicin, rituximab, bcl-2 antisense, cyclophosphamide, and several other drugs. 36, 118, 119, 120, 121, 122, 123 This sort of approach is now being studied in a variety of combination phase I and II studies in both hematologic and solid tumor malignancies. The results of such studies are eagerly anticipated.

Some encouraging clinical data in support of synergistic interactions have been reported by Orlowski et al.124 (2005), who have explored a combination of bortezomib and pegylated liposomal doxorubicin (PegLD). The basis of this particular synergistic interaction is derived from the observation that proteasome inhibitors transcriptionally induce the MKP-1 phosphatases a process which is antiapoptotic through its inactivation of JNK, while anthracyclines like doxorubicin appear to down-regulate MKP-1. 125, 126 In the phase I experience with this combination, bortezomib was administered on days 1, 4, 8, and 11 at doses ranging from 0.9 to 1.5 mg/m2, while the PegLD was administered at a fixed dose of 30 mg/m2 on day 4 every 21 days. The MTD was determined to be 1.3 mg/m2 of bortezomib with 30 mg/m2 of PegLD. Based on 22 evaluable patients, 8 patients with advanced multiple myeloma had a complete remission or near complete remission, including several with anthracycline-resistant myeloma. Another 8 myeloma patients experienced a partial remission. Additionally, one patient with refractory T-cell lymphoma achieved a complete remission, one patient with acute myeloid leukemia achieved a partial remission, and one patient with non-Hodgkin's lymphoma achieved a partial remission. The remarkable activity seen in myeloma has now led to what will likely be a large cooperative group trial of this combination in multiple myeloma.

The future development of bortezomib will depend on the toxicity and efficacy of this agent in combination with other conventional and investigational agents. Preclinical data support the notion that proteasome inhibitors may be synergistic with other novel classes of drugs, including histone deacetylase inhibitors, cyclin-dependent kinase inhibitors like flavopiridol, and the bcl-2 antisense molecule G3139 (Genasense).123, 127, 128 What remains to be clarified from a pharmacologic perspective is the importance of scheduling these agents and defining the optimal concentrations of these drugs for inhibiting their principal targets. For example, in some in vivo models of non-Hodgkin's lymphoma, the activity of the bcl-2 antisense molecule G3139 with cyclophosphamide and bortezomib was found to be very schedule dependent. Better results were seen using antisense bcl-2 first followed by cyclophosphamide and then followed 24 hours later by bortezomib.123 Combinations of many standard chemotherapy drugs are often given together. Such approaches, while certainly more convenient for the patient, often disregard compelling preclinical evidence of schedule dependency. The onus is now on both laboratory and physician scientists to understand these scheduling phenomena and then to elucidate the biological basis for the schedule dependency. In addition to sorting out these critical pharmacologic questions with this new class of molecules, it is apparent that understanding the molecular basis for the response or nonresponse of certain patients will be absolutely critical in the development of new classes of drugs.

FUTURE DIRECTIONS

Investigators have begun to explore the relationships between the response and gene expression profile of a particular myeloma. For example, in a recent study presented by Barlow et al.,129 different subsets of genes were identified that could be used to predict survival and event-free survival in myeloma patients receiving bortezomib. A subset of 10 genes were then identified that could predict poor survival, including genes involved in cell cycle control like CDC2, TYMS, BUBI, TOP2A, and Ki-67. Additional data presented by Richardson et al.130 have also established that both clinical and molecular information can be used to identify relatively good-risk and poor-risk populations of patients with respect to their ability to respond to bortezomib. The identification of patients most likely to benefit from a particular therapeutic approach is an important goal and may one day allow oncologists to construct drug cocktails capable of producing the best effects in particular patients.

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