refractory multiple myeloma

Leukemia Research 34 (2010) 1111–1118

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Invited review

Combined proteasome and histone deacetylase inhibition: A promising synergy for patients with relapsed/refractory multiple myeloma Sundar Jagannath a,∗ , Meletios A. Dimopoulos b , Sagar Lonial c a

St Vincent’s Catholic Medical Center, 325 W. 15th Street, New York, NY 10011-8202, USA Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece c Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA b

a r t i c l e

i n f o

Article history: Received 9 September 2009 Received in revised form 1 April 2010 Accepted 4 April 2010 Available online 15 May 2010 Keywords: Vorinostat Multiple myeloma Histone deacetylase inhibitor Proteasome inhibitor Bortezomib

a b s t r a c t Multiple myeloma (MM) is an incurable disease characterized by the accumulation of malignant plasma cells in the bone marrow. Recently, an improved understanding of the biology of the disease has led to the development of targeted agents such as the proteasome inhibitor bortezomib and the immunomodulatory agents thalidomide and lenalidomide; however, MM remains incurable. The combination of bortezomib and an HDAC inhibitor synergistically induces MM cell apoptosis and may be of value in the treatment of patients with relapsed/refractory MM. This review examines the potential of combined proteasome and HDAC inhibition in the treatment of relapsed/refractory MM. © 2010 Published by Elsevier Ltd.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1111 Bortezomib and the treatment of MM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1112 HDAC inhibition and DNA transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1112 Overcoming bortezomib resistance: synergistic mechanisms of combined proteasome and HDAC inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114 HDAC inhibition in MM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1116 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117

1. Introduction Multiple myeloma (MM) is a malignancy of plasma cells that accumulate in the bone marrow, where cytokines and growth factors promote plasma cell growth and resistance to therapy. MM accounts for 1% of all cancers and around 10% of all hematologic malignancies, and estimates suggest that 19,920 patients will be diagnosed with MM and 10,690 MM patients will die during 2008 in the United States [1]. Because of the effects of malignant plasma cells on the marrow and marrow microenvironment, patients present with anemia, predisposition to infection, bone pain, fractures, and elevated blood calcium. In addition, high lev-

∗ Corresponding author. Tel.: +1 212 604 6068; fax: +1 212 604 6029. E-mail address: [email protected] (S. Jagannath). 0145-2126/$ – see front matter © 2010 Published by Elsevier Ltd. doi:10.1016/j.leukres.2010.04.001

els of circulating light chain or protein produced by plasma cells can lead to significant renal dysfunction and primary amyloidosis. As such, MM is associated with significant morbidity. At present, treatment for MM focuses on the reduction of tumor growth and the treatment of symptoms. With an estimated overall survival rate of 54.4% and an event-free survival of 49.3% after 5 years, MM is still considered an incurable disease [2,3]. Currently, there is no single standard therapy for MM and treatment depends on patients’ age, complications, and comorbidities. Until recently, high-dose therapy and autologous peripheral blood stem cell (PBSC) transplant, cytotoxic agents, and steroids have provided the mainstay of MM treatment regimens [3]. However, not all patients are eligible for high-dose therapy and autologous PBSC transplant. While initial therapy can be successful, the emergence of a drug-resistant clone over time results in eventual loss of control of the disease [3]. Over recent years, a better understanding

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Table 1 Clinical studies examining the use of bortezomib in the treatment of MM [26]. [Reproduced from Reece et al. Treatment of relapsed and refractory myeloma. Leukemia & Lymphoma, 2008. Reprinted with permission of the publisher, Taylor & Francis Group, http://www.informaworld.com]. Study design

Single-agent trials 1 Phase III (APEX)

Regimen (dose)

N

Bortezomib (1.3 mg/m2 ) Dexamethasone

333 336

ORR (%)

CR (%)

Median survival (months) PFS (%)

OS (%)

43* 18

9* 1

6.2* (TTP) 3.5 (TTP)

29.8* 23.7

2

Randomized Phase II (CREST)

Bortezomib (1.3 mg/m2 ) Bortezomib (1.0 mg/m2 )

26 27

38 30

4 4

13.7 (TTP) 9.5 (TTP)

NR 26.7

3

Phase II (SUMMIT)

Bortezomib (1.3 mg/m2 )

193

28

4

7 (TTP)

17

Bortezomib/PLD Bortezomib (1.3 mg/m2 ) VDC BM (po) VMPT Weekly VMp ATO/B/AA

324 322 54 35 30 29 22

52* 44 76 47 67 62 9

9.3* (TTP) 6.5 (TTP) 12 (EFS) 8 61‡ 6.6 5

82* , † 75† 22 NR 84‡ 20.2 74‡

Combination trials 1 PHASE III (MMY-3001) 2 3 4 5 6

Phase II Phase I/II Phase I/II Phase II Phase I/II

4 2 15 6 17 3 0

Single agent trials: Study 1 [17,18], Study 2 [14], and Study 3 [15,16]. Combination trials: Study 1 [22,24], Study 2 [23], Study 3 [20], Study 4 [25], Study 5 [27], and Study 6 [21]. N, number; ORR, overall response rate; CR, complete response; PFS, progression-free survival; OS, overall survival; TTP, time to progression; PLD, pegylated liposomal doxorubicin; VDC, bortezomib, dexamethasone, cyclophosphamide; EFS, event-free survival; BM, bortezomib and melphalan; po, oral delivery; VMPT, bortezomib, melphalan, prednisone, thalidomide; VMp, bortezomib and methylprednisone; ATO/B/AA, arsenic trioxide, bortezomib, ascorbic acid. * p < 0.05. † At 14 months. ‡ At 1 year.

of the biology of the disease has led to the development of targeted agents such as the proteasome inhibitor bortezomib and the immunomodulatory agents thalidomide and lenalidomide. While these agents have prolonged the overall survival of MM patients, it is still generally accepted that almost all patients with MM will eventually develop resistance to these treatments [3]. Patients who relapse and are non-responsive to second-line treatment, or who exhibit disease progression within 60 days of therapy (refractory disease), have a particularly poor prognosis. Given the eventual development of refractory disease for nearly all patients, there is a need to develop specific new approaches that are active against the disease or agents that will enhance the efficacy of existing treatments. One promising approach is the inhibition of the enzyme histone deacetylase (HDAC) which has shown anti-myeloma activity in preclinical and clinical studies. This review examines the potential role of HDAC inhibitors when used in combination with bortezomib, in MM patients with relapsed or refractory disease following treatment with bortezomib monotherapy. 2. Bortezomib and the treatment of MM Bortezomib potently and selectively inhibits the function of the proteasome, a key regulator of intracellular protein degradation [4]. Proteasome inhibition results in the accumulation of mis-folded and damaged proteins, which, in turn, triggers a heatshock protein response leading to apoptosis [5]. Malignant plasma cells, as a result of increased immunoglobulin production, are more dependent upon protein regulation for homeostasis than other normal cells, rendering them more sensitive to the effects of proteasomal inhibition. Proteasome inhibition therefore appears to have a greater effect on MM cells compared with normal cells [6]. In patients with newly-diagnosed MM, including those with poor prognostic factors (advanced-stage disease, high tumor burden, renal impairment, and high-risk cytogenetics), bortezomib has consistently shown rapid and durable therapeutic benefit, with high rates of complete response (CR) both as monotherapy and when used in combination with other anti-MM agents [7–11]. Cur-

rently, in the United States bortezomib is approved for first-line treatment of MM [12]. In Europe, it is approved for use in combination with melphalan and prednisone for the treatment of patients with previously untreated MM who are not eligible for high-dose chemotherapy with peripheral blood stem cell (PBSC) transplant, or as monotherapy for the treatment of progressive MM in patients who have received at least one prior therapy and who have already undergone or are unsuitable for PBSC transplantation [13]. The efficacy of bortezomib monotherapy in patients with relapsed/refractory MM was reported in several trials including the SUMMIT (Study of Uncontrolled MM managed with proteasome Inhibition Therapy), CREST (Clinical Response and Efficacy Study of bortezomib in the Treatment of relapsing MM), and APEX (Assessment of Proteasome inhibition for Extending Remissions) trials (Table 1 [14–18]). Among patients with first relapse, the overall response rate (ORR) for single agent bortezomib was 50%, and based on the results of these studies, bortezomib was approved for the treatment of patients with relapsed and refractory MM. Although the use of bortezomib has resulted in high ORR, CR, and improvements in survival, many patients have either short duration or no response to bortezomib-based salvage therapy [19]. In order to overcome primary or acquired drug resistance, bortezomib has been combined with many other anti-MM agents leading to improved responses (Table 1 [14–18,20–28]). Currently, there is a need for novel treatment strategies that can improve the response to bortezomib in early relapse or among patients with bortezomib-resistant disease. Preclinical data have demonstrated robust in vitro responses for the combination of an HDAC inhibitor with bortezomib [29,30]. With ongoing preclinical and clinical experience, combined proteasome and HDAC inhibition has shown some promise in the treatment of relapsed/refractory MM. 3. HDAC inhibition and DNA transcription The transcription of DNA is, in part, regulated by the action of histone acetyltransferases (HAT) and HDAC enzymes, which act in conjunction to regulate the acetylation of histones. HAT enzymes work by transferring an acetyl group from acetyl CoA to a lysine

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Table 2 Classes of HDAC inhibitors. Classes of HDAC inhibitors

Agent

HDAC targets

Hydroxamic acids

Vorinostat

Pan-HDAC inhibitor

Panobinostat

Pan-HDAC inhibitor

Belinostat

Pan-HDAC inhibitor

Entinostat

Specific Class I inhibitor

Mocetinonostat

Specific Class I inhibitor

Romidepsin

Specific Class I inhibitor

Benzamides

Cyclic tetrapeptides

Pan-HDAC inhibitor: inhibitor of Class I and II HDACs; specific Class I inhibitor: inhibitor of primarily class I HDACs.

amino acid on the histone molecule to form ␧-N-acetyl lysine. This acetylation has the effect of neutralizing the overall positive charge of the histone molecule, reducing its affinity for binding to negatively charged DNA. By this process, HATs confer an open chromatin structure which renders DNA more accessible to transcription factors. In contrast, HDAC enzymes remove acetyl groups from ␧-N-acetyl lysine, increase the positive charge on the histone, and promote binding to DNA. This causes a condensing of the DNA, preventing gene transcription. By inhibiting HDAC enzyme activity, HDAC inhibitors modulate the expression of both pro- and nonapoptotic factors. In cancer cells where the balance favors cellular proliferation, HDAC inhibitors may alter the gene expression favoring growth arrest, differentiation or apoptosis. The transcription of pro-apoptotic genes subsequently leads to the suppression of anti-apoptotic proteins [31]. In addition, HDAC-inhibitor-mediated acetylation of transcription factors and other proteins has been

shown to alter gene expression [32,33]. For instance, acetylation of p53 opens the DNA binding domain of p53 and prevents association with MDM2 and ubiquitination and subsequent destruction [34]. As a result of these transcriptional and non-transcriptional events, HDAC inhibitors cause tumor cell differentiation, cell-cycle arrest and apoptosis [31,33]. Four classes of HDAC enzymes have been indentified. Classes I, II, and IV are Zn2+ -dependent enzymes whereas Class III are Zn2+ independent, NAD+ -dependent enzymes [35]. The Class I HDACS (HDAC1, HDAC2, HDAC3, and HDAC8) are primarily nuclear, ubiquitously expressed, and have histone protein substrates [35]. Class II enzymes (IIa; HDAC4, HDAC5, HDAC7, and HDAC9 and IIb; HDAC6 and HDAC10) are primarily localized in the cytoplasm, shuttle in and out of the nucleus, are expressed in a tissue specific manner, and have both histone and non-histone protein substrates [35,36]. Class III HDACs (SIRT 1–7) are sirutins, which are subcellu-

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Fig. 1. Potential mechanism of combined proteasome and HDAC inhibition in myeloma cells [30,43]. Proteasome inhibition results in the accumulation of large quantities of ubiquitin-conjugated proteins; MM cells organize these proteins into perinuclear aggresomes. The formation of perinuclear aggresomes requires HDAC 6 activity. It is thought that the disruption of bortezomib-induced aggresome formation and sensitisation of aggresome-positive cells to apoptosis by HDAC inhibition may mediate the synergistic effect of combined proteasome and HDAC inhibition. Reproduced with kind permission [30,43], © American Society of Hematology and © National Academy of Sciences, respectively.

larly located and have non-histone protein substrates [37]. Class IV comprises of HDAC 11 of which little is known [35]. The current HDAC inhibitors in the clinic comprise of two classes; non-specific pan-HDAC inhibitors and Class I HDAC inhibitors (Table 2). Vorinostat (suberoylanilide hydroxamic acid; Zolinza® ) is a nanomolar inhibitor of Class I and II HDAC enzymes, and was approved by the US Food and Drug Administration in October 2006 for the treatment of cutaneous manifestations of T-cell lymphoma, a type of non-Hodgkin’s lymphoma, in patients with progressive, persistent or recurrent disease on or following two

systemic therapies [38]. Since 2006, vorinostat has shown promising activity in the treatment of other hematologic malignancies as well as solid tumors. 4. Overcoming bortezomib resistance: synergistic mechanisms of combined proteasome and HDAC inhibition Resistance to bortezomib monotherapy presents a challenge to the successful treatment of relapsed/refractory MM. Although the exact mechanism underlying bortezomib resistance remains

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Table 3 Clinical studies examining the use of HDAC inhibitors in combination with bortezomib in the treatment of MM: preliminary results.

[54,55]

[49]

Study design

Treatment

N

Preliminary results

Phase I – ‘3+3’ dose escalation Relapsed/refractory MM

Vorinostat: 200 mg bid or 400 mg qd for 14 days Bortezomib: 0.7 or 0.9 mg/m2 on Days 4, 8, 11, and 15 OR 0.9, 1.1, or 1.3 mg/m2 on Days 1, 4, 8, and 11

Overall: 33 evaluable patients

12 (36.4%) PR 6 (18.2%) MR 13 (39.4%) SD

Prior bortezomib: 17 evaluable patients

6 (35.3%) PR 4 (23.5%) MR 7 (41.2%) SD 2 (9.5%) VGPR 7 (33.3%) PR 10 (47.6%) SD 2 (9.5%) PD

Phase I – dose escalation Relapsed/refractory MM

Overall: 21 evaluable patients

Vorinostat: 100–400 on Days 4–11 Bortezomib: 1–1.3 mg/m2 on Days 1, 4, 8, and 11

Prior bortezomib: 17 evaluable patients

[52]

Phase I/II study Relapsed/refractory MM

Romidepsin: 8–14 mg/m2 on Days 1, 8, and 15 Bortezomib: 1.3 mg/m2 on Days 1, 4, 8, and 11 Dexamethasone: 20 mg/m2 on Days 1, 2, 4, 5, 8, 9, 11, and 12

unknown, a number of pathways have been shown to promote myeloma cell survival; these include (i) the interaction between MM cells and host bone marrow microenvironment; (ii) upregulated expression of growth factor receptors and related signaling pathways; (iii) over-expression of anti-apoptotic proteins, such as Bcl-2; (iv) defects in drug-induced apoptotic signaling pathways, including those that occur at the level of mitochondria or endoplasmic reticulum; and (v) over-expression of P-glycoprotein [39]. More recently, heat shock protein 27 and aggresome formation have been cited as potential mediators of bortezomib resistance [39,40]. The mechanism by which synergistic activity of HDAC inhibition and bortezomib in MM cells acts is also unknown, but HDAC inhibitors exhibit a plethora of molecular mechanisms that may enhance the activity of bortezomib [29,41]. These mechanisms work on similar pathways implicated in bortezomib resistance. Initial studies in MM cell lines suggested that vorinostat suppressed the expression of proteasome subunits and several ubiquitin conjugating enzymes [29]. Further to this, the disruption of bortezomib-induced aggresome formation and sensitisation of aggresome-positive cells to apoptosis by vorinostat was reported in pancreatic cancer cells, suggesting a potential mechanism by which vorinostat could augment the activity of bortezomib [40,42]. Proteasome inhibition results in the accumulation of large quantities of ubiquitin-conjugated proteins. MM cells are thought to organize these proteins into perinuclear structures called aggresomes, which are cytoprotective [40]. The formation of aggresomes requires HDAC-6 activity so the addition of an HDAC inhibitor could provide a synergistic approach to treatment (Fig. 1 [30,43]). The inhibition of bortezomib-induced aggresome formation results in the dispersal of toxic microaggregates which in turn induce endoplasmic reticulum (ER) stress that may contribute to reactive oxygen species (ROS) generation [44]. This ROS generation, coupled with that simultaneously induced by the HDAC inhibitor, leads to overwhelming oxidative stress that has been associated with enhanced apoptosis [45]. Various preclinical models

Overall: 18 evaluable patients

1 (5.9%) VGPR 5 (29.4%) PR 9 (52.9%) SD 2(11.8%) PD Overall response rate: 67% (12 patients) 4 (22%) CR/near CR 4 (22%) VGPR 4 (22%) 4 PR 5 (28%) MR

have demonstrated that vorinostat in combination has resulted in synergistic apoptotic effects with associated increases in ROS and mitochondrial injury, caspase and poly (ADP-ribose) polymerase activation [29,45]. Similar results have been seen in MM cell lines, not only with vorinostat [40,42] but also with panobinostat, a hydroxamic acid pan-HDAC inhibitor [30] and the more specific HDAC-6 inhibitor tubacin [43]. In MM cells, vorinostat has also been shown to suppress the stimulation of interleukin-6 (IL-6) secretion triggered by MM cell adhesion to bone marrow stromal cells, downregulate IL-6 and insulin-like growth factor receptor signaling cascades, reduce the expression of anti-apoptotic members of the proto-oncogene Bcl-2 family, and downregulate the expression and activity of oncogenic kinases, DNA synthesis/repair enzymes and transcription factors [41,46]. Vorinostat has also been shown to enhance the anti-myeloma effect of other anti-cancer agents. In human MM cell lines, vorinostat enhanced the cytotoxic and apoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand [47], and reduced the viability of tumor cells isolated from patients with MM when combined with interferon ␣2b [48]. Furthermore, sequential exposure of human MM cell lines and primary patient-derived MM cells to bortezomib and vorinostat resulted in a marked increase in mitochondrial injury, caspase activation, and the synergistic induction of apoptosis [45]. Additionally, a subset analysis from a Phase I clinical trial showed reductions in nuclear factor-kappaB (NF-␬B), Bcl-2, Bcl-xl, cyclin-dependent kinase inhibitor 1A (p21) and X-linked inhibitor of apoptosis protein in patients with a treatment response to vorinostat in combination with bortezomib, as compared with those patients with PD or SD [49]. In addition to HDAC inhibition (Class I, excluding HDAC 8 and Class IIa excluding HDAC 9), valproic acid (VPA), a carboxylic acid HDAC inhibitor, has also been shown to decrease vascular endothelial growth factor secretion and inhibit angiogenesis (a mechanism essential for tumor growth), increase p21 accumulation, reduce cyclin D1, and induce G0/G1 cell-cycle arrest [50].

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5. HDAC inhibition in MM HDAC inhibitors have demonstrated preclinical and clinical activity in a number of malignancies [31] and, in combination with bortezomib, have shown potential in the treatment of MM (Table 3) [29,30,45,49,51–55]. As a single agent, vorinostat demonstrated the ability to induce early growth arrest and subsequent apoptosis in human MM cell lines and primary MM cells isolated from MM patients [56]. A Phase I study of oral vorinostat enrolled 13 heavily pretreated myeloma patients. All patients had previously received either dexamethasone or prednisone and the median number of prior systemic anti-cancer therapies received was 3, with a range of 1–10. Six patients had relapsed disease with the remaining 7 patients having relapsed and refractory disease. The results showed modest single-agent activity: of the 10 evaluable patients, 1 had a minimal response (MR) and 9 had SD, including 5 patients who were progression-free at 3 months, and 1 patient who was progressionfree at 6 months. Only small changes in the Eastern Cooperative Oncology Group (ECOG) performance status throughout the study were seen in the 9 patients with SD [57]. Vorinostat was generally well tolerated with the most common drug-related adverse events (AEs) reported being fatigue, anorexia, dehydration, diarrhea, and nausea; only 1 patient experienced a dose-limiting toxicity (DLT) (Grade 3 fatigue) [57]. Although there were no safety concerns, the study was terminated early due to a sponsor decision. In a Phase I combination study by Weber et al [55], patients with active relapsed or refractory MM were randomized in a conventional ‘3 + 3’ dose-escalation scheme to receive oral vorinostat (200 mg twice daily or 400 mg once daily for 14 days) in combination with bortezomib (0.7 or 0.9 mg/m2 on Days 4, 8, 11, and 15 or 0.9, 1.1, or 1.3 mg/m2 on Days 1, 4, 8, and 11) [55]. The best responses observed in the 33 evaluable patients were as follows; 12 (36.4%) patients had an partial response (PR), 6 (18.2%) patients exhibited a MR, and 13 (39.4%) patients had SD. Of 34 patients enrolled, a total of 29 (85.3%) patients discontinued treatment; 17 (50.0%) due to PD, 11 (32.4%) due to AEs, and 1 (2.9%) patient withdrew consent [55]. Within the treatment cohort, 17 patients had received prior treatment with bortezomib; the best responses observed in these patients were a PR in 6 patients, an MR in 4 patients, and SD in 7 patients [54]. Of these, 7 were considered refractory to bortezomib and the best responses observed were a PR in 2 patients, an MR in 2 patients, and SD in 3 patients [54]. In total, 14 patients previously treated with bortezomib discontinued treatment due to PD (9) and AEs (5). Two patients experienced a DLT; Grade 3 transient aspartate aminotransferase elevation in 1 patient receiving vorinostat 400 mg once daily and bortezomib 0.9 mg/m2 and Grade 4 thrombocytopenia in 1 patient receiving vorinostat 400 mg once daily and bortezomib 1.3 mg/m2 [55]. The maximum tolerated dose (MTD) was not reached (non-occurrence of ≥2 DLTs in 6 patients at any dose level); consequently, the highest dose level (vorinostat 400 mg once daily plus bortezomib 1.3 mg/m2 on Days 1, 4, 8, and 11) was considered the maximum administered dose and is being investigated in an additional 10 patients who have been enrolled in the expansion cohort [55]. The study by Badros et al. [49] has also shown promising responses to combined vorinostat and bortezomib treatment in patients with relapsed and refractory MM. Of the 21 patients evaluable for efficacy, the best response to vorinostat plus bortezomib was a very good partial response (VGPR) in 2 (9.5%) patients, a PR in 7 (33.3%) patients, SD in 10 (47.6%) patients, and PD in 2 (9.5%) patients [49]. Of 19 patients who had received prior bortezomib therapy, 17 were evaluable for efficacy. Of these, 1 (5.9%) patient achieved a best response of a VGPR, 5 (29.4%) patients had a PR, 9 (52.9%) patients had SD, and 2 (11.8%) patients had PD [49]. Eight of the 9 patients who had received prior bortezomib therapy and

were considered refractory were evaluable for efficacy; the best responses observed in these patients were a PR in 3 patients, SD in 4 patients, and PD in 1 patient [49]. The majority of AEs were hematologic and 2 patients experienced DLTs (Grade 3 fatigue and Grade 3 prolonged QTc interval). The MTD was established as vorinostat 400 mg once daily on Days 4–11 plus bortezomib 1.3 mg/m2 on Days 1, 4, 8, and 11 of a 21-day cycle [49]. These preliminary data from two Phase I studies suggest that the combination of vorinostat with bortezomib shows activity in patients with relapsed/refractory MM who had previously received or were naïve to treatment with bortezomib. The final results of these studies are eagerly awaited. Other HDAC inhibitors have shown activity in the treatment of MM. The anti-neoplastic activity of VPA was first reported in 1997 and was thought to be mediated through HDAC inhibition [58,59]. Although a relatively weak HDAC inhibitor [60], VPA has shown in vitro activity against various myeloma cell lines and primary MM cells [50,61,62]. The anti-myeloma effects of VPA are timeand dose-dependent and characterized by an accumulation of p21, reduced levels of cyclin D1 and G0/G1 cell-cycle arrest [50,61,62]. Interestingly, VPA has also been reported to reduce myeloma cell vascular endothelial growth factor secretion and inhibit angiogenesis, which is thought to be essential for tumor growth [50,61]. Moreover, the anti-angiogenic effects of VPA were potentiated in the presence of thalidomide [61]. VPA has also been shown to potentiate dexamethasone-mediated apoptosis in myeloma cells [61,63] and the effects of proteasome inhibition in leukemia cells [61,63]. In preclinical studies, romidepsin, a cyclic peptide HDAC inhibitor that preferentially inhibits Class I HDACs, induced apoptosis in MM cell lines, an effect that was potentiated by the addition of melphalan [64]. Other studies have shown that romidepsin in combination with bortezomib has potential for the treatment of leukemia [65]. Romidepsin has shown promise in the treatment of patients with relapsed/refractory MM when used in combination with bortezomib and dexamethasone [52]. Of 18 evaluable patients in a Phase I/II study, the combination of romidepsin, bortezomib, and dexamethasone was associated with an ORR of 67% (12 patients), with a CR or near CR, a VGPR, or a PR (in 22% [4 patients] each). An additional 28% (5 patients) experienced an MR [52]. The most common drug-related toxicities included fatigue (Grade 3, n = 2), neutropenia (Grade 3, n = 1), sepsis (Grade 3, n = 2), peripheral neuropathy (Grade 3, n = 1; Grade 2, n = 6), and nausea (Grade 2, n = 1) [52]. Belinostat is a low-molecular-weight hydroxamic acid HDAC (Class I/II) inhibitor with potent anti-proliferative and HDAC inhibitory activity. In preclinical studies, the combination of belinostat and bortezomib has resulted in selective synergistic anti-tumor activity and inhibition of osteoclast activity [51]. Increased oxidative stress, caspase activation and induction of apoptosis mediated the synergistic effects of exposure to this treatment regimen [51]. While results of a Phase I study in patients with advanced hematologic cancers indicated that single-agent belinostat may have activity in the treatment of MM [66], a Phase II study examining the efficacy and safety of belinostat in combination with bortezomib was recently terminated due to the development of DLTs [67]. The future of belinostat in the treatment of MM remains unknown. Finally, in vitro, panobinostat, a potent pan-HDAC inhibitor, induces both caspase-dependent and caspase-independent apoptosis and potentiates the effects of dexamethasone, melphalan, and bortezomib in drug-resistant MM cell lines and primary cells isolated from both treatment-naïve and treatment-refractory MM patients [53]. Panobinostat has also shown the ability to overcome bortezomib resistance by inhibiting the formation of aggresomes [30]. A Phase II/III study of panobinostat in relapsed/refractory MM has recently been terminated by the sponsor [68,69]. A Phase I

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study of panobinostat in combination with bortezomib is currently underway in patients with relapsed and refractory MM [70].

[12]

6. Conclusions Although MM treatment has improved in recent years with the introduction of new drugs such as bortezomib and lenalidomide, and older immunomodulatory agents such as thalidomide, many patients will ultimately relapse and others are refractory to these innovative agents. Therefore, there is a need for novel medications or new treatment combinations that can improve the treatment of MM. While the characterization of HDAC inhibitors in MM is still ongoing, results from recent studies suggest that combined HDAC and proteasome inhibition may have potential efficacy, even in patients with relapsed/refractory disease who have previously received bortezomib treatment. The HDAC inhibitors vorinostat and romidepsin have both shown anti-myeloma activity in the clinical trial setting when combined with bortezomib in patients with relapsed/refractory MM. Meanwhile, a Phase I study examining the activity of panobinostat in combination with bortezomib is currently underway. Vorinostat in combination with bortezomib has been shown to possess anti-myeloma activity in relapsed/refractory patients who have received prior bortezomib treatment. These study findings suggest that the combination of vorinostat and bortezomib has the potential to generate meaningful responses in patients with refractory disease. The final efficacy and safety results of Phase III trials examining combined vorinostat and bortezomib treatment in relapsed MM patients are eagerly awaited and will aid decisions regarding the future use of vorinostat in combination with bortezomib in the clinic.

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Acknowledgement [24]

The authors would like to thank Dr Steven G. Burke from Complete Medical Communications who provided medical writing support funded by Merck & Co., Inc.

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