Disrupting Polyamine Homeostasis As a Therapeutic Strategy for Neuroblastoma Nicholas F

Published OnlineFirst September 29, 2009; DOI: 10.1158/1078-0432.CCR-08-3213

Molecular Pathways

Disrupting Polyamine Homeostasis as a Therapeutic Strategy for Neuroblastoma Nicholas F. Evageliou and Michael D. Hogarty

Abstract MYC genes are deregulated in a plurality of human cancers. Through direct and indirect mechanisms, the MYC network regulates the expression of >15% of the human genome, including both protein-coding and noncoding RNAs. This complexity has complicated efforts to define the principal pathways mediating MYC's oncogenic activity. MYC plays a central role in providing for the bioenergetic and biomass needs of proliferating cells, and polyamines are essential cell constituents supporting many of these functions. The rate-limiting enzyme in polyamine biosynthesis, ODC, is a bona fide MYC target, as are other regulatory enzymes in this pathway. A wealth of data link enhanced polyamine biosynthesis to cancer progression, and polyamine depletion may limit the malignant transformation of preneoplastic lesions. Studies with transgenic cancer models also support the finding that the effect of MYC on tumor initiation and progression can be attenuated through the repression of polyamine production. High- risk neuroblastomas (an often lethal embryonal tumor in which MYC activation is paramount) deregulate numerous polyamine enzymes to promote the expansion of intracellular polyamine pools. Selective inhibition of key enzymes in this pathway, e.g., using DFMO and/or SAM486, reduces tumorigenesis and synergizes with chemotherapy to regress tumors in preclinical models. Here, we review the potential clinical application of these and additional polyamine depletion agents to neuroblastoma and other advanced cancers in which MYC is operative. (Clin Cancer Res 2009;15(19):5956–61)

Background in neural stem cells, for example, leads to an aberrant nuclear structure mimicking a heterochromatin state accompanied by The MYC proto-oncogenes, which include MYC, MYCN, and widespread histone modifications (5). Such higher-order regu- MYCL, are among the most frequently deregulated genes in latory activities may explain MYC's profound influence on tran- cancer. The MYC proto-oncogenes encode highly homolo- scription and the diversity in putative target genes identified gous basic-helix-loop-helix leucine zipper transcription factors across different model systems. that are biologically redundant, but differentially expressed spa- MYC activity is tightly regulated through transcriptional and tiotemporally. MYC genes function through heterodimerization posttranslational mechanisms, with rapid degradation of Myc with Max and operate within a network of related proteins to protein in concert with cell cycle exit. In many cancers, MYC regulate transcription through interactions at E-box sequences genes are deregulated through genomic translocation or ampli- within promoters of diverse target genes (1). Many estimates fication events that lead to supraphysiologic Myc expression. have the MYC network governing the expression of > 15% of Although mutations in Myc have been identified in Burkitt's all human genes (2, 3) and a growing roster of noncoding RNAs lymphoma cells (accompanying rather than replacing activating (4). A simplified gene-specific model of transcriptional regula- translocations; ref. 6), Myc oncogenesis typically results from tion has been expanded with the appreciation that MYC genes deregulated overexpression of wild-type protein. Such large- also contribute to global chromatin regulation. Loss of MYCN scale biological reprogramming of cells through enforced expression of this promiscuous transactivator and chromatin regulator is highly oncogenic, and the ubiquity of MYC activa-

Authors' Affiliations: Division of Oncology, The Children's Hospital of tion across tumor types makes it an attractive cancer target. Philadelphia and Department of Pediatrics, University of Pennsylvania Inhibiting grossly deregulated transcription factors remains a School of Medicine, Philadelphia, Pennsylvania daunting therapeutic challenge, yet pharmaceutical successes Received 5/4/09; revised 6/3/09; accepted 6/3/09; published OnlineFirst 9/29/09. continue to whittle away at the list of domains considered “un- Grant support: The author's laboratory is supported by National Institutes druggable”; thus, direct Myc antagonism may be an achievable of Health CA97323 and the Richard and Sheila Sanford Chair in Pediatric Oncology (M.D. Hogarty), and by the Children's Neuroblastoma Cancer goal. However, a secondary concern for such an approach is Foundation (N.F. Evageliou). that systemic interference with Myc might be quite toxic, as Requests for reprints: Michael D. Hogarty, Division of Oncology, The Chil- it is indispensable for cell cycle entry in response to mitogenic dren's Hospital of Philadelphia, 9 North ARC, Suite 902C, 3615 Civic Center signals. This fear has been partially allayed by evidence that a Boulevard, Philadelphia, PA 19104-4318. Phone: 215-590-3931; Fax: 215- 590-3770; E-mail: [email protected]. profound dominant-negative Myc contruct can be activated F 2009 American Association for Cancer Research. globally in mice without undue toxicity (7). An alternative ap- doi:10.1158/1078-0432.CCR-08-3213 proach to interfering directly with Myc is to define the critical

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Polyamine Depletion in Neuroblastoma downstream pathways necessary for its oncogenic activity. (OAZ1, OAZ2, OAZ3) are themselves responsive to intracellular Among these may be more immediately tractable drug targets polyamines to provide a negative feedback loop (20). Further- that exploit cancer-specific aspects of Myc activity with a greater more, two mammalian antizyme inhibitors have been identi- therapeutic index. fied [AZIN1 (ref. 21) and, more recently, ADC (ref. 22)] that An improved understanding of Myc biology has emerged encode enzymatically inactive Odc homologs that compete to from high-dimensional assays that provide global transcrip- neutralize the antizymes and therefore constitute a positive reg- tome and/or Myc-chromatin binding site data. These platforms ulator of Odc activity (23). This level of control underscores the have generated daunting lists of genes and chromatin binding importance of modulating Odc activity to ensure cellular fitness. sites that underscore the widespread involvement of Myc in Polyamine catabolism occurs via the acetylation of spermi- diverse biological processes. Still, patterns are discernible with- dine or spermine by the readily inducible spermidine/ in this complexity. The most conserved set of MYC target genes spermine-N-acetyltransferase, SAT1 (24). Acetylated polyamines functions in ribosomal biogenesis and protein metabolism may be exported from the cell through specific transmembrane and processing, and this is true for both MYC (3) and MYCN solute carriers to reduce intracellular levels (25). Alternatively, (8). Additionally, programs that direct carbon assimilation, an- acetylated spermine and spermidine may be converted through abolic pathways, and bioenergetics are all targeted by Myc (3). PAOX oxidase activity to spermidine and putrescine, respective- Thus, Myc orchestrates a program redirecting metabolism to ly, whereas SMOX oxidase activity can convert spermine to sper- provide for the energetic needs of the cell through augmented midine directly. These conversions allow for homeostatic aerobic glycolysis (9) and glutaminolysis (10), as well as the control over the repertoire of natural polyamines, which may biomass needs through enhanced synthesis and processing of be important for maintaining functions unique to select polya- RNA, DNA, protein, lipid, and polyamine precursors. mines [such as protein translation, in which spermidine acts as a Polyamines are multifunctional polycations found in nearly all gate-keeper by regulating the activity of eIF5A (ref. 26); Fig. 2]. living organisms. Polyamines support biological processes Finally, an as yet uncharacterized energy-dependent polyamine through the stabilization of anionic macromolecules and mod- transporter functions to selectively import polyamines present ulate DNA:protein and protein:protein interactions. A detailed in the microenvironment through dietary intake, export from understanding of polyamine activities is hampered by the fact neighboring cells, or synthesis by intestinal flora. This pathway that they participate in mainly transient ionic interactions that can restore polyamine levels under conditions of biosynthetic are difficult to study. Still, polyamine homeostasis is essential blockade and may potentially undermine therapeutic efforts for cell survival, and depletion activates cellular checkpoints to diminish intracellular polyamines. that constrain proliferation or induce apoptosis (11). Reduced This multitude of regulatory controls highlights the critical polyamines are seen in postmitotic and senescent cells, whereas need for polyamine homeostasis, and as polyamines operate enhanced polyamine biosynthesis accompanies normal as well downstream of Myc to support proliferation, they provide an as oncogenic proliferation (12, 13). That polyamine biosynthe- intriguing cancer target. Indeed, substantial effort has been sis may be instructive in the cancer process, rather than simply spent over 30 y to leverage polyamine disruption as an antican- a consequence of increased proliferation, emerged as molecular cer strategy with limited initial success. Inhibitors acting at studies linked numerous cancer genes directly to polyamine nearly every step in this pathway have been developed and metabolism (14, 15). For example, ornithine decarboxylase investigated, and despite encouraging preclinical results with (ODC1), the rate-limiting enzyme for polyamine biosynthesis, diverse agents and cancer models, translation to clinical utility is a MYC target gene (16) and a bona fide oncogene. Odc can has been slow (comprehensively reviewed in ref. 14). Initial substitute for Myc and cooperate with Ras to transform cells studies testing single polyamine-targeting drugs were disap- both in vitro (17) and in vivo (18). Thus, enhanced polyamine pointing, although not completely without successes, as activ- synthesis is essential to oncogenic signaling and may be specif- ity has been seen in hematolymphoid (27, 28) and CNS ically required to support Myc-governed functions. neoplasms (29). To date, however, little attention has been Intracellular polyamine levels are modulated through tightly paid to the potential for cancer genotype-specific responses, regulated synthetic, catabolic, uptake and export pathways analogous to synthetic lethal interactions in yeast. Studies with (Fig. 1; reviewed in refs. 14, 15, 19). The rate-limiting biosyn- transgenic cancer models suggest that Myc deregulation may thetic enzymes are ODC1 and S-adenosylmethionine decar- provide an Achilles' heel for cancer cells through a requirement boxylase (AMD1). Odc homodimers decarboxylate ornithine, for polyamine sufficiency that can be targeted therapeutically, a urea cycle product, to the diamine putrescine, whereas Amd and embryonal cancers may be particularly vulnerable. decarboxylates S-adenosylmethionine to provide the aminopropyl donor for the conversion of putrescine to spermidine Clinical-Translational Advances and spermine. These latter conversions are mediated by the aminopropyltransferases spermidine synthase (SRM) and sper- Neuroblastoma is a childhood embryonal tumor that frequently mine synthase (SMS), respectively. Multiple levels of control presents with high-risk clinical and genetic features. Despite are exercised over Odc and Amd activities. They are highly maximally intensive therapy, survival remains dismal and inno- transcriptionally regulated, with ODC1 being a direct MYC tar- vative treatment approaches are needed (reviewed in ref. 30). get (16); are posttranslationally regulated; and have the short- Several recurrent genomic alterations correlate with outcome, est half-lives (10-30 min) of any mammalian enzyme. Odc is and of these, MYCN amplification is most strongly correlated rapidly degraded through a process initiated by Odc antizymes with advanced disease and treatment failure (31, 32). In high- that bind monomeric Odc and present the protein to the 26S risk neuroblastomas that lack MYCN amplification, MYC is proteasome for degradation independent of ubiquitylation, frequently deregulated instead (33–35), suggesting that MYC sig- while also inhibiting polyamine uptake. These antizymes naling may be essential for the high-risk phenotype. Still, despite

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Molecular Pathways over 20 y of recognition that MYC deregulation is a seminal event genes, ODC1 expression is influenced by a functional promoter in neuroblastoma, no molecularly targeted therapy has emerged polymorphism at the A317G SNP (39, 40). In neuroblastoma, to leverage this discovery. Polyamine homeostasis, deregulated the higher-expressing genotypes have an inferior survival, partic- downstream of MYC genes, may provide such a target. ularly when analysis is restricted to the MYCN nonamplified tu- First, polyamine regulators are aberrantly expressed in high- mors, again supporting functional validity for this pathway (41). risk neuroblastomas to coordinately augment biosynthesis and Studies with a transgenic model of neuroblastoma support a re- reduce catabolism. ODC1 mRNA is significantly higher in high- quirement for polyamines in tumor initiation, progression, and risk tumors, whereas the antizyme OAZ2 is reduced, consistent therapy response. Mice carrying a neural crest-targeted MYCN with polyamine enhancement (note: unlike OAZ1, OAZ2 may transgene (TH-MYCN model) develop lethal neuroblastoma not deliver Odc to the proteasome for degradation, yet it is with complete penetrance in the homozygous state, and equipotent at inhibiting Odc activity and polyamine uptake; ∼40% penetrance in the hemizygous state (42, 43). Tumors ref. 36). Moreover, every prosynthetic enzyme (including arise stochastically within peripheral sympathetic ganglia and AMD1, SRM, and SMS) is markedly upregulated in the highest- recapitulate human neuroblastoma features, with cooperative risk subset with MYCN amplification, whereas, conversely, there genetic alterations at chromosome regions orthologous to those is reduced SMOX (37). A similar pattern is seen when evaluating in the human disease (42, 43). Importantly, tumors arise in an neuroblastoma cell lines in comparison with fetal adrenal or appropriate microenvironment to recapitulate the heterotypic neural tissues (37, 38). Second, ODC1 expression correlates with cell interactions important to cancer propagation and provide outcome in neuroblastoma, independent of MYCN amplifica- a relevant therapeutics-testing platform (44). As with human tion, supporting the finding that this upregulation has functional neuroblastomas, TH-MYCN tumors demonstrate altered consequences (37). In addition to direct transactivation by MYC polyamine regulator expression compared with sympathetic

Fig. 1. Schematic of polyamine metabolism required to support cell proliferation and therapeutic opportunities in this pathway. Putrescine (diamine), spermidine (triamine), and spermine (tetramine) are the major polyamines. Ornithine derived from the urea cycle provides the initial substrate for Odc-mediated decarboxylation to putrescine. Amd1 provides the aminopropyl donor to support SRM- and SMS-mediated conversion to higher-order polyamines. Pro-synthetic polyamine enzymes are shown in green; catabolic enzymes are shown in red (underlined enzymes are highly regulated and have among the shortest half-lives of any mammalian enzyme). Polyamine therapeutics and their sites of action are in blue (described in the text). Those shown are in preclinical or early phase clinical development as cancer therapeutics. ODC1, ornithine decarboxylase; SRM, spermidine synthase; SMS, spermidine synthase; AMD1, S-adenosylmethionine decarboxylase; AZIN1, Odc Antizyme inhibitor; SMOX, spermidine oxidase; PAOX, polyamine oxidase; SAT1, spermine/spermidine N-acetyltransferase; OAZ1,2,3, Odc Antizymes.

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Polyamine Depletion in Neuroblastoma

Fig. 2. Myc-polyamine axis and functional synergies. Myc genes govern diverse programs to support proliferation. Polyamines, mediated by Myc activity, support many Myc functions through ionic stabilization of key macromolecules in these processes. Spermidine has an additional vital function: eIF5A is a universal elongation factor that uniquely supports all three steps of protein translation (60), and it is active only after hypusination at lysine 51 (26). This posttranslational modification (in which spermidine alone acts as an aminobutyl donor) is not found in any other eukaryotic protein, and it is essential for eIF5A activity. This pathway links Myc-driven polyamine homeostasis to an essential role supporting protein translation. Additional roles for polyamines in free radical scavenging, formation of cytotoxic products, and additional covalent bond-mediated activities have been experimentally defined but are not shown (61). ganglia, with upregulated ODC1, AZIN, AMD1, SRM, and SMS that this strategy may have clinical relevance (37). Select DFMO and downregulated OAZ2, SMOX, and SAT1.1 Thus, the model and chemotherapy combinations improved survival as well, im- likely reflects the polyamine pools, pathway flux, and compen- plying that these synergistic effects went beyond cytostasis. satory mechanisms present in human neuroblastoma. Initial data with human neuroblastoma cell lines grown as Treating TH-MYCN mice with α-difluoromethylornithine xenografts in immunodeficient mice are similarly supportive (DFMO), a suicide inhibitor of Odc, increases tumor-free surviv- of the finding that polyamine disruption interferes with tumor al. Moreover, tumor penetrance is reduced in hemizygous mice progression1, although these studies will need to be extended to pre-emptively treated, supporting a requirement for Odc in tumor assess the role MYCN amplification plays in modulating sensi- initiation downstream of MycN (37, 38). Of note, no tumors tivity to these inhibitors. It is encouraging that both DFMO and arose after DFMO withdrawal, consistent with a finite vulnerable SAM486 inhibit neuroblastoma cell line growth in vitro inde- period for embryonal oncogenesis. This differs from the Eμ-Myc pendent of MYCN amplification (data not shown; ref. 37), like- lymphoma model in which protection from lymphomagenesis ly reflecting a commonality of Myc deregulation in the high-risk required persistent Odc inactivation (45). Neuroblastomas that phenotype (34). Mechanistically, Odc inhibition reduces Rb arise under DFMO exposure may activate compensatory mechan- phosphorylation at Ser795 and Ser807/811 through loss of isms to circumvent polyamine depletion. Since upregulated MycN-mediated repression of p27Kip1, leading to G1 growth Amd1 accompanies Odc inhibition in mammalian cells, as con- arrest. Coincident with this, Akt phosphorylation at Ser473 firmed in neuroblastoma (46), we tested the ability of DFMO and and GSK3B at Ser9 are induced, promoting survival (46, 48). ′ SAM486 (4-amidinoindan-1-one-2 -amidinhydrazone, a com- Although DFMO similarly abolished p27Kip1 induction in the petitive Amd1 inhibitor from Novartis) to synergize in this Eμ-Myc lymphoma model in vivo, the effects in the TH-MYCN model. Neuroblastoma penetrance was further reduced, includ- neuroblastoma model may instead be mediated through the ing in homozygous mice; thus, optimized polyamine depletion transcriptional upregulation of p21Cip1 (38). contributes to markedly improved efficacy (47). A more practical Strategies to integrate polyamine depletion therapeutics into test, however, requires treatment of established tumors. DFMO neuroblastoma treatment are warranted. It is unlikely that TH-MYCN treatment of mice harboring clinically detected neuro- traditional phase 1 and 2 studies for patients with relapsed or blastomas extends the time to tumor progression and augments refractory high-risk neuroblastoma with only polyamine- the efficacy of numerous chemotherapeutics, supporting the idea targeted agents would generate enthusiasm. These pathway- targeted compounds might best be tested on a backbone of 1 M. Hogarty, unpublished data. cytotoxic drugs to improve or restore chemoresponsiveness, as

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Molecular Pathways shown with the mouse model, and such clinical trials are in de- synergy for select polyamine depletion-chemotherapeutic com- velopment. However, maximizing the impact of such strategies binations (55). SAT1 acetylates polyamines and colocalizes requires a more complete understanding of tumor-specific poly- with the putative export solute transporter SLC3A2 to facilitate amine flux and the compensatory mechanisms used to escape export (25), leading to increased polyamine metabolic flux blockade in these pathways. For example, many tumor cells re- ∼five-fold through upregulated biosynthetic enzyme activity spond to Odc inhibition with enhanced polyamine uptake from in efforts to restore homeostasis (56). This provides another op- the microenvironment (49). Radiolabeled spermidine uptake portunity for synergy between SAT1 inducers (e.g., NSAIDs, cis- from neuroblastoma cell lines is not induced during DFMO or platinum) and biosynthesis inhibitors (e.g., DFMO, SAM486) SAM486 exposure, although it remains possible that this that prevent this response. represents accommodation to prolonged tissue culture, as poly- Finally, there is great interest in analogs that mimic native amine content is nominal in culture media. Neuroblastoma cell polyamines in homeostatic regulation. Such compounds can uti- lines established from the TH-MYCN model, however, do in- lize the polyamine transporter to concentrate in cancer cells, duce uptake from two- to six-fold under such conditions. De- leading to compensatory downregulation of polyamine biosyn- spite this, they remain sensitive to polyamine depletion in vivo. thesis and upregulation of catabolism, while not substituting There are potential opportunities for improving polyamine functionally for natural polyamines. Agents of this class include depletion responses for tumors with inducible polyamine up- assorted symmetrically and nonsymmetrically substituted ana- take. First, compounds that antagonize polyamine uptake, such logs, conformationally restricted analogs, oligoamines, and mac- 1 as D-lysine spermine (MQT-1426) and N -spermyl-L-lysinamide rocyclic polyamine analogs. These agents are reviewed in (14) (ORI202), are under preclinical development (50) and may co- and are in preclinical and clinical studies. Of these, PG-11047 operate with DFMO and other biosynthesis inhibitors to more (a second-generation conformationally restricted analog) has profoundly reduce polyamine levels and improve therapeutic re- been shown to have activity in vivo in preclinical non-small cell sponses in vivo (51). Alternatively, “Trojan horse” approaches lung carcinoma models (57), although recent testing against pe- with polyamine-chemotherapy conjugates have been used and diatric tumors through the Pediatric Preclinical Testing Program could cooperate with polyamine biosynthesis inhibitors. In this (58)2 showed minimal activity as a single agent against neuro- approach, DFMO or similar agents induce polyamine depletion blastoma (59). in cancer cells that subsequently upregulate polyamine trans- Optimism remains that polyamine depletion can be port. This targets the delivery of polyamine-chemotherapy con- exploited therapeutically, and the agents discussed herein rep- jugates preferentially to tumor cells. Numerous such conjugates resent only a partial list of those under active drug develop- have been developed, but a subsidiary benefit of delivering a ment. DFMO (Eflornithine) has gained FDA approval for DNA-interacting cytotoxic in this manner is that the polyamine trypanosomiasis and has been the most rigorously tested agent moiety not only enhances tumor-specific uptake, but also DNA in this class. Although it has demonstrated potent activity in co- binding through cationic interactions. The spermine-podophyl- lorectal adenoma chemoprevention, it has yet to demonstrate lotoxin conjugate F14512 (Pierre Fabre) has superior cytotoxicity potency against more advanced cancers. Whether combination in cells with enhanced polyamine uptake in vitro (IC50 in the na- therapies that deprive cancer cells of major compensatory path- nomolar range) and regressed breast carcinoma xenografts in vivo ways will synergize is not yet known, but many rational combi- (52), providing strong proof of principal for this approach. nations to do just that exist. Transitioning this class of agents Beyond inhibiting biosynthesis and impeding import, anoth- from scientific discovery to preclinical anticancer activity ap- er therapeutic option is to enhance catabolism and/or export of pears to have been achieved, but bridging the chasm to demon- polyamines. For example, NSAIDs influence polyamine acetyla- strate clinical utility remains a significant challenge. tion and export through the upregulation of SAT1 via PPARγ (53). Although anticancer effects attributable to this drug class Disclosure of Potential Conflicts of Interest are pleiotropic, cox-inhibitor-mediated apoptosis in cancer cells can be rescued by polyamine supplementation, implicating this No potential conflicts of interest were disclosed. pathway (53). This may have therapeutic relevance, because sulindac and DFMO together have documented efficacy in re- Acknowledgments ducing adenoma recurrence in an at-risk population, as demon- SAT1 The authors thank Susan Gilmour, Michelle Haber, Murray Norris, Glenn strated in a large chemoprevention trial (54). induction Marshall, and Andre Bachmann for helpful discussions. also occurs downstream of platinator and other chemotherapeutics, in association with additional polyamine regulator changes to repress polyamine content, and this may provide 2 pptp.stjude.org

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www.aacrjournals.org 5961 Clin Cancer Res 2009;15(19) October 1, 2009 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst September 29, 2009; DOI: 10.1158/1078-0432.CCR-08-3213

Disrupting Polyamine Homeostasis as a Therapeutic Strategy for Neuroblastoma

Nicholas F. Evageliou and Michael D. Hogarty

Clin Cancer Res 2009;15:5956-5961. Published OnlineFirst September 29, 2009.

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