	Printable version
	Version PDF

Proteasome inhibition: a new approach for the treatment of malignancies

Bulletin du Cancer. Volume 92, Number 11, 61-6, Novembre 2005, Electronic journal of oncology

Summary

Author(s) : Jean-Philippe Spano, Jacques-Olivier Bay, Jean-Yves Blay, Olivier Rixe , Département d’oncologie médicale, Groupe Hospitalier Pitié-salpêtrière, Assistance Publique/Hôpitaux de Paris, Département d’Oncologie Médicale, Groupe hospitalier Pitié-Salpêtrière, 47-83 bld de l’hôpital, Paris, France, Département d’oncologie médicale, centre Jean Perrin, Clermont-Ferrand, France, Département d’oncologie médicale, centre Léon Bérard, Lyon, France.

Summary : Since last years, the proteasome has emerged as a real and exciting target for anticancer therapy. Velcade ^® (bortezomib, formerly known as PS341) remains the first selective proteasome inhibitor that has demonstrated significant preclinical activity in several tumor models and a significant efficacy in patients with refractory or relapsed multiple myeloma, resulting in an accelerated approval in US and Europe in such a setting. The major biological effect of bortezomib is the inhibition of the nuclear transcription factor NFκB, with subsequent inhibition of the growth tumor cells, induction of apoptosis, inhibition of angiogenesis and of cellular adhesion. The better understanding of the role of proteasome in the regulation of tumor cell growth has led to the development of new therapeutic approaches, notably in patients with multiple myeloma but also seems to hold interesting promises in other hematologic malignancies and solid tumors. This review provides a summary of the rationale for using proteasome inhibitors and an update on available and ongoing clinical studies involving human malignancies.

Keywords : ubiquitin, proteasome, NFκB, bortezomib, lymphoma, solid tumors

ARTICLE

Auteur(s) : Jean-Philippe Spano¹, Jacques-Olivier Bay², Jean-Yves Blay³, Olivier Rixe¹

¹Département d’oncologie médicale, Groupe Hospitalier Pitié-salpêtrière, Assistance Publique/Hôpitaux de Paris, Département d’Oncologie Médicale, Groupe hospitalier Pitié-Salpêtrière, 47-83 bld de l’hôpital, Paris, France
²Département d’oncologie médicale, centre Jean Perrin, Clermont-Ferrand, France
³Département d’oncologie médicale, centre Léon Bérard, Lyon, France

The proteasome, is a multicatalytic complex present in all eucaryotic cells, an essential function of which is to degrade proteins involved in the regulation of cell cycle (for example, P53, P21 an P27). A large number of intracellular proteins are substrates for the proteasome, in particular those involved in apoptosis, angiogenesis, growth factors transcription, growth factor receptor production and signaling molecules [1]. This enzymatic complex is endowed with an ATP-dependent protease activity [2]. Thus, a dysregulation of these key proteins can stimulate cell proliferation, tumor growth angiogenesis and metastasis, warranting the strong focus put during recent years on proteasome inhibition as a novel approach to targeted therapies.Bortezomib or Velcade^®, initially designated PS341, is the first selective proteasome inhibitor to have reached clinical development. This drug has already demonstrated pre-clinical and clinical activity not only against solid tumorts but also in hematological malignancies. It can highly selectively inhibit the proteasome by reversibly stabilizing the 26S proteolytic subunit. Its impressive efficacy against multiple myeloma justified an accelerated authorization in 2003 by the Food and Drug Administration (FDA) for administration to such patients after other treatments had failed [3]. The purpose of this review is to emphasize proteasome involvement in some tumor types and the contribution of its inhibitors such as bortezumib in the management of these tumors.

Most relevant proteasome substrates and mechanism of action of inhibitors

The spectrum of substrates for proteasome is quite wide, most of them being involved in the regulation of cell cycle, apoptosis and angiogenesis. These include many cyclins (A, B, D, E) as well as the cyclin-dependent protein kinases (cdk) they regulate to control cell division. Moreover, proteins P21 and P27, whose function is to arrest the cell cycle by inhibiting cdk’s, can be transiently degraded by the proteasome [4, 5]. Proteasome inhibitors thus increase the levels of P21 and P27 thereby blocking the cell cycle in G1/S [6]. It was also demonstrated that a low level of P27 is associated with more agressive tumors and worse prognosis [1].

The tumor suppressor protein P53 is another target for proteasome [7]. P53 is a key regulator of cell cycle thru the transcription of many regulatory genes like P21, genes involved in DNA synthesis and repair, or in apoptosis induced by ionizing radiations [1, 7].

Of highest relevance for our purpose, proteasome targets IκB, the inhibitor of NFκB, an important nuclear transcription factor [8]. Indeed, under normal circumstances, NFκB and IκB form an inactive cytoplasmic complex. Upon cellular agression by chemotherapy, ionizing radiations or carcinogens, the IκB-NFκB complex dissociates, allowing the rapid degradation of IκB and nuclear transfer of NFκB where it activates anti-apoptotic genes, genes involved in cell proliferation, cytokine synthesis, cell adhesion and angiogenesis. All these effects cooperate to promote cell survival and endow with a malignant potential at risk of metastasis. NFκB also inhibits the pro-apoptotic function of TNFα [9, 10], ras oncogene [11], while favoring the production of anti-apoptotic factors like Bcl-2 [12]. NFκB induces the production of cell adhesion molecules like ICAM-1 (intracellular adhesion molecule), E-selectin, VCAM-1 (vascular cell adhesion molecule) [13], VEGF (vascular endothelial factor) and IL-6 [14]. As a matter of fact, NFκB exhibited particularly high levels of activity in many tumor models (prostate cancer, melanomas, myeloma, lymphomas, leukemias) [15] as confirmed by pre-clinical studies showing that proteasome inhibitors had a higher cytotoxicity against tumor cells (which contain high levels of both proteasome and NFκB) than normal ones [16]. Moreover, as compared to normal ones, tomor cells appear to be more sensitive to the pro-apoptotic effects of proteasome inhibitors [17]. Many pre-clinical models have also shown that proteasome inhibition could restore cell sensitivity to cytotoxic agents and enhance apoptosis by preventing NFκB activation [18].

Proteasome inhibition therefore emerges as a new therapeutic concept selectively targeting tumor cells with high levels of both proteasome and NFκB while reversing resistance to cytotoxic agents. As in multiple myeloma, proteasome inhibition not only affects malignant cells but also cells from their micro-environment which essential for their survival [19].

Pre-clinical development of proteasome inhibitors

Following identification of the biological target, many compounds were synthesized to selectively and revesibly inhibit proteasome activity. Less than 3 years after discovering the connection between proteasome and NFκB and the central role it played in controlling cell cycle and apoptosis, this search led to the preclinical identification of such inhibitors in 1999 [20]. These belonged to five classes among which dipeptide molecules, derived from boronic acid and inhibiting the protease activity, were selected for their antitumor activity.

An in silico system was recently developed to screen for inhibitors of NFκB activity, thereby allowing to explore their dynamic effects on the molecular network surrounding this target [21].

When tested against the NCI (National Cancer Institute) panel of 60 cell lines, 13 molecules belonging to the family of boronic acid derivatives were selected. Inhibitors from this family are the most potent and selective for the proteasome. Their spectrum of activity includes numerous types of tumors (small cell lung cancer, colon, central nervous system, ovary, prostate, breast and melanoma). For all 60 cell lines, the inhibition coefficient of these 13 peptides was directly correlated with cell growth inhibition. Among those, PS341 was rapidly singled out for its major antineoplastic activity [22]. It was the only one to go into clinical use. It stands out for an original activity profile against this cell lines panel, without any analogy with the 60,000 other compounds of the NCI databank.

In vitro studies on PC3 prostate cancer cell lines showed that PS341 increases intracellular levels of P21 independently of their P53 status. Accumulation of cells in G2/M clearly demonstrates that PS-341 acts on proteins controlling progression of the cell cycle while also affecting proteins involved in apoptosis like caspases and PARP.

In vivo studies using xenografts of PC3 cells in nude mice showed significant, dose-dependent, tumor regression after intravenous administration of PS341. The correlation of this effect with the decreased activity of the proteasome subunit was taken as a proof of concept in mouse. Many normal tissues exhibit this decreased activity except for brain and testis, suggesting a reduced penetration of the drug into the latter tissues.

PS341 blocks NFκB activation by TNFα in multiple myeloma cell lines, also in a dose-dependent manner, pointing to PS-341 as the major target. This inhibition however does not fully explain the activity of PS341 [23]. The related compound PS1145 also inhibits the IκB kinase IKK. Although both PS341 and PS-1145 block NFκB activation, PS341 completely suppresses tumor growth inhibition is only 20% for PS1145 [23]. Inhibition of tumor angiogenesis also contributes to bortezomib activity. Microvessels density is decreased in vivo as a result of proteasome inhibition in endothelial cells [24]. Modifications in the micro-environment induced by PS341also weakens the association between myeloma and stromal cells, reducing IL-6 secretion through NFκB inhibition [25]. In vitro, PS341 activity is independent of the P53 satus of tumor cells [26].

Bortezomib activity is additive to other therapeutic regimens in vitro and on animal models. Moreover, bortezomib has chemo-sensitizing properties allowing cytotoxic drugs to be used at concentrations smaller than in monotherapies. Such synergistic effects were observed with doxorubicine, melphalan and dexamethazone on myeloma cell lines, with irinotecan or iradiation on colon cancer xenografts, with gemcitabine on pancreatic cancer xenografts, with anti-IL-R2a antibodies on an adult T-cell leukemia, and with irradiation, cyclophosphamide ans cis-platin on a mammary carcinoma model [27-31]. Finally, bortezomib is able to restore sensitivity to cytotoxic agents in resistant cell lines. It was shown to circumvent acquired cell line resistance to doxorubicin, melphalan or mitoxantrone as well as de novo resistance to temozolomide in melanoma [32]. NFκB inhibition seems to play an essential role in this effect since it moodulates MDR1 expression [33].

The recent characterization of cell lines resistant to PS341 brought a lot of interesting informations. It revealed alterations of mitochondria and cytochrome c, quantitative modifications of caspases 8, 9, 3 and PARP as well as of the JNK (c-Jun NH2 terminal kinase) pathway [34-36] leading to altered apoptotic signals. Moreover, the identification of alternative pathways to proteasome degradation of ubiquitinylated proteins involving the « aggresome » provides a rationale for the pharmacological modulation of bortezomib : « aggresome » inhibitors like tubacin are indeed strongly synergistic [35].

The toxicity profile of PS341, established in rodents and primates includes anorexia, nausea and diarrhea in a dose-dependent manner, with no observed medullar toxicity.

Bortezomib use in onco-hematology

Pharmacological data

In order to facilitate the finding of optimal doses for phase I and II trials, a functional test was designed to determine proteasome activity in white cells as well as in tumor samples [37]. Minor variations in proteasome activity in healthy patients were observed. After exposure to PS341, proteasome activity goes back to basal level in 48 to 72 hrs [38]. Gastro-intestinal toxicity of PS341 is dose-dependent. Tolerance is good if proteasome inhibition does not exceed 80% of the basal level, beyond which modifications in blood pressure and cardiac frequency add up to gastro-intestinal problems.

The dose of PS341 corresponding to 80% inhibition opf proteasome activity was estimated to be 1.96 mg/m². This bioassay is an important tool to define the optimal dose and was used in several phase I and phase II trials [39].

Interest for multiple myeloma treatment

The best recognized indication of bortezomib in onco-hematology remains multiple myeloma (MM). It is undoubtedly the pathology where most data have accrued from phase I and phase II trials. In MM, although bortezomib has multiple relevant targets, the most significant one is the JNK pathway. This drug induces apoptosis through the mitochondrial accumulation of two activating molecules : SMAC (second mitochondria-derived activator of caspases) and cytochrome c [40, 41]. But bortezomib also ointerferes with other cellular processes : it blocks IL-6 mediated signaling whose activating role was demonstrated in a number of cancers where it cleaves IL-6 receptor at the surface of tumor cells [42]. Similarly, bortezomib induces P53 accumulation and phosphorylation at Ser 15 leading to dissociation from its inhibitor MDM2.

Apoptosis is thus induced by the c-Jun pathway through caspases 3 and 8 activation.

Finally, DNA repair is inhibited by cleavage of DNA/PK and ATM/ATR complexes [43].

Doses defined after phase I studies were from 0.15 to 2 mg/m² once or twice a week. The work by Orlowski et al. [44] showed a good tolerance a 1.04 mg/m² and they observed 9 complete and 8 significant remissions in refractory MM patients. Phase II trials by Richardson et al. [45] followed with the inclusion of 202 MM patients refractory to conventional treatments. A dose of 1.3 mg/m² was used twice a week for two weeks (D1, D4, D8 and D11) followed by a week without treatment. This cycle was repeated 8 to 10 times. There was an overall 35% with 4% complete, 6% quasi-complete and 17% partial remission. The disease was stabilized in 24% of patients. Median time until progression was 7 months, median response time 12 months and median survival 16 months. Reported toxic effects were moderate with essentially thrombopenia, constipation, and peripheral neuropathies.

These results were confirmed by a second phase II trial. Jagannath et al. [46] included 54 refractory patients, one group receiving 1 mg/m² and the other 1.3 mg/m². The administration regimen was the same as in the first trial [45]. Overall response rates were respectively 33 and 55% suggesting a dose effect. Patients which concommitantly received dexamethazone at 40 mg per day had a better response (62 versus 44%). In addition to a dose effect of bortezomib, there might be an additional effect with dexamethazone. Median time until progression was 11 months.

The same team as above engaged into a phase II trial randomizing bortezomib at 1.3 mg/m² following the same administration regimen with 40 mg dexamethazone per day [47]. A total of 669 patients were included revealing an unequivocal advantage for patients receiving bortezomib (38%) over dexamethazone (18%) with p < 0.001. Median times until progression were respectively 13.5 versus 6.2 months. One year survival was 80% for bortezomib patients and 66% for others (p < 0.001). Patients refractory to dexamethazone might therefore benefit from bortezomib.Toxic effects were moderate but a better tolerance for bortezomib with fewer surinfection episodes and a smaller mortality rate associated to treatment.

Another recent study by Richardson et al. [48] was aimed at defining predictive factors for response to bortezomib. Classical criteria for poor prognosis like deletions on chromosome 13 and β2-microglobulin elevation were not predictors of a bad response to bortezomib, at variance with the large tumor mass and age over 65 which remained factors of poor prognosis.

Although the efficacy of bortezomib seems now established for refractory MM, there remain many open questions with respect with its use during the course of the disease. Should it be used as first line therapy at the time of diagnosis? If so, associated with which other drugs? Could bortezomib be included in conditioning protocols for autografts or ex vivo on grafts? Considering the development of non myeloablative conditioning protocols followed by allografts of hematopoietic stem cells in MM, would it be possible to uuse bortezomib in post-graft or in case of relapse?

Somer answers have already been reported, albeit mostly in preliminary form. As a first line therapy, it seems possible to use bortezomib either alone or in combination with dexamethazone with or without an anthracycline. Along this line, data from Richardson et al. [49] are interesting since they are the only one to date to have used bortezomib alone at the time of diagnosis. 28 patients were included in this phase II trial, 27 being evaluable. Overall response was 45% (one patient in complete, 11 in partial remission). If one adds 6 patients with limited improvement, the response rate reaches 67%. The interest of this study resided also in teh assessment of therapeutic tolerance in patients without previous specific treatments. Reported undesirable effects were : peripheral nneuropathies (22%, of which only one patient reached grade 3 requiring cessation of treatment), digestive problems, asthenia and skin rashes.

Concerning refractory MM, Richardson et al. showed an enhanced efficacy of dexamethazone-bortezomib association [47]. A similar trend was also noted by Jagannath et al. [46] foor first line treatment with bortezomib. A preliminary analysis of the first 23 patients included in this phase II study revealed a partial response in 83% of patients with variations depending upon the number of cures received. It was noteworthy that, although 43% of patients showed an early response (after 2 cures), 13% responded much later with a maximiuum effect after 6 cures. 14 patients received a secondary administration of dexamethazone which seemed to confer an additional benefit. Preliminary studies from the French Institute of Myeloma on 18 patients reached essentially the same conclusions [50]. Adding an anthracyclin to the regimen in the trial by Cavanagh et al. [51] further increased the initial response rate up to 95% (20 of 21 patients). In addition to bortezomib and dexamethazone (40 mg), these patients received either 4.5 or 9 mg/m² of doxorubicin. Albeit preliminary, these phase II results appear very encouraging. As of now, no information is available concerning the possible effect of bortezomib with or without melphalan and prednisone, especially in older patients.

It should be noted that, in all three trials above [46, 49, 51], peripheral hematopoietic stem cells were sampled after injection of an hematopoietic growth factor. The quality of these stem cells remained unaffected and first line bortezomib administration does not seem to preclude later use of melphalan et high doses. The same conclusion was reached by the preliminary results of another phase II trial conducted by Sureda et al. [52]. Bortezomib dose was 1 or 1.3 mg/m² at D-4 and D-1 before melphalan administration at high dose. The authors claim a good feasibility wirth no additional toxicity being observed in the 37 evaluable patients. Final results of this study will be interesting but confirmation of the therapeutic advantage brought about by bortezomib gives a therapeutic edge without additional toxic side effects.

There has been so far only one report on the use of bortezomib after hematopoietic stem cells allograft in 9 patients in progression [53]. In 6 of these 9 patients, the monoclonal component was reduced by over 50% with no noticeable effect on the incidence of severity of graft versus host (GVH) reactions despite the in vitro observations by Sun et al. [54] that bortezomib can induce apoptosis of alloreactive T cells known to induce GVH. In vivo data in mice confirm the reduced GVH incidence. It seems however that the graft versus leukemia (GVL) effect is conserved [54]. These results are particularly interesting and should deserve studies in man since they imply the possibility of reducing toxicity without compromising the immunological therapeutic efficacy.

Bortezomib and other hematologic malignancies

New perspectives seem to open especially for some lymphomas following two recent reports on tolerance and efficacy of bortezomib in non-Hodgkin malignant lymphoma (NHML) patients (B phenotype or mantel cell lymphomas in relapse or refractory to conventional therapies, irrespective of the number of therapeutic lines received). Preclinical animal models and phase I trials had already shown interesting responses [39, 44].

The study by Guy et al. [55] on 60 patients treated (33 mantel lymphomas, and 27 B phenotype NHML) is quite stimulating in this respect. 12 of 29 evaluated patients with mantel cell lymphoma exhibited an objective response, 6 of which with complete remission. After a median followup of 9.3 months (1.4-24 months), median time until progression had not yet been reached. 4 (19%) of 21 evaluable B phenotype NHML were brought into complete remission. Grade 3 toxicities observed were thrombopenia (47%), asthenia (13%), neutropenia (10%) and peripheral neuropathy (5%). They are quite similar to that observed for MM with no toxic death.

Another study by O’Connor et al. [56] 26 patients with low grade B phenotype NHML or mantel lymphomas. 24 cases could be evaluated with an overall objective remission rate of 58%, including one complete remission, another non confirmed one and 4 partial among follicular lymphomas patients with responses extending from 3 to 24 mois. For patients with mantel cell lymphoma, one non-confirmed complete remission, 4 partial and 4 stabilizations were obtained with expected toxicities. For one of these patients, bortezomib could be kept in remission for 19 months.

Other potential applications can be envisionned. Preclinical and phase I studies suggested that additional hematopoietic malignancies like Hodgkin disease ou chronic lymphocytic leukemias (CLL) couls also benefit from bortezomib treatment. A recent in vitro study by Duechler et al. [57] showed a cytoxic effect of bortezomib in association with purine analogs on lymphocytes from CLL.

Bortezomib and solid tumors

Three phase I studies have been published to date with PS-341, enrolling over 200 patients with solid tumors. Three main regimens were tested: one weekly injection for 4 of 6 weeks, or for 2 of 3 weeks, or two weekly injections for 2 of 3 weeks. The recommended regimen and main secondary effects are summarized in table 1( Table 1 ).

The first of these trials, conducted at MD Anderson by Papandreou et al. [58] used a weekly scheme for 4 weeks, followed by 2 weeks rest, with bortezomib doses above 1 mg/m². Main side effects were fever and asthenia after several cycles, moderate thrombopenia, moderate diarrhea easily controlled by loperamide and peripheral neuropathies among patients previously exposed to high doses of platin and taxanes.

The second study by Aghajanian et al. [59] reported objective responses in non small cell lung carcinom (NSCLC), hormone-resistant prostate cancer and myeloma.

The third study by Dy et al. [60] showed a dose-dependent effect of proteasome inhibition as assayed in peripheral lymphocytes (75% inhibition at 1.25 mg/m²)

From the above trials, it emerges that a dose of 1.5 mg/m²,twice a week, administered 2 weeks of 3 apperas the most interesting with regard to the dose used, its measured biological activity and the observed tolerance.

Several phase II trials designed to look for a therapeutic activity were recently reported.

No objective response was noted in 21 patients treated for metastatic kidney cancer [61] neither in 16 metastatic, well differenciated, neuroendocrine tumors, despite a clearly documented effect on proteasome inhibition [62]. The same situation was observed for 27 metastatic melanoma patients, although the scheme used (2 weekly injections for 2 of 3 weeks) resulted in proteasome inhibition in peripheral blood [63].

Finally, a study including 21 patients with osteosarcomas, Ewing sarcomas or soft tissues sarcomas allowed to note only one partial response in a leiomyosarcoma patient [64].

No response also was observed for 19 metastatic colo-rectal cancer [65]. However, a significant increase of intra-tumoral HIF-1a was reported in this study, without modification of P53, NFκB or IκB expression suggesting that proteasome inhibition alters the response to tumor hypoxia.

Based on a strong pre-clinical background, there are many ongoing phase I-II trials using bortezomib in combination with cytotoxic agents like docetaxel, paclitaxel, gemcitabine, CPT-11, Pt salts or anthracyclins [66]. Also worth mentioning is the NCI phase I trial testing bortezomib in association with re-irradiation in Ear-Nose-Throat (ENT) cancers.

Proteasome inhibition is not modified in patients with impaired renal function and neither is there any increase in toxicity [67].
Table 1 Summary of phase I studies on solid tumors

Author	Regimen (weekly)	Number of patients	Limiting toxicities	Recommended dose	Activity
Papandreou [58]	4 weeks/6	53	Diarrhea, low blood pressure	1.6 mg/m²	prostate
Aghajanian [59]	2 weeks /3	43	diarrhée, neuropathie	1.56 mg/m²	lung
Dy [60]	Twice weekly, 2 weeks/3	73	diarrhea, nauseas, asthenia, hypoglycemia, low blood pressure	1.5 mg/m²	myeloma

Conclusion

Clinical development of proteasome inhibitors followed an accelerated process thanks to strong collaboration between biologiste and clinicians, allowing now the routine use of PS-541 (bortezomib or Velcade^®) in multiple myeloma. It took only 5 years between the first research data in 1994 [68]and the report of phase III survival data in myeloma [47], with the first screening of drug targeting the proteasome in 1999.

Not surprisingly, though, many issues remain to be addressed : place of Velcade® among other myeloma treatments, combination studies especially with chemotherapy. In the case of solid tumors, the first results with Velcade^® alone are not encouraging in colon, kindney cancers, sarcomas and melanomas. Conversely, the strong pre-clinical data and the first objective biological responses already observed in phase I trials warrant a strong interest for further development in hormone-resistant prostate cancer as welle as in NSCLC [69].

Association of bortezomib with conventional chemotherapy seems especially interesting to circumvent intrinsic or acquired resistance to cytotoxic drugs, the more so as observed toxicities remain quite acceptable. Ongoing clinical trials are currently addressing this issue as well as bortezomib association with radiotherapy.

A convincing proof-of-concept of the therapeutic virtue of proteasome inhibition has been obtained with PS-541which remains until now the only drug of this category to be used or further developed in onco-hematology. There is therefore room for the development of other new or related drugs with a different spectrum of activities in the hope to be able to target solid tumors.

Références

1 Park DJ, Lenz HJ. The role of proteasome inhibitors in solid tumors. Ann Med 2004; 36: 296-303.

2 King RW, Deshaies RJ, Peters JM, et al. How proteolysis drives the cell cycle. Science 1996; 274: 1652-9.

3 Kane RC, Bross PF, Farrell AT, Pazdur R. Velcade: US FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist 2003; 8: 508-13.

4 Blagosklonny MV, Wu GS, Omura S, El-Deiry WS. Proteosome-dependant regulation of p21 WAF/CIP1 expression. Biochem Biophys Res Com 1996; 227: 564-9.

5 Sherr CJ. Cancer cell cycles. Science 1996; 274: 1672-7.

6 Machiels BM, Henfling ME, Gerards WL, et al. Detailed analysis of cell cycle kinetics upon proteasome inhibition. Cytometry 1997; 28: 243-52.

7 Maki CG, Huibregtse JM, Howley PM. In vivo ubiquitination and proteasome-mediated degradation of p53. Cancer Res 1996; 56: 2649-54.

8 Palombella VJ, Rando OJ, Goldberg AL, Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-κB B1 precursor protein and the activation of NFκB. Cell 1994; 78: 773-85.

9 Beg AA, Baltimore D. An essential role for NFκB in preventing TNF-alpha induced cell death. Science 1996; 274: 782-4.

10 Wang CY, Mayo MW, Baldwin Jr. AS. TNFα and cancer therapy-induced apoptosis: potentiation by inhibition of NFκB. Science 1996; 274: 784-7.

11 Mayo MW, Wang CY, Cogswell PC, et al. Requirement of NFκB activation to suppress p53-independant apoptosis induced by oncogenic ras. Science 1997; 278: 1812-5.

12 Zong WX, Edelstein LC, Chen C, Bash J, Gelinas C. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-κB that blocks TNFalpha-induced apoptosis. Genes Dev 1999; 13: 382-7.

13 Zetter BR. Adhesion molecules in tumor metastasis. Semin Cancer Biol 1993; 4: 219-22.

14 Huang S, DeGuzman A, Bucana CD, Fidler IJ. Nuclear factor-kappa B activity correlates with growth, angiogenesis, and metastasis of human melanoma cells in nude mice. Clin Cancer Res 2000; 6: 2573-81.

15 Bours V, Dejardin E, Goujon-Letawe F, Merville MP, Castronovo V. The NF-κB transcription factor and cancer: high expression of NFκB and IκB-related proteins in tumor cell lines. Biochem Pharmacol 1994; 47: 145-9.

16 Adams J. The proteasome: structure, function, and role in the cell. Cancer Treat Rev 2003; 29(Suppl. 1): 3-9.

17 Richardson PG, Hideshima T, Anderson KC. Bortezomib (PS-341): a novel, first-in-class proteasome inhibitor for the treatment of multiple myeloma and other cancer. Cancer Control 2003; 10: 361-9.

18 Ma MH, Parker KM, Manyak S, et al. Proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma cells to chemotherapeutic agents and overcomes chemo-resistance through inhibition of the NFκB pathway. American Society of Hematology, 2001; abstract 1978.

19 Dalton WS. The tumor microenvironment: focus on myeloma. Cancer Treat Rev 2003; 29(Suppl. 1): 11-9.

20 Adams J. The proteasome: a suitable antineoplastic target. Nat Rev Cancer 2004; 4: 349-60.

21 Sung MH, Simon R. In silico simulation of inhibitor drug effects on nuclear factor-kappaB pathway dynamics. Mol Pharmacol 2004; 66: 70-5.

22 Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999; 59: 2615-22.

23 Hideshima T, Chauhan D, Richardson P, Mitsiades C, Mitsiades N, Hayashi T, et al. NF-kappa B as a therapeutic target in multiple myeloma. J Biol Chem 2002; 277: 16639-47.

24 Williams S, Pettaway C, Song R, Papandreou C, Logothetis C, McConkey DJ. Differential effects of the proteasome inhibitor bortezomib on apoptosis and angiogenesis in human prostate tumor xenografts. Mol Cancer Ther 2003; 2: 835-43.

25 Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ, Adams J, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res 2001; 61: 3071-6.

26 Drexler HC. Activation of the cell death program by inhibition of proteasome function. Proc Natl Acad Sci USA 1997; 94: 855-60.

27 Mitsiades N, Mitsiades CS, Richardson PG, Poulaki V, Tai YT, Chauhan D, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood 2003; 101: 2377-80.

28 Russo SM, Tepper JE, Baldwin Jr. AS, Liu R, Adams J, Elliott P, et al. Enhancement of radiosensitivity by proteasome inhibition: implications for a role of NF-kappaB. Int J Radiat Oncol Biol Phys 2001; 50: 183-93.

29 Bold RJ, Virudachalam S, McConkey DJ. Chemosensitization of pancreatic cancer by inhibition of the 26S proteasome. J Surg Res 2001; 100: 11-7.

30 Tan C, Waldmann TA. Proteasome inhibitor PS-341, a potential therapeutic agent for adult T-cell leukemia. Cancer Res 2002; 15: 1083-6.

31 Teicher BA, Ara G, Herbst R, Palombella VJ, Adams J. The proteasome inhibitor PS-341 in cancer therapy. Clin Cancer Res 1999; 5: 2638-45.

32 Amiri KI, Horton LW, LaFleur BJ, Sosman JA, Richmond A. Augmenting chemosensitivity of malignant melanoma tumors via proteasome inhibition: implication for bortezomib (Velcade, PS-341) as a therapeutic agent for malignant melanoma. Cancer Res 2004; 15: 4912-8.

33 Fujita T, Washio K, Takabatake D, Takahashi H, Yoshitomi S, Tsukuda K, et al. Proteasome inhibitors can alter the signaling pathways and attenuate the P-glycoprotein-mediated multidrug resistance. Int J Cancer 2005; 117: 670-82.

34 Ling YH, Liebes L, Zou Y, Perez-Soler R. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells. J Biol Chem 2003; 278: 33714-23.

35 Hideshima T, Chauhan D, Ishitsuka K, Yasui H, Raje N, Kumar S, et al. Molecular characterization of PS-341 (bortezomib) resistance: implications for overcoming resistance using lysophosphatidic acid acyltransferase (LPAAT)-betainhibitors. Oncogene 2005; 24: 3121-9.

36 Chauhan D, Li G, Podar K, Hideshima T, Mitsiades C, Schlossman R, et al. Targeting mitochondria to overcome conventional and bortezomib/proteasome inhibitor PS-341 resistance in multiple myeloma (MM) cells. Blood 2004; 104: 2458-66.

37 Lightcap ES, McCormack TA, Pien CS, Chau V, Adams J, Elliott PJ. Proteasome inhibition measurements: clinical application. Clin Chem 2000; 46: 673-83.

38 Adams J. Preclinical and clinical evaluation of proteasome inhibitor PS-341 for the treatment of cancer. Curr Opin Chem Biol 2002; 6: 493-500.

39 Adams J. Development of the proteasome inhibitor PS-341. Oncologist 2002; 7: 9-16.

40 Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000; 288: 870-4.

41 Chauhan D, Li G, Hideshima T, Podar K, Mitsiades C, Mitsiades N, et al. JNK-dependent release of mitochondrial protein, Smac, during apoptosis in multiple myeloma (MM) cells. J Biol Chem 2003; 278: 17593-6.

42 Hideshima T, Chauhan D, Hayashi T, Akiyama M, Mitsiades N, Mitsiades C, et al. Proteasome inhibitor PS-341 abrogates IL-6 triggered signaling cascades via caspase-dependent downregulation of gp130 in multiple myeloma. Oncogene 2003; 20: 8386-93.

43 Hideshima T, Mitsiades C, Akiyama M, Hayashi T, Chauhan D, Richardson P, et al. Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341. Blood 2003; 101: 1530-4.

44 Orlowski RZ, Stinchcombe TE, Mitchell BS, Shea TC, Baldwin AS, Stahl S, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol 2002; 15: 4420-7.

45 Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 2003; 348: 2609-17.

46 Jagannath S, Brian D, Wolf JL, Camacho E, Irwin D, Lutzky J, et al. A phase 2 study of bortezomid as firts-line therapy in patients with multiple myeloma. Blood 2004; 104: 98a; (abstract 333).

47 Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T, et al. Assessment of Proteasome Inhibition for Extending Remissions (Apex) Investigators. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med 2005; 352: 2487-98.

48 Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, et al. Clinical factors predictive of outcome with bortezomib in patients with relapsed, refractory multiple myeloma. Blood 2005; (in press).

49 Richardson PG, Chanan-Khan A, Schlossman RL, Munshi NC, Wen P, Briemberg H, et al. Phase II trial of single agent bortezomid (Velcade) in patients with previously untreated multiple myeloma. Blood 2004; 104: 100a; (abstract 336).

50 Harousseau JL, Attal M, Leleu X, et al. Bortezomib (Velcade^®) plus dexamethasone as induction treatment prior to autologuous stem cell transplantation in patients with newly diagnosed multiple myeloma: preliminary results of an IFM phase II study. Proceed ASH 2004; 1490.

51 Cavenagh JD, Popat R, Curry N, Stec J, Morris TC, Drake M, et al. PAD combinaison therapy (PS-341/Bortezomid, adriamycin and dexamethasone) for previuosly untreated patients with multiple myeloma. Blood 2004; 104: 413a; (abstract 1478).

52 Hollmig K, Stover J, Talamo G, Zangari M, Thertulien R, van Rhee F, et al. Addition of bortezomi (Velcade) to high dose melphalan (VelMel) as an effective conditionning regimen with autologous stem cell support in multiple myeloma (MM). Blood 2004; 104: 266a; (abstract 929).

53 Giralt S, Aleman A, Lei X, Davis M, Mickler K, Weber D, et al. Results of bortezomid (BTZ) therapy for myeloma (MM) patients relapsing after an allogeneic transplant. Preliminary results show efficacy without induction of GVHD. Blood 2004; 104: 459a; (abstract 1651).

54 Sun K, Welniak LA, Panoskaltsis-Mortari A, O’Shaughnessy MJ, Liu H, Barao I, et al. Inhibition of acute graft-versus-host disease with retention of graft-versus-tumor effects by the proteasome inhibitor bortezomib. Proc Natl Acad Sci USA 2004; 101: 8120-5.

55 Goy A, Younes A, McLaughlin P. Phase II study of proteasome inhibitor bortezomib in relapsed or refractory B-cell non-hodgkin’s lymphoma. J Clin Oncol 2005; 23: 667-75.

56 O’Connor OA, Wright J, Moskowitz C. Phase II clinical experience with the novel proteasome inhibitor bortezomib in patients with indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. J Clin Oncol 2005; 23: 676-84.

57 Duechler M, Linke A, Cebula B, Shehata M, Schwarzmeier JD, Robak T, et al. In vitro cytotoxic effect of proteasome inhibitor bortezomib in combination with purine nucleoside analogues on chronic lymphocytic leukaemia cells. Eur J Haematol 2005; 74: 407-17.

58 Papandreou CN, Daliani DD, Nix D, Yang H, Madden T, Wang X, et al. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 2004; 22: 2108-21.

59 Aghajanian C, Soignet S, Dizon DS, Pien CS, Adams J, Elliott PJ, et al. A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies. Clin Cancer Res 2002; 8: 2505-11.

60 Dy GK, Thomas JP, Wilding G, Bruzek L, Mandrekar S, Erlichman C, et al. A phase I and pharmacologic trial of two schedules of the proteasome inhibitor, PS-341 (bortezomib, velcade), in patients with advanced cancer. Clin Cancer Res 2005; 11: 3410-6.

61 Davis NB, Taber DA, Ansari RH, Ryan CW, George C, Vokes EE, et al. Phase II trial of PS-341 in patients with renal cell cancer: a University of Chicago phase II consortium study. J Clin Oncol 2004; 22: 115-9.

62 Shah MH, Young D, Kindler HL, Webb I, Kleiber B, Wright J, et al. Phase II study of the proteasome inhibitor bortezomib (PS-341) in patients with metastatic neuroendocrine tumors. Clin Cancer Res 2004; 10: 6111-8.

63 Markovic SN, Geyer SM, Dawkins F, Sharfman W, Albertini M, Maples W, et al. A phase II study of bortezomib in the treatment of metastatic malignant melanoma. Cancer 2005; 103: 2584-9.

64 Maki RG, Kraft AS, Scheu K, Yamada J, Wadler S, Antonescu CR, et al. A multicenter phase II study of bortezomib in recurrent or metastatic sarcomas. Cancer 2005; 10: 1431-8.

65 Mackay H, Hedley D, Major P, Townsley C, Mackenzie M, Vincent M, et al. A phase II trial with pharmacodynamic endpoints of the proteasome inhibitor bortezomib in patients with metastatic colorectal cancer. Clin Cancer Res 2005; 11: 5526-33.

66 Voorhees PM, Dees EC, O’Neil B, Orlowski RZ. The proteasome as a target for cancer therapy. Clin Cancer Res 2003; 9: 6316-25.

67 Jagannath S, Barlogie B, Berenson JR, Singhal S, Alexanian R, Srkalovic G, et al. Summit/Crest Investigators. Bortezomib in recurrent and/or refractory multiple myeloma. Initial clinical experience in patients with impared renal function. Cancer 2005; 103: 1195-200.

68 Palombella VJ, Rando OJ, Goldberg AL, Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell 1994; 78: 773-85.

69 Harousseau JL. Methods for evaluating biological response modifiers. Bull Cancer 1998; 85: 993-6.