Development of the pan-DAC inhibitor panobinostat (LBH589): Successes and challenges

Cancer Letters 280 (2009) 233–241

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

Development of the pan-DAC inhibitor panobinostat (LBH589): Successes and challenges Peter Atadja * Novartis Institutes for Biomedical Research, Cambridge, MA, USA

a r t i c l e

i n f o

Article history: Received 23 December 2008 Received in revised form 3 February 2009 Accepted 9 February 2009

Keywords: Panobinostat LBH589 Histone deacetylase inhibitors Pharmacodynamics Hematologic malignancies Solid tumors

a b s t r a c t The histone deacetylase (HDAC) inhibitors are emerging as a highly useful class of anticancer agents that inhibit the enzyme HDAC involved in the deacetylation of histone and nonhistone cellular proteins. The HDAC inhibitor, panobinostat (LBH589, Novartis Pharmaceuticals), achieves potent inhibition of all HDAC enzymes implicated in cancer and has demonstrated potent anti-tumor activity in preclinical models and promising clinical efficacy in cancer patients. In this review we discuss the successes and challenges surrounding the development of panobinostat, focusing on its proposed mechanism of action, preclinical anti-tumor activity, and early clinical efficacy in hematologic and solid tumors. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In recent years, considerable evidence has emerged to suggest that, in addition to genetic mutations, epigenetic changes also play a critical role in the onset of cancer and its progression [1]. Epigenetic changes are defined as heritable changes in gene expression that are not due to any alteration in the DNA sequence, and most commonly include increased methylation of CpG islands within DNA gene promoter regions and post-translational modifications on histone proteins [1–3]. Changes to the patterns of epigenetic modification are common in cancer and evidence suggests that epigenetic dysregulation may be a preliminary transforming event frequently observed in benign neoplasias and early stage tumors [4,5]. DNA and histones provide the main building blocks for nucleosomes, the structural units of chromatin that are important for packaging eukaryotic DNA. Changes in the structural configuration of chromatin to a relatively active (open) or inactive (condensed) form alters the accessibility * Tel.: +1 862 871 3447; fax: +1 862 871 3453. E-mail address: [email protected]. 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.02.019

of DNA for transcription, ultimately affecting gene expression [6]. One of the major ways by which transcription factor binding to DNA is regulated is through changes in chromatin conformation, which in turn is governed by chemical modifications such as the acetylation and deacetylation of lysine residues in the amino terminal tails of the core nucleosomal histones. The changes to core histone acetylation is under the control of opposing activities of the enzymes histone deacetylase (HDAC) and histone acetylase (HAT), leading to changes in gene expression, which includes genes involved in cell cycle regulation, differentiation and apoptosis. Acetylation is generally linked to an ‘open’ chromatin state that is ready for transcription or that corresponds to actively transcribed genomic regions, whereas deacetylation is associated with a closed or inactive state, leading to gene repression. The relative degree of histone acetylation and deacetylation therefore controls the level at which a gene is transcribed. Although histone proteins were traditionally considered to be the primary focus for HDAC and HAT activities, there is now increasing evidence to suggest that acetylation also plays a crucial role in contexts other than histone and DNAdependent processes. A considerable number of non-histone

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proteins that play an important role in cell cycle proliferation and apoptosis have been identified as regulated by HAT and HDAC. These include transcription factors such as p53, NF-jB and E2F1, which play key roles in tumorigenesis and anti-tumor response, as well as proteins that do not directly regulate gene expression but instead regulate DNA repair (Ku70), the cellular cytoskeleton (a-tubulin) and protein stabilisation (Hsp90) [7]. Notably, among non-histone HDAC substrates, Hsp90 plays a major role in the proper folding and stability of several major oncoproteins. HDAC activity also regulates cell protein turnover via the aggresome pathway, which if disrupted, results in the accumulation of polyubiquitinated misfolded protein aggregates, leading to cell stress and caspase-dependent apoptosis [8]. These observations have extended the mechanism of anti-tumor activity of deacetylase inhibitors to include effects on non-histone proteins, implicated in multiple oncogenic pathways, in conjunction with epigenetic changes. 2. HDAC as a potential target for the discovery of anticancer drugs HDACs can be divided into two groups: the zinc-dependent HDACs comprising Class I (HDACs 1, 2, 3 and 8 localized to the nucleus), Class II a/b (HDACs 4, 5, 6, 7, 9 and 10 found in the nucleus and cytoplasm) and Class IV (HDAC11); and the zinc-independent, NAD-dependent Class III sirtuin enzymes [9]. Important targets for Class I HDACs are histones and other proteins, such as the transcription factor p53. Class II HDACs act predominantly on non-histone proteins, including Hsp90, which is the target of deacetylation by HDAC6 [10]. The Class IV HDAC (HDAC11) displays features of both Class I and II HDACs. Increased HDAC expression has been reported in several human tumors and cancer cell lines. For example, HDAC1 is overexpressed in prostate, gastric, colon and breast tumors [11]. Furthermore, HDAC6, in addition to its role in Hsp90 function, acts as a downstream effector of estrogen signaling in breast cancer [12], and HDAC2 is a downstream effector of adenomatosis polyposis coli aberrations known to occur in colon cancers [13]. Deregulation of HDAC activity in association with chromosomal translocated proteins have been closely implicated in the silencing of differentiation and tumor suppressor genes, resulting in the promotion of oncogenesis, particularly leukemias [14]. Because of this important link, the use of HDAC inhibitors to reverse aberrant epigenetic changes in neoplastic cells has emerged as a potential strategy for the treatment of hematopoetic malignancies. Furthermore, because of the additional activity of deacetylases on non-histone proteins, the discovery of HDAC inhibitors provides the opportunity to prevent and reverse the effects of aberrant deacetylation through epigenetic modifications and also via effects on non-histone protein targets implicated in oncogenesis.

3. Panobinostat – a novel HDAC inhibitor The HDAC inhibitors are a group of structurally diverse, targeted anticancer agents of which several are currently

in clinical development. HDAC inhibitors are characterized as Class I-specific or as pan-deacetylase (pan-DAC) inhibitors, denoting activity against both Classes I and II HDACs [15]. Class I-specific inhibitors include MGCD0103, MS275 and romidepsin (depsipeptide). Pan-DAC inhibitors include panobinostat (LBH589), vorinostat (suberoylanilide hydroxamic acid, SAHA) and belinostat (PXD101). The structurally diverse nature of this group of compounds, which includes hydroxamic acid-derived compounds (panobinostat, vorinostat and belinostat), cyclic peptides (romidepsin) and benzamides (MS-275, MGCD0103), is illustrated in Fig. 1. Based on their demonstrable in vitro and in vivo preclinical activity in a wide range of malignancies, HDAC inhibitors have undergone a rapid phase of clinical development in recent years, with many entering Phase I–III clinical trials. Among the pan-DAC inhibitors, vorinostat is currently the most extensively studied and has received approval by the US Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (CTCL) [16]. More recently, the hydroxamic acid pan-DAC inhibitor, panobinostat, has demonstrated promising activity as an anticancer therapeutic agent. In this review, we discuss the development of panobinostat, focusing on its proposed mechanism of action, preclinical anti-tumor activity and early clinical efficacy in hematologic and solid tumors. 3.1. Panobinostat as a potent pan-DAC inhibitor Panobinostat has potent inhibitory activity at low nanomolar concentrations against all Class I, II and IV purified recombinant HDAC enzymes, suggesting true pan-DAC activity [17]. In studies using enzymatic assays, panobinostat IC50 values (half maximal inhibitory concentration) were in the low nanomolar range (613.2 nM) for all HDAC enzymes, with the exception of HDAC4, HDAC7 and HDAC8, which had values in the mid nanomolar range (203–531 nM) (Table 1). Panobinostat IC50 values were also consistently lower than those for the pan-DAC inhibitors, vorinostat and belinostat, and the selective Class I HDAC inhibitor, MGCD0103. As a pan-DAC inhibitor, panobinostat was at least 10-fold more potent than vorinostat [17] and appears to be the most potent among the panDAC inhibitors in development. 3.2. Potency of panobinostat as an inhibitor of tumor cell growth In line with its highly potent HDAC inhibition, panobinostat has demonstrated potent antiproliferative and cytotoxic activity in a variety of cancer cell lines while exerting minimal toxicity on all normal cells tested. At low nanomolar concentrations (concentration necessary to produce 90% cell death [LD90] 14–541 nM), panobinostat-induced growth inhibition and cytotoxicity across CTCL, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), Hodgkin lymphoma, and breast, prostate, colon and pancreatic cancer cell lines [17]. In the HuT78, Hut102, MJ and HH CTCL cell lines, panobinostat-induced growth inhibition was evident at sub-nanomolar IC50 concentrations and significant cell death was observed in HH

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Fig. 1. Chemical structures of HDAC inhibitors.

Table 1 In vitro activity profile of panobinostat in comparison with other histone deacetylase inhibitors. Inhibition of enzyme activity IC50 (nM)

Panobinostat (LBH589)

Vorinostat (SAHA)

Belinostat (PXD-101)

MGCD0103

HDAC1 HDAC2 HDAC3 HDAC4 HDAC5 HDAC6 HDAC7 HDAC8 HDAC9 HDAC10 HDAC11

2.5 13.2 2.1 203 7.8 10.5 531 277 5.7 2.3 2.7

75.5 362 57.4 15,056 163 27.1 12522 1069 78.1 88.4 109

17.6 33.3 21.1 1236 76.3 14.5 598 157 44.2 31.3 44.2

142 147 205 >30,000 1889 >30,000 >30,000 28,167 1177 54.9 104

and HuT78 cells, with LD90 values in the nanomolar range (Shao et al., submitted for publication). Although the in vitro antiproliferative effect of panobinostat is similar across all cell lines, differential sensitivities across cancer cell lines have been observed for panobinostat-induced cell death. Higher LD90 values were reported for breast, colon and pancreatic cell lines compared with AML, CML, Hodgkin lymphoma and CTCL cell lines (306–541 versus 14–57.5 nM). Notably, normal cells

(human mammary and renal epithelial cells) were shown to be particularly resistant to panobinostat cytotoxicity with LD90 values >5 lM, indicating that panobinostat might induce cancer cell-specific cytotoxicity [17]. The differential toxicity of panobinostat for tumor cells versus normal cells was further evaluated by assessing the activation of the apoptosis mediators, caspases, in response to panobinostat in both cancer cells (K562s, HH and HCT116 cells) and normal cell lines (HMEC, HRE, human fetal lung cells and peripheral blood mononuclear cells). Treatment of normal cells with panobinostat 100 nM for 24 h did not result in induction of caspase 3/7 activity compared with vehicle control, whereas panobinostat produced an approximately 10–11-fold induction of caspase activity compared with control treatment in the cancer cell lines K562, HH and HCT116, further supporting the notion that panobinostat selectively induces apoptosis in cancer cells but not in normal cells [17] (Atadja et al., in preparation). Compared with other deacetylase inhibitors, panobinostat exhibited greater anti-tumor potency in terms of inhibition of cancer cell proliferation and viability. For example, in HCT116 cells, BT474 cells and the CTCL line HH, cell proliferation and cell viability were inhibited by panobinostat at nanomolar concentrations that were up to 100-fold lower than the inhibitory concentrations of

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Table 2 Effect of panobinostat on cancer cell proliferation and viability in comparison with vorinostat and MGCD0103. Inhibition of cell proliferation and viability

Panobinostat

Vorinostat

MGCD0103

IC50 [nM]

LD50 [nM]

IC50 [nM]

LD50 [nM]

IC50 [nM]

LD50 [nM]

HH HCT116 BT474

1.8 7.1 2.6

8.3 51.7 22.4

647 1040 1152

1497 3660 5195

244.5 419.8 702.5

677.5 1411 1335

MGCD0103 and vorinostat (Table 2) [17] (Atadja et al., in preparation). Notably, there is good consistency between the concentrations of panobinostat required for HDAC inhibition and antiproliferative activity (IC50 for HDAC inhibition and antiproliferative activity: 2.0–530 and 1.8– 15.9 nM, respectively). This may give panobinostat a potential advantage versus other HDAC inhibitors in terms of relating anti-tumor activity to on-target effects. 3.3. Increased protein acetylation as a biomarker of cellular HDAC inhibition Consistent with its excellent in vitro cellular potency, panobinostat demonstrated effective and dose-dependent acetylation of histone proteins in the human colon carcinoma cell line HCT116. Levels of the acetylated proteins H3 and H4 were increased, in line with Class I HDAC inhibition [17] (Atadja et al., in preparation). The concentration of panobinostat (10 nM) required for histone acetylation was much lower than the concentrations of belinostat, vorinostat and MGCD0103 required for a similar effect. In addition, accumulation of acetylated histone was reported as early as 2 h after treatment with panobinostat (100 nM) and was maintained at 6 and 24 h with continuous treatment [17] (Atadja et al., in preparation). Panobinostat induced histone H3 acetylation in a dosedependent manner. Doses that did not induce histone acetylation were not antiproliferative, suggesting a relevance of the target inhibition in cells to cell growth inhibition in sensitive cells. However, even in non-sensitive cells, increased H3 acetylation was observed, indicating that the differential sensitivity may be due to other non-histone targets or differential effects downstream of histone acetylation in sensitive versus insensitive cells (Shao et al., submitted for publication). 3.4. Effect of panobinostat in tumor-bearing animals Panobinostat has low oral bioavailability in rodents, therefore intravenous (i.v.) and intraperitoneal administration was used to study the drug in tumor-bearing mice and rats. Conversely, the oral bioavailability of panobinostat in dogs is markedly higher than that in rats (6 and 33–50%, respectively) and is comparable to that in humans (49%) (Novartis Pharmaceuticals, data on file). In HCT-116 tumor-bearing mice, administration of a single i.v. dose of panobinostat (25 mg/kg) revealed rapid and preferential uptake and accumulation of panobinostat in tumor cells (Fig. 2) (Novartis Pharmaceuticals, data on file). Concentrations of panobinostat were higher in the tumor compared

Fig. 2. Plasma and tumor exposure levels to panobinostat after administration of a single intravenous dose of panobinostat (25 mg/kg) to HCT116 tumor-bearing mice.

with plasma, suggesting the potential for anti-tumor efficacy with decreased systemic toxicity. The preferential uptake of panobinostat into tumors is also associated with sustained target (HDAC) inhibition in animal models. Robust and durable H3 and H4 acetylation was reported after single-dose i.v. administration of panobinostat to PC3M2AC6 orthotopic tumor-bearing mice, and this increase in histone acetylation persisted for at least 48 h (Atadja et al., in preparation). This finding was indicative of a sustained target effect during dose intermission, providing the opportunity for less-frequent dosing with panobinostat. The potency of panobinostat against tumor cell proliferation and survival in vitro, combined with possible effects on histone and non-histone proteins implicated in oncogenesis, have translated into both potent and broad anti-tumor activity in vivo and synergy in combination with other agents in various tumor xenograft models, including CTCL, multiple myeloma, colon, breast, and pancreatic cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) [17–23] (Shao et al., submitted for publication). In an HH CTCL mouse xenograft model, panobinostat achieved significant tumor regression of up to 94% relative to vehicle-treated animals (versus growth inhibition of 24% with 5-fluorouracil). This was associated with rapid upregulation of acetylated histone H3 and H4 proteins within 1 h, which was sustained after 24 h, suggesting persistent HDAC inhibitory action in vivo [21] (Shao et al., submitted for publication). Panobinostat demonstrated anti-myeloma activity across a range of human myeloma cell lines [20] and significantly inhibited the growth of multiple myeloma cell lines and fresh cells from multiple myeloma patients (IC50 <40 nmol/L), including cells resistant to standard chemotherapeutic agents [24,25]. In line with its in vitro activity, panobinostat achieved a dose-related reduction in tumor burden in a multiple myeloma xenograft mouse model, which was associated with a delay in the onset of clinical symptoms [20]. Bone density loss was also reduced in this model as indicated by a decrease in trabecular and cortical bone damage in the panobinostat compared with the vehicle-treated animals [20] (Ocio et al., in preparation). Notably, combination of panobinostat with bortezomib was associated with greater anti-tumor activity, in terms of a reduction in tumor burden, disease progression and trabecular bone loss, than either agent alone.

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In the HCT116 colon xenograft mouse model, i.v. panobinostat (5–20 mg/kg, five-times weekly for 3 weeks) demonstrated dose-dependent inhibition of tumor growth; panobinostat 10 mg/kg was equivalent to the standard chemotherapeutic agent 5-fluorouracil (75 mg/kg, once weekly for 3 weeks), but at a dose of 20 mg/kg, 8% tumor regression (percent change in final tumor volume at the end of study versus starting tumor volume) was reported without measurable toxicity [17]. In the Bx-PC3 pancreatic xenograft model, panobinostat (30 mg/kg, three-times weekly for 3 weeks) inhibited tumor growth and produced tumor regression of 13% without evidence of significant general cytotoxicity. Paclitaxel was used as a comparator agent in this study and had a minimal effect on the BxPC3 xenograft model. However, combination of panobinostat with paclitaxel resulted in a greater anti-tumor effect than panobinostat alone– with 20% tumor regression and minimal cytotoxicity [17]. At clinically attainable concentrations, panobinostat also induced potent tumor regression in primary tumor xenograft models of SCLC [18]. Inhibition of tumor growth by panobinostat was superior to that of the standard of care agents, cisplatin or cisplatin plus etoposide, in xenografts of patient-derived primary SCLC tumors, and enhanced anti-tumor activity was reported in the H69 SCLC xenograft tumor model with the combination of panobinostat plus etoposide [18]. Similarly, profound tumor regression was reported with panobinostat in a H146 SCLC xenograft mouse tumor model, which was superior to that achieved with chemotherapeutic agents [18]. 3.5. Indication and tumor selection for panobinostat development Both empirical and mechanism-based approaches were adopted when selecting the indications for further development of panobinostat. Assessment of the in vitro antiproliferative activity of panobinostat against a large panel of cell lines from a variety of tumor types formed the basis for the empirical strategy. As already discussed, panobinostat inhibited the proliferation of all tumor cell lines treated with high potency, as assessed by the IC50 values; however, when the effect on tumor cell viability was measured, a range of LD50 values from single digit nanomolar concentrations to >1000 nM were observed (Atadja et al., in preparation). As a consequence, the cell lines were reclassified according to their sensitivity to panobinostat-induced cell death. All cell lines from hematologic tumor types were subsequently found to exhibit uniformly high sensitivity to panobinostat in this respect, providing a sound rationale for further evaluation of panobinostat in hematologic malignancies. Interestingly, as in hematologic malignant cell lines, solid tumor SCLC cell lines also exhibited a uniformly high sensitivity to panobinostat, which subsequently translated into potent in vivo tumor regression. In contrast, cell lines from other solid tumors including NSCLC, breast and prostate cancer, exhibited a range of sensitivities to panobinostat (LD50 < 10- > 1000 nM) (Novartis Pharmaceuticals. Data on file); however, when breast and prostate cancer cell lines were evaluated further, it was noted that those cell lines with high levels of human

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epidermal growth factor receptor type 2 (HER-2/neu) and androgen receptor (AR), respectively, (see below) exhibited the highest sensitivity to panobinostat-induced cell death [22,26]. Mechanism-based approaches for selecting tumors for panobinostat development have centered around the effects of panobinostat on non-histone proteins, such as Hsp90, aggresome formation and angiogenesis. Acetylated Hsp90 binds ATP less efficiently and functions poorly as a molecular chaperone [10]. Thus, deacetylation of the Hsp90 chaperone is crucial for the stability of hormone receptors, such as AR, estrogen receptor (ER), HER-2, and epidermal growth factor receptor (EGFR), which drive cancer cell growth and survival in several tumors, including breast, prostate and lung. When prostate cancer cell lines were examined in vitro, panobinostat depleted AR and HER-2 in both AR+ androgen-dependent and -independent cell lines [26], and in the hormone-refractory prostate cancer (HRPC) CWR22Rv1 tumor model, single agent panobinostat-induced prolonged tumor stasis with concomitant depletion of AR from tumor tissue. Notably, although both AR-positive and AR-negative prostate cancer cells were sensitive to the antiproliferative effects of panobinostat, AR-positive cells were markedly more sensitive to panobinostat-induced cell death than AR-deficient cells (LD50 20– 81.9 nM versus >1000 nM) [26]. Combination of panobinostat with docetaxel also resulted in enhanced anti-tumor effects and delay of tumor progression in the HRPC cancer CWR22Rv1 tumor model [26]. Similarly, breast cancer cells that were positive for HER2 and ER were more sensitive to panobinostat-induced cell death than deficient cell types [27], and synergistic cytotoxic activity was reported with panobinostat in combination with the HER-2 inhibitor trastuzumab in the BT-474 breast cancer cell line [27]. Through its ability to regulate the accessibility of DNA for transcription via histone acetylation, panobinostat also selectively modulated human aromatase gene expression in breast cancer cells [28]. In addition, expression of the silenced ER alpha gene, associated with insensitivity to endocrine therapy in human breast cancer patients, was restored following panobinostat treatment [29]. Expression of ER mRNA, which persisted for at least 96 h after cessation of panobinostat treatment, was associated with creation of an active chromatin structure at the ER promoter via accumulation of acetylated histones H3 and H4 [29]. Furthermore, in the ER-negative human breast cancer cell line, MDA-MB-231, treatment with panobinostat for 24 h was associated with enhanced sensitivity to 4-hydroxy-tamoxifen [29]. In a study in EGFR-dependent human lung cancer cells, panobinostat-induced acetylation of Hsp90 resulted in reduced association of Hsp90 with the protein EGFR. This was accompanied by decreased Hsp90 association with other key chaperone proteins, such as c-Src, signal transducers and activators of transcription (STAT)-3 and Akt, together with a depletion of STAT3-dependent survival proteins (Bcl-xL, Mcl-1 and Bcl-2) [30]. In addition to its Hsp90-mediated effects on hormone receptor stability, panobinostat also disrupted the chaperone function of Hsp90, leading to destabilization and degradation of growth factors and their downstream signaling

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effectors. Bcr-Abl is an activated tyrosine kinase and a client protein for Hsp90 that controls the growth, proliferation and survival of CML cells through its ability to activate signal transducers, such as CRKL, AKT, Ras/Raf and ERK 1/2 kinases and the transcriptional activators STAT5 and NF-jB [31]. By inhibiting Hsp90 chaperone activity, panobinostat has been shown to have activity against both wild-type and imatinib-resistant mutant forms of Bcr-Abl [10,31,32]. In human leukemia cells, panobinostat also downregulated levels of Flt-3, another Hsp90 client protein involved in the regulation of hematopoietic cell differentiation, proliferation and survival [31]. Furthermore, as well as its effects on tumor-specific oncoprotein targets, such as Bcr-Abl, panobinostat also decreased levels of generic oncogenic targets, such as c-Raf and phosphorylated (activated) AKT, which affect cellular proliferation downstream of signaling pathways, such as Bcr-Abl [17,32,33]. Targeting of the protein degradation machinery has proven clinically successful in multiple myeloma. The aggresome is an alternative misfolded protein processing organelle in myeloma cells that may act as a bypass resistance mechanism to proteosome inhibitors such as bortezomib [25]. HDAC6 is required for the formation of aggresomes and because, as a pan-DAC inhibitor, panobinostat potently inhibits HDAC6, we figured that it would be effective either as a single agent or in combination with proteasome inhibitors in multiple myeloma. Thus, multiple myeloma was selected as an important indication for panobinostat development. Previous studies in multiple myeloma indicate that inhibition of proteosomal degradation of ubiquitinated proteins by the proteasome inhibitor, bortezomib, results in increased aggresomal activity, whereas blockade of the aggresome cascade by the tubulin deacetylase inhibitor, tubacin, initiates a compensatory increase in proteasomal degradation of ubiquitinated proteins [25,34]. The combination of panobinostat and bortezomib is associated with synergistic cytotoxicity against myeloma cells and patient cells, including those sensitive and resistant to conventional and novel therapies, suggesting that the coadministration of panobinostat may overcome intrinsic or acquired resistance to bortezomib [25]. In addition, aggresome induction by bortezomib was also reported in pancreatic cancer cells, and this effect was inhibited by HDAC6 small interfering RNA or HDAC inhibitors, resulting in synergistic cytotoxicity [35]. Among its many functions, the nuclear transcription factor, HIF-1a, activates several target genes involved in the promotion of angiogenesis and regulates the proangiogenic factor, vascular endothelial growth factor in many solid tumors [36]. Direct deacetylation of HIF-1a, as well as deacetylation of Hsp90, which chaperones HIF1a into its active conformation, are required for pro-angiogenic gene transcription [10,37]. Pan-DAC inhibition would therefore be expected to decrease HIF-1a gene expression and inhibit angiogenesis. Consistent with this theory, panobinostat reduced HIF-1a protein levels in human umbilical vein endothelial cells and also blocked new blood vessel formation in human prostate carcinoma cell PC-3 xenografts [15]. The angiogenesis pathway has been iden-

tified as a therapeutic target in renal cell carcinoma and may be a potential indication for the exploration of the anti-angiogenic activity of panobinostat. In addition to HIF-1a, panobinostat also has an effect on another transcription factor, p53, which is a significant tumor suppressor involved in the regulation of the cell cycle and DNA damage response. Acetylation has been shown to promote p53 stability, leading to tumor suppressor activity via transcriptional activation of anti-mitogenic genes, such as the cell cycle inhibitor CDKNIA (p21). Induction of p21 gene expression is a key feature of HDAC inhibition [38], and panobinostat has been reported to increase p53 protein levels and increase the transcription of target genes such as p21 [17,24,31,39]. 3.6. Panobinostat in the clinical setting Based on its potent anticancer efficacy across a wide range of tumors in preclinical studies, panobinostat has entered clinical development in a number of indications, including CTCL, Hodgkin lymphoma, AML, multiple myeloma and HRPC. Data obtained from hematologic cell lines underscore the clinical activity of single-agent panobinostat demonstrated in a number of hematologic malignancies to date. In a Phase I study in patients with advanced stage CTCL, treatment with oral panobinostat (20 mg, three-times weekly) was associated with a response in six of 10 patients (two complete responses, four partial responses) as confirmed by stringent CTCL-specific response criteria. Among the responding patients were two patients with disease progression while on panobinostat who achieved complete remission several weeks after treatment cessation. Consistent with the proposed mechanism of action of panobinostat, the investigators also reported hyperacetylation of histone H3 in tumor cells just 4 h after administration of panobinostat and also in peripheral blood mononuclear cells. In addition, microarray analysis of tumor samples indicated rapid changes in gene expression following panobinostat treatment, with the majority of genes repressed rather than activated [40]. Similarly, significant increases in acetylation of H2B and H3 histones were reported in leukemic blast (CD34+) cells in a Phase I study of patients with refractory hematologic malignancies [41]. Also in this study, panobinostat was reported to demonstrate transient antileukemic activity with reduction in blasts in the peripheral blood in seven of 11 patients, which rebounded shortly after completion of treatment. Further confirmation of the efficacy of panobinostat in CTCL was provided recently by the interim data from an ongoing Phase II study [42]. Oral panobinostat has also been investigated in an ongoing Phase I study in patients with advanced hematologic malignancies, including relapsed/refractory Hodgkin’s lymphoma and AML. Patients with Hodgkin lymphoma received panobinostat P30 mg, three-times weekly (every week or every other week); preliminary data show that among 28 evaluable patients 16 (57%) achieved a partial response and one (3.5%) achieved a complete response assessed by computed tomography, and ten (36%) and one (3.5%) patient, respectively, achieved partial

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and complete metabolic responses as assessed by positron emission tomography (PET) scan. Among 36 evaluable patients with AML enrolled thus far (treated with panobinostat P40 mg, three-times weekly), encouraging biologic activity has been reported, including two complete remissions and two reports of prolonged stable disease [43]. In a Phase II study of patients with advanced refractory multiple myeloma (n = 38), treatment with oral panobinostat (20 mg, three-times weekly) was well tolerated and safe. One confirmed partial response along with stabilization of previously progressive bone lesions was reported in a patient who had previously progressed on lenalidomide/dexamethasone [44]. The relatively low response rates seen in this study may have been attributable to the use of a sub-optimal dose of panobinostat, as subsequent data from other studies suggest that oral panobinostat doses greater than 20 mg may represent a more optimal dosing schedule [44]. Oral panobinostat is also under investigation in two ongoing Phase I studies to determine the maximum tolerated dose in combination with bortezomib [45] or lenalidomide/dexamethasone in patients with relapsed multiple myeloma.

4. Conclusion The development of HDAC inhibitors has been and still is associated with a number of challenges. As a multi-class, multi-member target family, the specific HDAC class or isoforms responsible for tumorigenesis have yet to be elucidated and, in terms of the pan-DAC inhibitors, it is unclear which isoform(s) are responsible for mediating antitumor activity. Furthermore, modulation of epigenetic and multiple non-epigenetic mechanisms hamper the identification of specific mechanistic actions involved in the mediation of anti-tumor activity. Other challenges include the absence of known genetic aberrations (i.e. mutations and amplifications) of HDAC, making it difficult to stratify tumors for treatment with HDAC inhibitors and also a lack of biomarkers that correlate with the degree of HDAC inhibitor anti-tumor activity. However, certain characteristics of the pan-DAC inhibitors make them particularly attractive for development as anticancer agents. Their effect on multiple tumorigenic pathways provides the opportunity for anti-tumor activity in a wide variety of clinical indications, and their differential sensitivity towards tumor versus normal cells provides anti-tumor activity at tolerable doses. The effect of HDAC inhibitors on multiple pathways also allows for good complementary activity during combination with other anti-tumor agents, leading to synergy. Furthermore, histone acetylation as a biomarker can be used to identify biologically effective doses to determine the window between the potential minimally effective dose and maximum tolerated dose. In addition, the duration of effect on histone acetylation can be used to guide frequency of dosing. As an HDAC inhibitor, panobinostat is emerging as a highly valuable therapeutic option. Panobinostat is a highly potent inhibitor of all HDAC enzymes implicated in cancer development and progression, and compared with the pan-DAC inhibitors, vorinostat and belinostat, dis-

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play the most potent and broadest inhibitory activity at clinically achievable concentrations. Furthermore, through its effects on histone acetylation and gene expression, as well as on the oncogenic function of non-histone proteins such as Hsp90, panobinostat offers a multifaceted approach for the inhibition of cancer cell proliferation and survival, a desirable feature when treating cancers with complex biology. The potent preclinical cytotoxicity of panobinostat demonstrated against a wide range of cancer cell lines and tumor models is now being translated into the clinical setting with panobinostat demonstrating good anti-tumor activity against both hematologic malignancies and solid tumors (in combination). However, further studies are required to determine the optimum panobinostat dose and schedule for both the i.v. and oral formulations. The greatest clinical utility for panobinostat is likely to be in combination with other therapeutic agents that synergize with the epigenetic regulation mediated by panobinostat. This is suggested by the preliminary results of a study evaluating panobinostat in combination with bortezomib in multiple myeloma (MM). It is as yet unclear which drugs will be the ideal combination partners for panobinostat. For example, should panobinostat be combined with drugs that have an overlapping mechanism of action (e.g. an Hsp90 inhibitor in prostate cancer) or with drugs that affect the same target or pathway through a complementary mechanism of action (e.g. an AR antagonist in prostate cancer or Her2 antagonist in breast cancer). By more fully elucidating the effect of panobinostat on the regulation of gene expression and non-epigenetic targets, a more comprehensive picture of the role of panobinostat as an anticancer agent should emerge, providing further direction for the rationale combination of panobinostat with other anticancer agents. Conflicts of Interest Peter Atadja is an employee of Novartis Institutes for Biomedical Research, a division of Novartis AG and the developer of panobinostat. Acknowledgements I would like to thank all the panobinostat team members (past and present) at Novartis AG, the study investigators and study patients for the important contributions that they have made during the development of panobinostat. In addition, I would like to acknowledge Dina Marenstein of Chameleon Communications International, who provided editorial support with funding from Novartis Oncology. References [1] P.A. Jones, S.B. Baylin, The epigenomics of cancer, Cell 128 (2007) 683–692. [2] S. Thiagalingam, K.H. Cheng, H.J. Lee, N. Mineva, A. Thiagalingam, J.F. Ponte, Histone deacetylases: unique players in shaping the epigenetic histone code, Ann. N. Y. Acad. Sci. 983 (2003) 84–100. [3] M. Esteller, Epigenetics in cancer, New Engl. J Med. 358 (2008) 1148– 1159.

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P. Atadja / Cancer Letters 280 (2009) 233–241

[4] A.P. Feinberg, R. Ohlsson, S. Henikoff, The epigenetic progenitor origin of human cancer, Nat. Rev. Genet. 7 (2006) 21–33. [5] M. Esteller, Cancer epigenomics: DNA methylomes and histonemodification maps, Nat. Rev. Genet. 8 (2007) 286–298. [6] T. Kouzarides, Chromatin modifications and their function, Cell 128 (2007) 693–705. [7] J.E. Bolden, M.J. Peart, R.W. Johnstone, Anticancer activities of histone deacetylase inhibitors, Nat. Rev. Drug Discov. 5 (2006) 769–784. [8] A. Rodriguez-Gonzalez, T. Lin, A.K. Ikeda, T. Simms-Waldrip, C. Fu, K.M. Sakamoto, Role of the aggresome pathway in cancer: targeting histone deacetylase 6-dependent protein degradation, Cancer Res. 68 (2008) 2557–2560. [9] M.A. Glozak, E. Seto, Histone deacetylases and cancer, Oncogene 26 (2007) 5420–5432. [10] P. Bali, M. Pranpat, J. Bradner, M. Balasis, W. Fiskus, F. Guo, K. Rocha, S. Kumaraswamy, S. Boyapalle, P. Atadja, E. Seto, K. Bhalla, Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors, J. Biol. Chem. 280 (2005) 26729–26734. [11] M. Nakagawa, Y. Oda, T. Eguchi, S. Aishima, T. Yao, F. Hosoi, Y. Basaki, M. Ono, M. Kuwano, M. Tanaka, M. Tsuneyoshi, Expression profile of class I histone deacetylases in human cancer tissues, Oncol Rep. 18 (2007) 769–774. [12] S. Saji, M. Kawakami, S. Hayashi, N. Yoshida, M. Hirose, S. Horiguchi, A. Itoh, N. Funata, S.L. Schreiber, M. Yoshida, M. Toi, Significance of HDAC6 regulation via estrogen signaling for cell motility and prognosis in estrogen receptor-positive breast cancer, Oncogene 24 (2005) 4531–4539. [13] P. Zhu, E. Martin, J. Mengwasser, P. Schlag, K.P. Janssen, M. Gottlicher, Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis, Cancer Cell 5 (2004) 455–463. [14] R.J. Lin, T. Sternsdorf, M. Tini, R.M. Evans, Transcriptional regulation in acute promyelocytic leukemia, Oncogene 20 (2001) 7204–7215. [15] D.Z. Qian, Y. Kato, S. Shabbeer, Y. Wei, H.M. Verheul, B. Salumbides, T. Sanni, P. Atadja, R. Pili, Targeting tumor angiogenesis with histone deacetylase inhibitors: the hydroxamic acid derivative LBH589, Clin. Cancer Res. 12 (2006) 634–642. [16] US Food and Drug Administration. Center for Drug Evaluation, FDA approves vorinostat (Zolinza) for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma (CTCL), October 10, 2006. . [17] W. Shao, J.D. Growney, Y. Feng, P. Wang, Y. Yan-Neale, G. O’Connor, Potent anticancer activity of the pan-deacetylase inhibitor panobinostat (LBH589) as a single agent in in vitro and in vivo tumor models, in: 99th American Association of Cancer Research Annual Meeting, April 12–16, 2008, San Diego, CA, 2008, Abstract 6244. [18] P. Atadja, W. Shao, Y. Wang, J.D. Growney, J. Cheng, C. Kowal, Y. Feng, G. O’Connor, Potent anticancer activity of panobinostat (LBH589) in small-cell lung cancer in both in vitro and in vivo tumor models, Ann. Oncol. 2008, viii50, Abstract 86P. [19] P. Atadja, Y. Wang, J.D. Growney, Y. Feng, Y.-M. Yao, A.S. Wallace, M.C. Crisanti, S. Fawell, S. Albelda, Efficacy of panobinostat (LBH589) in lung cancer: potent anticancer activity in both in vitro and in vivo tumor models, in: EORTC-AACR-NCI 2008 Symposium, October 21– 24, Geneva, Switzerland, 2008, Abstract 151. [20] J.D. Growney, W. Shao, Y. Wang, M. Pu, B. Firestone, J. Cheng, C. Kowal, C. Miller, J. Eckman, Y.-M. Yao, S. Fawell, A. Spencer, J.F. San Miguel, K.C. Anderson, Efficacy of panobinostat (LBH589) in multiple myeloma cell lines and a mouse xenograft model: anti-tumor and anti-osteolytic effects in multiple myeloma, in: Blood (ASH Annual Meeting Abstracts), 2007, Abstract 1510. [21] W. Shao, J.D. Growney, Y. Feng, G. O’Connor, M. Pu, Y.-M. Yao, S. Fawell, P. Atadja, Efficacy of panobinostat (LBH589) in CTCL cell lines and a murine xenograft model: defining molecular pathways of panobinostat activity in CTCL, in: Blood (ASH Annual Meeting Abstracts), 2007. Abstract 1375. [22] W. Shao, J.D. Growney, Y. Feng, G. O’Connor, P. Kwon, Y.-M. Yao, S. Fawell, P. Atadja, S. Fruchtman, Evaluation of the antitumor activity of LBH589 antitumor activity in HER2 + breast cancer models and of potential synergy in combination with HER2 targeted therapy, in: ASCO Breast Cancer Symposium, September 5–7 2007, Washington, DC, 2007, Abstract 173. [23] Y. Wang, J.D. Growney, J. Cheng, C. Kowal, G. Yang, R. Mosher, R. Meyer, Y. Yan-Neale, M. Pu, W. Shao, Y. Feng, P. Kwon, P. Atadja, S. Fawell, Y.-M. Yao, Potent anticancer activity of the deacetylase

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

inhibitor panobinostat (LBH589) in colon cancer cell lines and patient-derived primary colon cancer xenografts, in: 99th American Association of Cancer Research Annual Meeting, April 12–16, 2008, San Diego, CA 2008, Abstract 2442. P. Maiso, X. Carvajal-Vergara, E.M. Ocio, R. Lopez-Perez, G. Mateo, N. Gutierrez, P. Atadja, A. Pandiella, J.F. San Miguel, The histone deacetylase inhibitor LBH589 is a potent antimyeloma agent that overcomes drug resistance, Cancer Res. 66 (2006) 5781–5789. L. Catley, E. Weisberg, T. Kiziltepe, Y.T. Tai, T. Hideshima, P. Neri, P. Tassone, P. Atadja, D. Chauhan, N.C. Munshi, K.C. Anderson, Aggresome induction by proteasome inhibitor bortezomib and alpha-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells, Blood 108 (2006) 3441–3449. W. Shao, J.D. Growney, G. O’Connor, Y. Feng, H. Scher, Y. Yao, S. Fawell, P. Atadja, Efficacy of panobinostat (LBH589) in prostate cancer cell models: targeting the androgen receptor in hormonerefractory prostate cancer (HRPC), in: ASCO Genitourinary Cancers Symposium, February 14–16, 2008, SanFrancisco, CA, 2008, Abstract 216. R.S. Finn, W. Shao, J. Dering, C. Gither, O. Kalous, D. Conklin, Panobinostat (LBH589), a pan-DAC inhibitor, induces cell death in ER+ and HER2 amplified cell lines in vitro and is synergistic in vivo with trastuzumab, San Antonio Breast Cancer Meeting, December 11–15, 2008, in: San Antonio, Texas, USA, 2008. S. Chen, J. Ye, D.B. Evans, I. Kijima, A synergistic suppression of aromatase by letrozole and the DAC inhibitor panobinostat (LBH589), an inhibitory modulator of aromatase gene expression, in: San Antonio Breast Cancer Meeting, December 11–15, 2008, San Antonio, Texas, USA, 2008. Q. Zhou, P. Atadja, N.E. Davidson, Histone deacetylase inhibitor LBH589 reactivates silenced estrogen receptor alpha (ER) gene expression without loss of DNA hypermethylation, Cancer Biol. Ther. 6 (2007) 64–69. A. Edwards, J. Li, P. Atadja, K. Bhalla, E.B. Haura, Effect of the histone deacetylase inhibitor LBH589 against epidermal growth factor receptor dependent human lung cancer cells, Mol. Cancer Ther. 6 (2007) 2515–2524. P. George, P. Bali, S. Annavarapu, A. Scuto, W. Fiskus, F. Guo, C. Sigua, G. Sondarva, L. Moscinski, P. Atadja, K. Bhalla, Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3, Blood 105 (2005) 1768– 1776. W. Fiskus, M. Pranpat, P. Bali, M. Balasis, S. Kumaraswamy, S. Boyapalle, K. Rocha, J. Wu, F. Giles, P.W. Manley, P. Atadja, K. Bhalla, Combined effects of novel tyrosine kinase inhibitor AMN107 and histone deacetylase inhibitor LBH589 against Bcr-Abl-expressing human leukemia cells, Blood 108 (2006) 645–652. W. Fiskus, Y. Ren, A. Mohapatra, P. Bali, A. Mandawat, R. Rao, B. Herger, Y. Yang, P. Atadja, J. Wu, K. Bhalla, Hydroxamic acid analogue histone deacetylase inhibitors attenuate estrogen receptor-{alpha} levels and transcriptional activity: a result of hyperacetylation and inhibition of chaperone function of heat shock protein 90, Clin. Cancer Res. 13 (2007) 4882–4890. T. Hideshima, J.E. Bradner, J. Wong, D. Chauhan, P. Richardson, S.L. Schreiber, K.C. Anderson, Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma: therapeutic implications, Proc. Natl. Acad. Sci. USA 102 (2005) 8567–8572. S.T. Nawrocki, J.S. Carew, M.S. Pino, R.A. Highshaw, R.H. Andtbacka, K. Dunner Jr., A. Pal, W.G. Bornmann, P.J. Chiao, P. Huang, H. Xiong, J.L. Abbruzzese, D.J. McConkey, Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells, Cancer Res. 66 (2006) 3773–3781. G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003) 721–732. D.Z. Qian, S.K. Kachhap, S.J. Collis, H.M. Verheul, M.A. Carducci, P. Atadja, R. Pili, Class II histone deacetylases are associated with VHLindependent regulation of hypoxia-inducible factor 1{alpha}, Cancer Res. 66 (2006) 8814–8821. L.C. Sambucetti, D.D. Fischer, S. Zabludoff, P.O. Kwon, H. Chamberlin, N. Trogani, H. Xu, D. Cohen, Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects, J. Biol. Chem. 274 (1999) 34940–34947. T. Bluethner, M. Niederhagen, K. Caca, F. Serr, H. Witzigmann, C. Moebius, J. Mossner, M. Wiedmann, Inhibition of histone deacetylase for the treatment of biliary tract cancer: a new

P. Atadja / Cancer Letters 280 (2009) 233–241 effective pharmacological approach, World J. Gastroenterol. 13 (2007) 4761–4770. [40] L. Ellis, Y. Pan, G.K. Smyth, D.J. George, C. McCormack, R. WilliamsTruax, M. Mita, J. Beck, H. Burris, G. Ryan, P. Atadja, D. Butterfoss, M. Dugan, K. Culver, R.W. Johnstone, H.M. Prince, Histone deacetylase inhibitor panobinostat induces clinical responses with associated alterations in gene expression profiles in cutaneous T-cell lymphoma, Clin. Cancer Res. 14 (2008) 4500–4510. [41] F. Giles, T. Fischer, J. Cortes, G. Garcia-Manero, J. Beck, F. Ravandi, E. Masson, P. Rae, G. Laird, S. Sharma, H. Kantarjian, M. Dugan, M. Albitar, K. Bhalla, A phase I study of intravenous LBH589, a novel cinnamic hydroxamic acid analogue histone deacetylase inhibitor in patients with refractory hematologic malignancies, Clin. Cancer Res. 12 (2006) 4628–4635. [42] M. Duvic, J.C. Becker, S. Dalle, F. Vanaclocha, M. Grazia Bernengo, C. Lebbe, R. Dummer, S. Hirawat, L. Zhang, M. Marshood, G. Laird, H.M. Prince, Phase II trial of oral panobinostat (LBH589) in Patients with refractory cutaneous T-Cell lymphoma (CTCL), in: Blood (ASH Annual Meeting Abstracts), 2008, Abstract 1005.

241

[43] O.G. Ottmann, A. Spencer, H.M. Prince, K.N. Bhalla, T. Fischer, A. Liu, K. Parker, M. Jalaluddin, G. Laird, M. Woo, J.W. Scott, D.J. Deangelo, Study of oral panobinostat (LBH589), a novel pan-deacteylase inhibitor (DACi) demonstrating efficacy in patients with advanced hematologic malignancies, in: Blood (ASH Annual Meeting Abstracts), 2008, Abstract 958. [44] J.L. Wolf, D. Siegel, J. Matous, S. Lonial, H. Goldschmidt, S. Schmitt, R. Vij, M. De Malgalhaes-Silverman, R. Abonour, M. Jalaluddin, M. Li, K. Hazell, P.M. Bourquelot, M.-V. Mateos, K.C. Anderson, A. Spencer, J.-L. Harousseau, J. Bladé, A phase II study of oral panobinostat (LBH589) in adult patients with advanced refractory multiple myeloma, in: Blood (ASH Annual Meeting Abstracts), 2008, Abstract 2774. [45] D. Siegel, O. Sezer, J.F. San Miguel, M.-V. Mateos, I. Prosser, M. Cavo, M. Jalaluddin, K. Hazell, P.M. Bourquelot, K.C. Anderson, A Phase IB, multicenter, open-label, dose-escalation study of oral panobinostat (LBH589) and IV bortezomib in patients with relapsed multiple myeloma, in: Blood (ASH Annual Meeting Abstracts), 2008, Abstract 2781.