mechanisms of resistance to histone deacetylase...

Mechanisms of Resistance to Histone Deacetylase InhibitorsandTheir Therapeutic ImplicationsValeria R. Fantin and Victoria M. Richon

Abstract Histone deacetylase inhibitors (HDI) are a promising new approach to the treatment of cancer.HDIs have been shown to induce differentiation, cell cycle arrest, and apoptosis in a variety oftransformed cell lines; inhibit tumor growth in animal models; and show antitumor activity inclinical trials.Vorinostat, which has shown clinical responses in f30% of patients with advancedcutaneousT-cell lymphoma, is the first HDI approved for the treatment of cancer, and it is currentlybeing evaluated in other indications. A better understanding of the molecular determinants ofresistance to HDIs may provide the basis for therapeutic combinations with improved clinicalefficacy. Poor response to treatment could be linked to systemic factors like pharmacokinetics orto tumor-specific factors both at the level of the malignant cells (tumor intrinsic) or the tumormicroenvironment. This review focuses on the tumor intrinsic mechanisms of drug resistance(excluding mechanism of acquired resistance due to chronic exposure). In particular, attention isgiven to selected mechanisms that are relevant across chemical classes of HDIs and that can aid inthe design of rational combination strategies.

Histone deacetylases (HDAC) represent a new target for cancertherapeutics. Four classes of HDACs have been identified (1).Class I human HDACs are homologous to the yeast HDACRpd3 and include HDAC1, HDAC2, HDAC3, and HDAC8.Class II HDACs include HDAC4, HDAC5, HDAC6, HDAC7,HDAC9, and HDAC10. HDAC11 makes up class IV HDACs.The key catalytic residues have been conserved in both class Iand class II and IV HDACs. The third class of human HDACsconsists of homologues of yeast Sir2. Class III HDACs requirenicotinamide-adenine dinucleotide NAD+ for activity and differfrom classes I and II in their catalytic site. Class I and II HDACshave been shown to be overexpressed, aberrantly recruitedto oncogenic transcription factors, and mutated in cancer, thusrepresenting potential targets for small-molecule inhibitors.Several structural classes of HDAC inhibitors (HDI) arecurrently undergoing evaluation in the clinic and include theshort-chain fatty acids (e.g., valproate and phenylbutyrate);hydroxamic acid derivatives [e.g., vorinostat (suberoylanilidehydroxamic acid), belinostat (PXD-101), and panobinostat(LBH-589)]; benzamides [e.g., SNDX-275 (MS-275) andMGCD0103]; and cyclic tetrapeptides [e.g., romidepsin(FK-228); refs. 2, 3]. These HDIs under clinical developmentdo not inhibit the class III enzymes. Instead, they either inhibitin general both class I and II enzymes (hydroxamatederivatives) or selectively inhibit class I enzymes (benzamides).Although the precise mechanism of action of HDIs has not

been completely elucidated, at the molecular level, inhibition

of HDAC activity leads to accumulation of acetylated proteinsincluding histones, transcription factors, tubulin, and heatshock protein 90 (4, 5). The increase in the acetylation state ofthese proteins alters their function, leading to alterations intranscription, mitosis, and protein stability. Collectively, thesechanges interfere with tumor cell proliferation, survival, andmaintenance. In addition, HDIs have shown antiangiogenicand immunomodulatory activity that may play an importantrole in mediating their antitumor effects. Indeed, the mecha-nism of action of HDIs is complex, and the mechanisms ofresistance to HDIs described to date are diverse becausealterations at various cellular levels can potentially block theiractivity.

Mechanisms of Resistance to HDAC Inhibitors

Inhibition of HDACs trigger a number of cellular responsesthat interfere with growth-promoting signals, increase cellularstress, and ultimately lead to tumor cell death. Cancer cells,however, are highly adaptable in their ability to respond andresist growth inhibitory and damaging factors. In fact, theprocess of tumorigenesis is characterized by constant selectionof those malignant cells that adapt to cope with damagingagents and environmental constraints and to become moreindependent from external proliferative/survival stimuli. Inaddition to these general mechanisms of survival or resistanceto death that arise as a consequence of natural selection, stablealterations that may hinder the mode of action of HDIs can alsobecome determinants of response. For instance, DNA hyper-methylation represents a mechanism of resistance uniquelyrelevant to HDIs, which interferes with their ability to inducetranscriptional activation of silenced tumor suppressor genes.Furthermore, exposure of tumor cells to first-line therapeutictreatments such as chemotherapy and radiotherapy may selectfor those with increased capacity to deal with high levels of

Molecular Pathways

Authors’Affiliation: Merck Research Laboratories, Boston, MassachusettsReceived 8/31/07; accepted 9/26/07.Requests for reprints: Victoria M. Richon, Cancer Biology and Therapeutics,Merck Research Laboratories, Merck & Co., Inc., 33 Avenue Louis Pasteur,Boston, MA 02115. Phone: 617-992-2044; Fax : 617-992-2412; E-mail :[email protected].

F2007 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-07-2114

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DNA damage and oxidative stress. Ultimately, those adapta-tions could also contribute to block cell death and interferewith the activity of HDIs.The antitumor activity of small-molecule compounds can be

affected by efflux mechanisms, drug deactivation, status of drugtarget, bypass/repair mechanisms for drug-induced damage, andalterations in the cell death pathway. Cellular factors that havebeen implicated as determinants of resistance in HDIs include(a) drug efflux, (b) target overexpression and desensitization, (c)chromatin/epigenetic alterations, (d) stress response mecha-nisms, and (e) antiapoptotic/prosurvival mechanisms (Fig. 1).Effect of drug efflux mechanisms on sensitivity to HDIs. One

of the molecular alterations most commonly associated withthe multidrug resistance phenotype in cancer cells is thatmediated by overexpression of efflux pumps in the ATP-bindingcassette transporter family. The antiproliferative activities ofvorinostat, belinostat, AN9, and romidepsin have beenexamined in multidrug-resistant human cancer cell lines withhigh levels of P-glycoprotein, the product of the multidrug

resistance-1 gene (MDR1) as well as the multidrug resistanceprotein 1 (6–10). To date, romidepsin is the only HDI that hasbeen established as a substrate for P-glycoprotein andmultidrug resistance–associated protein 1. Overall, multidrugresistance mediated by efflux mechanisms does not seem toconstitute a major driver of tumor cell resistance to HDIs thatbelong to either the hydroxamate or carboxylic acid class. Ofnote, the combination of romidepsin and all-trans retinoic acidwas shown to up-regulate P-glycoprotein, inducing resistance todoxorubicin in promyelocytic cells (11). This effect was due, inpart, to an increase in histone acetylation and chromatinremodeling at the MDR1 promoter. In addition to romidepsin,other HDIs including sodium butyrate have been shown toactivate MDR1 transcription (12). Further studies will help toclarify the effect of other HDIs on P-glycoprotein expressionto inform combination studies involving chemotoxins thatare substrates of drug efflux pumps.Alterations in HDACs and their effect on response to

HDIs. The first indication that resistance to HDIs could be

Fig. 1. Mechanisms that block apoptosis of cancer cells in response to HDIs.The schematic diagram depicts the cascade of events and outcomes following inhibition ofHDAC activity in vitro. Several factors that confer resistance along the pathway are indicated.

Molecular Pathways

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traced to alterations at the level of their molecular target camefrom studies done with the hydroxamate derivative trichostatinA (13). Subsequent studies in melanoma cells established thatoverexpression of HDAC1 was sufficient to confer resistance tosodium butyrate (14). More recently, an inactivating mutationin HDAC2 was identified in various human colon andendometrial cancer cell lines with microsatellite instability(15). Treatment of HDAC2-deficient RKO and Co115 cells withtrichostatin A failed to induce histone acetylation and inhibitproliferation. Interestingly, these cells remain sensitive tovalproate and butyrate. The selective resistance to trichostatinA is intriguing, and additional studies are necessary to gainfurther insight into the molecular basis of this phenomenon.Noteworthy, the same frameshift mutation found in cell lineswas also present in 48 of 228 (21%) tumor samples analyzed,suggesting that alterations in HDAC2 expression may play arole in clinical response to selective HDIs.Epigenetic and chromatin alterations as determinants of

response. In addition to genetic alterations, epigenetic mech-anisms contribute to malignant transformation (16, 17). DNAmethylation and histone acetylation alter the nucleosomestructure to activate or repress gene transcription (18). Silencingof tumor suppressor genes is frequently mediated by DNAmethylation at the promoter regions. Acetylation of histones,linked to methylation sites through the recruitment of HDACsby methylcytosine binding proteins (e.g., MeCP-2), constitutesa second level of regulation of epigenetic silencing (19–21).In addition, HDAC1 and HDAC2 have been shown to interactwith DNA methyltransferases. The evidence thus far indicatesthat these two biochemical processes cooperate to induce genesilencing (22). Indeed, DNA methylation and hypoacetylationof histones H3 and H4 are frequently associated with silentgenes. There are a number of cases of restoration of theexpression of tumor suppressor genes after HDI treatment.Some studies, however, have shown examples of dominance ofDNA methylation over histone repressive marks in cancer cells(23, 24). From that stand point, methylation of DNA hindersthe ability of HDIs to fully restore expression of epigeneticallysilenced genes and represents a mechanism of resistance to HDItreatment. For instance, HDAC inhibition was not sufficient torestore expression of the tumor suppressor genes hMHL1 andTIPM3. Minimal methylation after a low dose of 5-aza-2¶-deoxycytidine was required for robust reexpression of thesegenes in response to trichostatin A. Furthermore, cases in whicheither treatment alone was insufficient to induce significantreexpression of epigenetically silenced genes have beenreported. Among those, synergistic reexpression of the MAGEfamily in testicular cancers and TMSC1 in hepatocellularcarcinoma occurs only in response to the combination of5-aza-2¶-deoxycytidine and HDIs (25, 26). Although emergingnew data suggest that in some cases HDIs can affect global andgene-specific DNA methylation (27, 28), most studies indicatethat this repressive mark cannot be overcome by the singleaction of HDIs. These observations provided the rationale forthe clinical evaluation of HDIs in combination with hypo-methylating agents.Cellular polyamines like spermidine and spermine are

cationic molecules that regulate gene expression by affectingthe chromatin microenvironment (29). The relative abundanceof polyamines is controlled by ornithine decarboxylase, therate-limiting enzyme in the process of polyamine biosynthesis.

In vitro , polyamines have been shown to condense chromatinfibers and facilitate oligomerization of nucleosomal arrays(30, 31). A relationship between high polyamine levels andaltered acetylation of core histones seems to be linked withproliferation. The basis for this correlation is not fully under-stood. Enhanced histone acetyltransferase activity, however,was detected in skin samples and skin fibroblasts from K6-ODCtransgenic mice (29). Recently, the contribution of polyaminesto the response of human cancer cell lines to inhibition ofHDACs was evaluated (32). These studies showed that poly-amines modulate the response to structurally diverse HDIsincluding trichostatin A, trapoxin A, and sodium butyrate,and that their depletion increases resistance to apoptosisin response to treatment. Examination of the correlationbetween polyamine levels and clinical response to HDI meritstesting. Furthermore, the investigators proposed that elevatedornithine decarboxylase activity could represent a biomarker ofresponse to HDIs.

Mechanism of protection against oxidative stress as adeterminant of resistance. Treatment of cancer cells withHDIs including vorinostat, SNDX-275, sodium butyrate,panobinostat, and romidepsin results in oxidative stresscharacterized by increased production of reactive oxygenspecies and decreased ratio of reduced to oxidized glutathione(33–37). Furthermore, cell death in response to several HDIscan be partially rescued by preincubation of cells with theglutathione precursor and free radical scavenging N-acetylcys-teine (38). These data implicate oxidative damage as asignificant component of the antiproliferative effect triggeredby HDIs. Cellular redox homeostasis is maintained througha complex set of reactions, and the thioredoxin family par-ticipates in this process. Thioredoxins are proteins withdisulfide reducing activity that provide reducing equivalentsto ribonucleotide reductase for DNA synthesis and toperoxiredoxins for scavenging reactive oxygen species.Through these activities, thioredoxins play a role in prolifer-ation, protection against oxidative damage, and inhibition ofstress-induced cell death (39). The activity of thioredoxin-1 ismodulated by thioredoxin-binding protein 2, which binds tothe reduced from of the enzyme and interferes with itsfunction. Response to vorinostat has also been associated withits ability to induce the expression of thioredoxin-bindingprotein 2 and down-regulate the expression of thioredoxin incell lines that are sensitive to the compound (40). In addition,up-regulation of thioredoxin has been proposed to constitutea defense mechanism against HDI-induced increase in reactiveoxygen species levels in nontransformed cells (41). Inaddition, thioredoxin was found to be differentially expressedacross prostate cancer cell lines with diverse vorinostatsensitivity (42). In tumors, high levels of thioredoxin-1 havebeen associated with grade and resistance to standard therapy(39). Based on the existing in vitro data, it is tempting tospeculate that mechanisms that contribute to alleviate cellularoxidative stress may antagonize the activity of HDIs. Thus,tumors with inherent high levels of antioxidants, includingenzymes involved in the maintenance of cysteine thiolhomeostasis, may be able to overcome HDI-mediated damage.In this scenario, proteins that participate in the stress responseto oxidative damage could constitute determinants of clinicalresistance to HDIs. This is an intriguing hypothesis thatwarrants rigorous clinical evaluation.

Resistance to HDAC Inhibitors




Role of mediators of cell death pathway and of autophagicresponse in treatment outcome. In vitro , HDIs elicit apoptosis ofa variety of malignant cells (43–47). Cell death proceedsmainly via the intrinsic apoptotic pathway and is characterizedby mitochondrial damage, cytochrome c release, and produc-tion of reactive oxygen species (33, 48). Consistently, over-expression of antiapoptotic Bcl-2 or Bcl-XL is sufficient torender transformed cells resistant to HDIs (48–51). Therapeu-tic efficacy of HDIs was correlated with induction of apoptosisin mice bearing EA-Myc lymphoma cells (52). Extensiveanalysis of the role of the tumor suppressor p53 andintermediates involved in the mitochondrial apoptotic cascadeon the response of the B-cell lymphomas indicates that theHDI-induced death is independent of p53 activity, requires theproapoptotic BH3-only proteins Bim and Bid, and can beblocked by Bcl-2. Because Bcl-2 overexpression occurs inleukemias and lymphomas, and loss of Bim and Bid has beenreported in human cancers, it is possible that alterations inthese proteins may play a role in clinical response to HDIs.Future studies will provide clarity.The antiapoptotic transcription nuclear factor nB (NF-nB) has

been identified as a mediator of resistance to HDI treatment.Inhibition of HDACs stimulates transcriptional activation ofNF-nB through acetylation of the RelA/p65 subunit andinduction of an array of genes that mediate protection fromcell death (53). The activation of NF-nB by HDIs interfereswith their ability to trigger cell death in non–small cell lungcancer lines and leukemia cell lines (54, 55). In fact, inhibitionof NF-nB activation by the compound BAY-11-7085 sensitizesthe malignant cells to death in response to inhibition ofHDACs. Because several HDIs are at various stages of clinicalevaluation in these indications, it is important to understandthe relevance of the pathway as a determinant of responseto treatment.Recently, we have uncovered a correlation between constitu-

tive activation of various members of the signal transducerand activator of transcription family and lack of response tovorinostat in patients with cutaneous T-cell lymphoma.1 In vitrostudies suggest that the protective role of these proteins ismediated, at least in part, by transcriptional control of severalantiapoptotic genes.In addition to its well-established role as a negative

regulator of the cell cycle, the cyclin-dependent kinase inhibitorp21(CIP1/WAF1) has been implicated in the regulation ofprogrammed cell death (56). Several studies have shown thatHDIs induce the expression of p21(CIP1/WAF1) in a p53-dependent and p53-independent way, and that p21(CIP1/WAF1)

up-regulation in response to HDI treatment mediates cell cyclearrest and differentiation or apoptosis (57–59). Thus, the roleof p21(CIP1/WAF1) as a determinant of response is not totallyunderstood. Early studies in U937 leukemia cells showed thatHDI-mediated p21(CIP1/WAF1) induction protected cells fromapoptosis, and that blocking it resulted in an increase in celldeath (58). Subsequent studies with SNDX-275, however,suggest that the p21(CIP1/WAF1)-mediated protection is dosedependent. In this case, low concentrations of SNDX-275induced cell cycle arrest in leukemia cells, and that effect wasovercome at high concentrations of HDI (34).

The role of autophagy in the response of cancer cells totherapy is a matter of intense investigation. Autophagy is aprocess in which intracellular membranes sequester proteinsand organelles (autophagosome) that are subsequentlydelivered to the lysosome for degradation (60). This cellularprogram enables elimination and/or recycling of proteins andintracellular components in response to starvation and stress.The contribution of autophagy to the response to anticancertherapy seems to depend on the cellular context. Whereas insome cancer cells activation of autophagy results in cell deathand correlates with therapeutic efficacy, in others autophagyplays a protective role by which cells cope with therapy-induced bioenergetic stress and cellular damage (61, 62). Inthis second scenario, therapeutic strategies that incorporateinhibitors of autophagy may enhance the efficacy ofanticancer therapy (63). Treatment of imatinib-resistantchronic myeloid leukemia with chloroquine, which blocksfusion of the autophagosome and lysosome, results inenhanced antineoplastic activity of the HDI vorinostat (64).This study underscores the role of protective autophagy as adeterminant of response to HDI treatment in a definedsubpopulation of chronic myeloid leukemia cells. Additionalwork will help to understand the relevance of this cellularprocess for other cancer types.

Clinical-Translational Implications

Undoubtedly, characterization of the molecular determi-nants of resistance to HDIs will help to stratify patientsaccording to their likelihood of responding to treatment andto develop combination strategies to circumvent them.It is important to emphasize that most of the hypotheses

drawn from preclinical studies on the mechanism ofresistance to HDIs await clinical evaluation. One practicalconsideration is that a number of pharmacologic agents thatcan counteract the in vitro mechanisms previously describedare in the discovery phase and are not available for clinicaluse. Inhibitors of efflux pumps (tariquidar and laniquidar),antiapoptotic Bcl-2 (oblimersen and ABT-263), and nuclearfactor nB (oligonucleotide decoys), however, are at advancedstages of clinical development and could be evaluated incombination with HDIs in the near future.As discussed earlier, certain tumor cells may have a high

antioxidant protective barrier that undermines the antineoplas-tic activity of HDIs. Thus, one possible strategy to maximize thetherapeutic efficacy of HDIs is to combine them with agents thatalso elicit oxidative damage as part of their mechanism ofresponse and to potentiate this effect. This approach may sufferfrom high toxicity. Tolerability profiles for combinations of thisnature may vary with the complementary compound selected.Whenever tolerated, however, pro-oxidant combinations maystress cells above their resistance threshold and push them downthe apoptotic pathway. One of the combinations that has beenexplored preclinically involves the typical pro-oxidant agent2-methoxyestradiol, which directly affects the mitochondria, themajor source of cellular reactive oxygen species (65). A numberof clinically approved compounds induce oxidative damage aspart of their mode of action. Among them, the activity ofbortezomib and doxorubicin may synergize with that of HDIs atvarious levels, including the increase in reactive oxygen speciesand depletion of reduced glutathione (66, 67). Clinical trials1 V.R. Fantin andV.M. Richon, unpublished data.

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with these two agents are under way and rigorous testing of thehypotheses may be possible.The use of inhibitors of DNA methylation in combination

with HDIs as a means to reactivate genes with tumor suppressoractivity is one of the hypotheses that quickly progressed fromthe preclinical to the clinical setting. In addition to the strongmechanistic rationale for these studies, the availability ofclinically approved hypomethylating agents and HDIs madeit possible to reduce the concept to practice. The tolerabilityand efficacy of the combinations of valproic acid/5-aza-2¶-deoxycytidine and sodium phenylbutyrate/5-azacitidine wereevaluated in patients with leukemia or myeloid dysplasticsyndrome (68, 69). Both clinical trials showed that thecombinations were well tolerated and active. Correlative studieshave shown reversal of epigenetic gene silencing. The activity ofHDI/hypomethylating agent combination strategy highlights

the potential of epigenetic therapy and warrants developmentof future trials to explore additional HDIs in combination withhypomethylating agents.

Conclusion

The molecular heterogeneity of tumor cells, together with thepleiotropic nature of the cellular response triggered byinhibition of HDACs, poses a major challenge in the questfor determinants of resistance. It is clear from preclinical studiesthat resistance to inhibitors of HDACs is multifactorial, andthat the dominant pathway of resistance in tumors may bedependent on tissue of origin and genetic context. In the future,availability of large enough sets of clinical samples will enableus to further evaluate the clinical significance of thesepreclinical hypotheses of resistance.

Resistance to HDAC Inhibitors


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2007;13:7237-7242. Clin Cancer Res Valeria R. Fantin and Victoria M. Richon and Their Therapeutic ImplicationsMechanisms of Resistance to Histone Deacetylase Inhibitors

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