Drug Discovery Today: Technologies
Vol. 11, 2014
Editors-in-Chief Kelvin Lam – Simplex Pharma Advisors, Inc., Arlington, MA, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Drug resistance
Resistance to minor groove binders Benedetta Colmegna, Sarah Uboldi, Eugenio Erba, Maurizio D’Incalci* Department of Oncology, Laboratory of Cancer Pharmacology, IRCCS – Istituto di Ricerche Farmacologiche ‘Mario Negri’, via La Masa 19, Milan 20156, Italy
In this paper multiple resistance mechanisms to minor groove binders (MGBs) are overviewed. MGBs with antitumor properties are natural products or their derivatives and, as expected, they are all substrates of P-glycoprotein (P-gp). However, a moderate expression of P-gp does not appear to reduce the sensitivity to trabectedin, the only MGB so far approved for clinical use. Resistance to this drug is often related to transcriptional mechanisms and to DNA repair pathways,
Section editors: Ju¨rgen Moll – Boehringer-Ingelheim, Vienna, Austria. Gemma Texido´ – Nerviano Medical Sciences S.r.l, Nerviano, Italy. them have been reported. Here we review the knowledge of the resistance mechanisms to the MGBs with particular focus on trabectedin, which is approved for clinical use in Europe and several other countries for the therapy of soft tissue sarcomas and ovarian cancer.
particularly defects in transcription-coupled nucleotide excision repair (TC-NER). Therefore tumors resistant to trabectedin may become hypersensitive to UV rays and other DNA damaging agents acting in the major groove, such as Platinum (Pt) complexes. If this is confirmed in clinic, that will provide the rationale to combine trabectedin sequentially with Pt derivates. Introduction The discovery that many tumors have defects in DNA repair mechanisms and in checkpoints of the cell cycle explains why DNA-damaging agents have high antitumor selectivity. Minor groove alkylating agents are a distinct class of anticancer drugs, derived originally from natural products that interact with DNA, unlike conventional alkylators, explaining why their spectra of activity and resistance patterns do not overlap. The DNA minor groove binders (MGBs) can be divided into two classes: compounds that covalently bind to adenines at the N3 position in adenine-thymine (AT)-rich sequences of DNA and compounds that bind to guanines at the N2 position. Both have been extensively investigated in preclinical systems and several mechanisms of resistance to *Corresponding author.: M. D’Incalci ([email protected]) 1740-6749/$ ß 2014 Elsevier Ltd. All rights reserved.
Mechanisms of resistance of minor groove binders that alkylate N3 of adenines The chemical structures of the most representative antitumor agents that act by binding adenines at N3 position in the minor groove of DNA are shown in Fig. 1. Tallimustine, N-deformylN-4-N,N-bis(2-chloroethylamino)benzoyl distamycin-A, like the parent compound distamycin-A [1], binds preferentially to (AT)-rich sequences in the minor groove of B-DNA. Both distamycin-A and tallimustine in vitro inhibit the binding of transcription factors that recognize cis-elements rich in AT, whereas they have no effect on the binding of transcription factors that recognize guanine-cytosine-rich boxes. However, tallimustine had a potent cytotoxic and antitumor effect against several cancer cell lines grown in vitro and in mouse tumor models, whereas distamycin was inactive. Thus the alkylation of adenine caused by tallimustine, but not by distamycin, is crucial for its antitumor activity. Resistance to tallimustine was investigated in cell lines made resistant by prolonged exposure to this compound. In some cases the resistance was related to lower intracellular retention of tallimustine, because of overexpression of the multi-drug resistance (MDR1) gene encoding for P-gp (P-170) involved in drug efflux from the cell. In these cases the resistance was reversed by P-gp inhibitors [2]. In other cases
http://dx.doi.org/10.1016/j.ddtec.2014.03.001
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(a)
NH2 H
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Figure 1. MGBs that bind N3-adenine of DNA. Panel A: the chemical structures of distamycin and two derivatives, tallimustine and brostallicin. Panel B: the structures of the natural product CC-1065 and derivatives containing cyclopropylpirrolo(e)indolone such as adozelesin, carzelesin and bizelesin.
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resistance appeared to be associated with an increase in glutathione (GSH), although blocking the synthesis of GSH by buthionine sulphoximine (BSO) only partially reversed the resistance, suggesting that other mechanisms were involved. Some studies with a murine leukemia cell line resistant to tallimustine, L1210/24517 [3] showed cross-resistance to other MGBs like CC-1065, which can bind adenine N-3 in DNA with high sequence-specificity [4,5]. This cell line was also crossresistant to distamycin which is toxic only at a high concentration without producing any alkylation in DNA, thus excluding that the cross-resistance between tallimustine and CC1065 was due to efficient removal of the alkylated bases by DNA repair mechanisms. Studies in a cell line resistant to Hoechst 33342 and distamycin-A [6], which bind the DNA minor groove without forming stable adducts in DNA, indicated an enhanced ability to remove ligand molecules from cellular DNA through a still poorly understood pathway that can be blocked by topoisomerase II poisons. This invites speculation on the existence of a repair system that recognizes the change in DNA structure produced by MGBs, that might be important for the sensitivity to these agents. Mismatch repair deficiency has been reported in the resistance to compounds acting by selectively alkylating adenine N3 in AT-rich sequences. This was demonstrated in a HCT-116 cell line with a mutation of hMLH1 gene that was resistant to tallimustine, carzelesin and CC-1065 [7]. When hMLH1 was restored by chromosome 3 transfer, the resistance was lost. Further support of the involvement of mismatch repair in the sensitivity to N3 adenine alkylators is the finding that two ovarian carcinoma cell lines, A2780/CP70 and A2780/MCP-1, resistant to cisplatin because of a defect in hMLH1, were resistant to MGBs that alkylate adenines N3. This mechanism does not apply to the bromoacryloyl derivative of distamycin A, Brostallicin, that was reported to retain sensitivity in DNA mismatch repair-deficient tumor cells [8]. Brostallicin was also different from the other distamycins, being more cytotoxic against cells with a high GSH content, a potentially interesting property considering there are cases in which the low sensitivity to DNA-interacting compounds is related to high expression of the glutathione S-transferase (GST)/GSH system. Although several compounds that bind selectively to N3 of adenines, such as tallimustine, carzelesin, adozelesin and bizelesin [9,10], have been tested in initial clinical investigations, none has been developed further, mainly because of their severe bone marrow toxicity and low therapeutic index [11,12]; therefore no data are available on the clinical relevance of the findings related to resistance mechanisms.
Mechanisms of resistance to minor groove binders that alkylate N2 of guanine Unlike the N3-adenine alkylators, it appears that compounds that selective alkylate guanine N2 in the minor groove of DNA, like trabectedin, can be safely used at doses effective
Drug Discovery Today: Technologies | Drug resistance
against at least some malignancies such as soft tissue sarcomas and ovarian cancer. Since to the best of our knowledge trabectedin is the only MGB that specifically alkylates N2 of guanine that is approved for clinical use, so we will overview the literature on this compound, although some other compounds will be mentioned too. Exposing Igrov-1 human ovarian cancer cells to increasing concentrations of trabectedin for short or long times, Erba et al. [13] obtained Igrov-1 sublines very resistant to trabectedin (i.e. approximately 50-fold), which overexpress P-gp; there was no increase in other multi-drug resistance-related proteins, such as MRP or LRP. Beumer et al. [14] have unambiguously shown that trabectedin is a P-gp substrate by investigating the vectorial transport in LLC-PK1 pig kidney, Madine-Darby canine kidney (MDCK) subclones and in the murine mdr1a and/or human MDR1 transfected subclones. The LLC-PK1 cells transfected with either mdr1a or MDR1 genes showed much higher trabectedin transport out of the cells than control untransfected cell lines. The same results were obtained by transfecting MDCK cells. In both models the P-gp transport system was blocked by LY335979 (a P-gp inhibitor) only in the MDR1 transfected cells, not in the control lines. The same authors examined a series of cell lines with high or moderate P-gp expression levels and found that trabectedin displayed the typical MDR phenotype only in highly P-gp expressing cells, but not in lines with moderate expression levels, more similar to clinical samples. In keeping with these observations were the reports by Scotlandi et al. [15,16] who found trabectedin was effective in a series of osteosarcoma and Ewing sarcoma cell lines resistant to doxorubicin, expressing moderate levels of P-gp. Since trabectedin is not a preferential substrate of P-gp, cell lines with a constitutive moderate expression of the MDR1 gene (sensitive to trabectedin) appeared resistant to anthracyclines or taxanes, the major substrates of P-gp. Since in some cases the MDR1 gene can be transcriptionally induced it could be interesting to investigate whether trabectedin inhibits this mechanism. Trabectedin can inhibit the transcription of some rapidly inducible genes such as heat shock proteins [17] or cell cycle genes [18]. Elegant in vitro experiments by Jin et al. [19] showed that trabectedin blocks the activation of transcription of the MDR1 gene promoter, induced by histone deacetylase inhibitors. In line with this observation Kanzaki et al. [20] reported that exposure to trabectedin caused downregulation of the MDR1 gene, enhancing the cytotoxicity and cellular accumulation of doxorubicin and vincristine in cell lines overexpressing P-gp. Duan et al. [21] investigated two chondrosarcoma cell lines made resistant to trabectedin or to Zalypsis1 – which has some structural analogies to trabectedin (Fig. 2) – exposing the cells to increasing concentrations. Analysis of differentially expressed genes showed that some zinc finger proteins, such www.drugdiscoverytoday.com
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HO NH
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Figure 2. MGBs that bind N2-guanine of DNA. The chemical structure of trabectedin and its derivatives Zalypsis1 and lurbinectedin.
as ZNF93 and ZNF43, were over-expressed in the resistant lines; silencing these genes reversed the resistance to trabectedin or Zalypsis1, suggesting their role in the resistance mechanisms. Marchini et al. [22] compared the gene expression profiles of trabectedin-resistant cell lines derived from an ovarian cancer or a chondrosarcoma cell line. Genes involved in the cytoskeleton dynamics such as g-actin, Rho E, Rac-b or other small GTPases were downregulated much more in the resistant than in the sensitive clones. In addition, in the two cell systems, the a-2 chain of collagen IV gene was down-regulated in the resistant line. These findings were consistent with data obtained in other cellular systems [23] suggesting extracellular matrix proteins are involved in the resistance to trabectedin. Takebayashi [24] isolated a HCT116 cell line resistant to trabectedin (HCT116/ER5); evidence that this line was also hypersensitive to UV radiation stimulated the investigation of the DNA repair pathways potentially involved. They found that functional XPG protein was not expressed in HCT116/ER5 cells because of a point mutation in the XPG gene, which resulted in a premature termination codon. These results are in keeping with reports [25–28] that cells deficient in NER, which are generally more sensitive to conventional alkylating agents and Pt complexes, are resistant to trabectedin. 76
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Tavecchio et al. [29] used an isogenic NER-proficient cellular system (CHO-AA8) and an NER-deficient one (CHO-UV-96) lacking functional ERCC-1, which are hypersensitive to UV light, and a UV-96 cell clone stably transfected with ERCC1(ERA-5) that shows the same sensitivity to UV light as AA8 cells. UV-96 cells were four times as resistant to trabectedin than AA8 or ERA-5 cells. Trabectedin different induced cell cycle perturbations in NER-deficient and NER-proficient UV-96 cells, even when treated with equitoxic drug concentrations (i.e. four times higher for NER-deficient cells). Biparametric flow cytometric methods indicated that trabectedin caused a block of cells in G2-M in NER-proficient AA8 or ERA5 cells and not in NER-deficient UV-96 cells. Applying a computer simulation method it was found that the dynamics of the cell cycle perturbations were complex, with blocks in both G1 and G2 and a delayed S phase [30]. In cells with functional NER, exit from G1 block was faster than in NER-deficient cells, then cells progressed slowly through S phase and were subsequently blocked irreversibly in G2-M phase, indicating they were unable to repair the damage [29]. These studies indicate that the resistance to trabectedin is associated with a different cell response from trabectedin-induced DNA damage, resulting in different cell cycle perturbations in NER-deficient or -proficient cells.
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Resistance mechanism to trabectedin related to the absence of NER has been observed in several cell lines with mutations of one of the proteins involved in NER, such as XPG or XPD or XPA [24]. Is this pattern of resistance unique to trabectedin? Another example has been reported for an anthracycline derivative that probably acts by a different mechanism from doxorubicin [31]. In a cell line made resistant to nemorubicin (30 -deamino-30 (2-(S)-methoxy-4-morpholinyl), a doxorubicin derivative that forms a metabolite, 30 -deamino-30 ,40 -anhidro-(20 (S)-methoxy-30 (R)-hydroxy-40 -morpholinyl) doxorubicin, which is 1000 times more potent than doxorubicin, the resistance appears to be related to the lack of XPG expression. Interestingly a cell line that did not express XPG because of hypermethylation of its promoter was cross-resistant with trabectedin, indicating that there may be an epigenetic mechanism of resistance to the drug [31]. Several laboratories have shown that cells made resistant to trabectedin by prolonged exposure often become hypersensitive to UV light, because they have a defect in the NER pathway, the main mechanism of repair of UV-induced DNA damage. Recently D’Incalci et al. (D’Incalci et al., abstract C93, AACRNCI-EORTC, Boston, October 2013) isolated a myxoid liposarcoma and an ovarian cancer cell line resistant to trabectedin, both lacking XPG expression. As expected, the resistant cells were hypersensitive to UV light. Since several reports had indicated that the sensitivity to cisplatin was increased in NERdeficient cell lines [26], the response to cisplatin was compared in the resistant and sensitive cell lines. Both trabectedin-resistant cell lines that do not express XPG were much more sensitive to cisplatin than the parental cell lines. These in vitro findings have been confirmed in vivo in immunodeficient mice. Xenografts from the trabectedinresistant ovarian cancer cell line were more sensitive to cisplatin, T/C 18% (where T and C are respectively the mean tumor weight of cisplatin-treated and control groups), than the parental cell line, T/C 64.2% (unpublished data obtained in our laboratory). These results provide the rationale for sequential use of trabectedin and Pt complexes in ovarian cancer (see the section ‘Clinical implications’). Although trabectedin was effective in several types of soft tissue sarcoma the most sensitive was certainly myxoid liposarcoma, in which the rate of objective responses as more than 50% in patients resistant to doxorubicin and ifosfamide [32,33]. The mechanism of sensitivity appears to be related to the ability of trabectedin to block the transactivating function of the fusion gene product FUS-CHOP, that is the pathogenic lesion of this type of liposarcoma, demonstrated both in vitro [34] and in vivo [35] in tumor biopsies from xenografts with myxoid liposarcomas. Uboldi et al. [36] isolated and characterised a trabectedin-resistant myxoid liposarcoma cell line, 402-91/ET, that appeared to be resistant to trabectedin by more than one mechanism. A mutation of XPG was one of the
Drug Discovery Today: Technologies | Drug resistance
mechanisms, also associated with increased sensitivity to UV radiation. However, when the 402-91/ET cell line was transfected with an expression vector encoding for XPG cDNA, the sensitivity to UV light was markedly reduced, with only partial reversion of the resistance to trabectedin, suggesting that other mechanisms play a role in the resistance. Chromatin-Immunoprecipitation (ChIP) assays showed that in the resistant cell line the FUS-CHOP was no longer able to transactivate its target promoters, suggesting that the mechanism of resistance was related to the change in the target of the drug action. This cell line showed cross-resistance to lurbinectedin and Zalypsis1 (Fig. 2) and collateral sensitivity to methylating agents, due to the lack of expression of the repair protein O6 methyl-guanineDNA methyl transferase (MGMT). In 402-91/ET cells, but not in the parental cell line, the MGMT gene promoter was highly methylated. The peculiar mechanism of resistance to trabectedin, involving regulation of transcription, prompted the authors to conduct a more detailed investigation of the behaviour of genes, microRNAs and proteins, in order to reconstruct in silico the regulatory networks leading to the resistance to trabectedin [37]. The combination of transcriptional, posttranscriptional and translational regulation made it possible to reconstruct putative gene–microRNA–protein circuits with altered trabectedin resistance. The transcriptome and proteome data agreed in recognizing anti-apoptosis and cell cycle regulation as the main biological processes distinguishing the sensitive and resistant sarcoma cell lines. This study illustrates how system biology approaches help clarify complex mechanisms of resistance to anticancer drugs. Although these studies provide interesting new molecular pharmacology data on cellular resistance to trabectedin, they require in vivo validation. This is particular so for trabectedin considering the recent findings by Germano et al. [38] who reported that a fibrosarcoma made resistant to trabectedin in vitro by prolonged drug exposure was no longer resistant when transplanted in mice. When the cells were regrown in vitro they were still resistant to trabectedin, suggesting that the in vivo antitumor activity was not due to a direct cytotoxic effect against cancer cells but was host-mediated. Recent evidence suggests in fact that trabectedin can reduce the number of tumor-associated macrophages (TAM) which have a pro-tumoral effect by secreting growth and angiogenic factors [38]. A decrease in the levels of some cytokines and chemokines was found in tumors of mice treated given therapeutic doses of trabectedin, suggesting that the modification of the tumor micro-environment is an important part of the mechanism of action of this drug.
Clinical implications There are many possible mechanisms of resistance to trabectedin, including overexpression of transport proteins, DNA repair, genetic and epigenetic mechanisms related to DNA www.drugdiscoverytoday.com
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Table 1. Cell lines made resistant to MGBs that bind N3-adenine or N2-guanine. Cell line
Drug
N3-Adenine LOVO/24517 L1210/245171 MCF-7/24517 (1)
TALLIMUSTINE TALLIMUSTINE TALLIMUSTINE
N2-Guanine 402-91/ET Igrov-1/25 ET HCT-116/ER5 SW480/ER0.5 Et-743 resistant CS-1 CS-1/ER CS-1/PR TC/ET 3nM TC/ET 6nM TC/ET 12nM
Fold resistance
Mechanisms involved in the resistance
References
56 48 10
P-gp overexpression P-gp overexpression GSH and unknown
[2] [3] [40]
TRABECTEDIN
10
[36]
TRABECTEDIN TRABECTEDIN TRABECTEDIN TRABECTEDIN TRABECTEDIN ZALYPSIS1 TRABECTEDIN TRABECTEDIN TRABECTEDIN
50 23 10 36 12 20 28 47 102
Defects in NER system (lack of XPG); change of FUS-CHOP promoter targeting P-gp overexpression Defects in NER system (lack of XPG) Defects in NER system (lack of XPG) Reorganization and repartitioning of actin cytoskeleton Overexpression ZNF93 Overexpression ZNF93 P-gp and IGF-1R overexpression P-gp and IGF-1R overexpression P-gp and IGF-1R overexpression
response pathways and transcription regulation. Although the very high level of expression of P-gp was reported to confer resistance to trabectedin, moderate overexpression, commonly observed in clinical tumor biopsies, does not cause significantly reduce the activity. The specific functional inhibition of a transcription factor by trabectedin for some sarcomas that express a fusion gene acting as an aberrant transcription factor explains why the mechanism of resistance of the drug seems to be related to changes in transcription regulation. Whether this also occurs for other tumors remains to be ascertained. The evidence that DNA repair, particularly TC-NER, is involved in the resistance mechanisms to trabectedin is quite strong in many preclinical systems [24,26,27,29], and confirmed in clinical studies [39]. The lack of expression of proteins involved in NER, for example, XPG, was related to mutations or epigenetic silencing [24,36]. A unique peculiar aspect of trabectedin molecular pharmacology is that cells deficient in NER are resistant to the drug, while they are hypersensitive to UV rays and other DNA damaging agents such as Pt complexes. Although the molecular mechanism of this paradoxical form of resistance has not been fully elucidated, it provides an opportunity to exploit collateral sensitivity to Pt complexes in patients treated with trabectedin who have become resistant. Trabectedin has been approved by EMA in Europe for the treatment of ovarian cancer patients who have relapsed six months after Pt-based therapies in combination with pegylated liposomal doxorubicin. After trabectedin treatment, these patients are eligible for other regimes containing Pt complexes that are potentially effective. Therefore the finding that the resistance to trabectedin, arising after prolonged treatment is often associated with a NER defect with a consequential increase in sensitivity to Pt derivatives, is of great clinical importance. Preliminary clinical data suggest that the sequence of trabectedin and carboplatin is in fact very effective, as indicated by 78
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[13] [24] [24] [23] [21] [21] [16] [16] [16]
patients’ overall survival (Callata H et al., abstract 144, 18th International Meeting, ESGO, Liverpool, 2013).
Conclusions Like for most active anticancer drugs, for MGBs too resistance is due to multiple mechanisms (Table 1). Transport and DNA repair pathways are the major ones. For trabectedin, so far the only MGB anticancer drug approved for clinical use, the resistance mechanism is related to transcriptional mechanisms and often to NER deficiency, so resistance might be related to collateral sensitivity to conventional alkylators such as Pt complexes. These data provide a rationale for clinical trials with trabectedin given before conventional DNA damaging agents.
Conflict of interest The authors have no conflict of interest to declare.
Acknowledgement Most of the experimental research performed on MGBs by this laboratory was supported by the Italian Association for Cancer Research (AIRC).
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40.
upon transcription-coupled nucleotide-excision repair. Nat Med 2001;7:961–6. Zewail-Foote M, Hurley LH. Differential rates of reversibility of ecteinascidin 743-DNA covalent adducts from different sequences lead to migration to favored bonding sites. J Am Chem Soc 2001;123:6485–95. Damia G, Silvestri S, Carrassa L, Filiberti L, Faircloth GT, Liberi G, et al. Unique pattern of ET-743 activity in different cellular systems with defined deficiencies in DNA-repair pathways. Int J Cancer 2001;92:583–8. Erba E, Bergamaschi D, Bassano L, Damia G, Ronzoni S, Faircloth GT, et al. Ecteinascidin-743 (ET-743), a natural marine compound, with a unique mechanism of action. Eur J Cancer 2001;37:97–105. Tavecchio M, Simone M, Erba E, Chiolo I, Liberi G, Foiani M, et al. Role of homologous recombination in trabectedin-induced DNA damage. Eur J Cancer 2008;44:609–18. Tavecchio M, Natoli C, Ubezio P, Erba E, D’Incalci M. Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach. Cell Prolif 2007;40:885–904. Falcetta F, Lupi M, Colombo V, Ubezio P. Dynamic rendering of the heterogeneous cell response to anticancer treatments. PLoS Comput Biol 2013;9:e1003293. Sabatino MA, Marabese M, Ganzinelli M, Caiola E, Geroni C, Broggini M. Down-regulation of the nucleotide excision repair gene XPG as a new mechanism of drug resistance in human and murine cancer cells. Mol Cancer 2010;9:259. Grosso F, Jones RL, Demetri GD, Judson IR, Blay JY, Le Cesne A, et al. Efficacy of trabectedin (ecteinascidin-743) in advanced pretreated myxoid liposarcomas: a retrospective study. Lancet Oncol 2007;8:595–602. Grosso F, Sanfilippo R, Virdis E, Piovesan C, Collini P, Dileo P, et al. Trabectedin in myxoid liposarcomas (MLS): a long-term analysis of a single-institution series. Ann Oncol 2009;20:1439–44. Forni C, Minuzzo M, Virdis E, Tamborini E, Simone M, Tavecchio M, et al. Trabectedin (ET-743) promotes differentiation in myxoid liposarcoma tumors. Mol Cancer Ther 2009;8:449–57. Di Giandomenico S, Frapolli R, Bello E, Uboldi S, Licandro SA, Marchini S, et al. Mode of action of trabectedin in myxoid liposarcomas. Oncogene 2013. http://dx.doi.org/10.1038/onc.2013.462, http://nature.com. This paper shows that the selective mechanism of action of trabectedin in myxoid liposarcomas is related to its specific effect on inhibition of the trans-activating function of FUS-CHOP chimera.. Uboldi S, Bernasconi S, Romano M, Marchini S, Fuso Nerini I, Damia G, et al. Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents. Int J Cancer 2012;131:59–69. This paper shows that resistance of myxoid liposarcoma cells is related to alterations of regulation of gene transcription. Uboldi S, Calura E, Beltrame L, Fuso Nerini I, Marchini S, Cavalieri D, et al. A systems biology approach to characterize the regulatory networks leading to trabectedin resistance in an in vitro model of myxoid liposarcoma. PLoS ONE 2012;7:e35423. By a system biology approach that combines transcriptional, post-transcriptional and translational regulation, the regulation networks leading to trabectedin resistance that involves anti-apoptosis and cell cycle regulation were elucidated in an in vitro model of myxoid liposarcoma. Germano G, Frapolli R, Belgiovine C, Anselmo A, Pesce S, Liguori M, et al. Role of macrophage targeting in the antitumor activity of trabectedin. Cancer Cell 2013;23:249–62. This paper shows that the antitumor activity of trabectedin is partially related to its ability to decrease the tumor associated macrophages and to down-regulate the production of cytokines, chemokines and angiogenic factors.. ¨ ffski P, Taron M, Jimeno J, Grosso F, Sanfilipio R, Casali PG, et al. Scho Predictive impact of DNA repair functionality on clinical outcome of advanced sarcoma patients treated with trabectedin: a retrospective multicentric study. Eur J Cancer 2011;47:1006–12. Salvatore C, Bigioni M, Iafrate EM, Cianfriglia M, Manzini S. FCE 24517resistant MCF-7 human breast cancer cell line: selection and characterization. Anticancer Drugs 1999;10:663–9.
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Editors-in-Chief Kelvin Lam – Simplex Pharma Advisors, Inc., Arlington, MA, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Drug resistance
Resistance to minor groove binders Benedetta Colmegna, Sarah Uboldi, Eugenio Erba, Maurizio D’Incalci* Department of Oncology, Laboratory of Cancer Pharmacology, IRCCS – Istituto di Ricerche Farmacologiche ‘Mario Negri’, via La Masa 19, Milan 20156, Italy
In this paper multiple resistance mechanisms to minor groove binders (MGBs) are overviewed. MGBs with antitumor properties are natural products or their derivatives and, as expected, they are all substrates of P-glycoprotein (P-gp). However, a moderate expression of P-gp does not appear to reduce the sensitivity to trabectedin, the only MGB so far approved for clinical use. Resistance to this drug is often related to transcriptional mechanisms and to DNA repair pathways,
Section editors: Ju¨rgen Moll – Boehringer-Ingelheim, Vienna, Austria. Gemma Texido´ – Nerviano Medical Sciences S.r.l, Nerviano, Italy. them have been reported. Here we review the knowledge of the resistance mechanisms to the MGBs with particular focus on trabectedin, which is approved for clinical use in Europe and several other countries for the therapy of soft tissue sarcomas and ovarian cancer.
particularly defects in transcription-coupled nucleotide excision repair (TC-NER). Therefore tumors resistant to trabectedin may become hypersensitive to UV rays and other DNA damaging agents acting in the major groove, such as Platinum (Pt) complexes. If this is confirmed in clinic, that will provide the rationale to combine trabectedin sequentially with Pt derivates. Introduction The discovery that many tumors have defects in DNA repair mechanisms and in checkpoints of the cell cycle explains why DNA-damaging agents have high antitumor selectivity. Minor groove alkylating agents are a distinct class of anticancer drugs, derived originally from natural products that interact with DNA, unlike conventional alkylators, explaining why their spectra of activity and resistance patterns do not overlap. The DNA minor groove binders (MGBs) can be divided into two classes: compounds that covalently bind to adenines at the N3 position in adenine-thymine (AT)-rich sequences of DNA and compounds that bind to guanines at the N2 position. Both have been extensively investigated in preclinical systems and several mechanisms of resistance to *Corresponding author.: M. D’Incalci ([email protected]) 1740-6749/$ ß 2014 Elsevier Ltd. All rights reserved.
Mechanisms of resistance of minor groove binders that alkylate N3 of adenines The chemical structures of the most representative antitumor agents that act by binding adenines at N3 position in the minor groove of DNA are shown in Fig. 1. Tallimustine, N-deformylN-4-N,N-bis(2-chloroethylamino)benzoyl distamycin-A, like the parent compound distamycin-A [1], binds preferentially to (AT)-rich sequences in the minor groove of B-DNA. Both distamycin-A and tallimustine in vitro inhibit the binding of transcription factors that recognize cis-elements rich in AT, whereas they have no effect on the binding of transcription factors that recognize guanine-cytosine-rich boxes. However, tallimustine had a potent cytotoxic and antitumor effect against several cancer cell lines grown in vitro and in mouse tumor models, whereas distamycin was inactive. Thus the alkylation of adenine caused by tallimustine, but not by distamycin, is crucial for its antitumor activity. Resistance to tallimustine was investigated in cell lines made resistant by prolonged exposure to this compound. In some cases the resistance was related to lower intracellular retention of tallimustine, because of overexpression of the multi-drug resistance (MDR1) gene encoding for P-gp (P-170) involved in drug efflux from the cell. In these cases the resistance was reversed by P-gp inhibitors [2]. In other cases
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Figure 1. MGBs that bind N3-adenine of DNA. Panel A: the chemical structures of distamycin and two derivatives, tallimustine and brostallicin. Panel B: the structures of the natural product CC-1065 and derivatives containing cyclopropylpirrolo(e)indolone such as adozelesin, carzelesin and bizelesin.
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resistance appeared to be associated with an increase in glutathione (GSH), although blocking the synthesis of GSH by buthionine sulphoximine (BSO) only partially reversed the resistance, suggesting that other mechanisms were involved. Some studies with a murine leukemia cell line resistant to tallimustine, L1210/24517 [3] showed cross-resistance to other MGBs like CC-1065, which can bind adenine N-3 in DNA with high sequence-specificity [4,5]. This cell line was also crossresistant to distamycin which is toxic only at a high concentration without producing any alkylation in DNA, thus excluding that the cross-resistance between tallimustine and CC1065 was due to efficient removal of the alkylated bases by DNA repair mechanisms. Studies in a cell line resistant to Hoechst 33342 and distamycin-A [6], which bind the DNA minor groove without forming stable adducts in DNA, indicated an enhanced ability to remove ligand molecules from cellular DNA through a still poorly understood pathway that can be blocked by topoisomerase II poisons. This invites speculation on the existence of a repair system that recognizes the change in DNA structure produced by MGBs, that might be important for the sensitivity to these agents. Mismatch repair deficiency has been reported in the resistance to compounds acting by selectively alkylating adenine N3 in AT-rich sequences. This was demonstrated in a HCT-116 cell line with a mutation of hMLH1 gene that was resistant to tallimustine, carzelesin and CC-1065 [7]. When hMLH1 was restored by chromosome 3 transfer, the resistance was lost. Further support of the involvement of mismatch repair in the sensitivity to N3 adenine alkylators is the finding that two ovarian carcinoma cell lines, A2780/CP70 and A2780/MCP-1, resistant to cisplatin because of a defect in hMLH1, were resistant to MGBs that alkylate adenines N3. This mechanism does not apply to the bromoacryloyl derivative of distamycin A, Brostallicin, that was reported to retain sensitivity in DNA mismatch repair-deficient tumor cells [8]. Brostallicin was also different from the other distamycins, being more cytotoxic against cells with a high GSH content, a potentially interesting property considering there are cases in which the low sensitivity to DNA-interacting compounds is related to high expression of the glutathione S-transferase (GST)/GSH system. Although several compounds that bind selectively to N3 of adenines, such as tallimustine, carzelesin, adozelesin and bizelesin [9,10], have been tested in initial clinical investigations, none has been developed further, mainly because of their severe bone marrow toxicity and low therapeutic index [11,12]; therefore no data are available on the clinical relevance of the findings related to resistance mechanisms.
Mechanisms of resistance to minor groove binders that alkylate N2 of guanine Unlike the N3-adenine alkylators, it appears that compounds that selective alkylate guanine N2 in the minor groove of DNA, like trabectedin, can be safely used at doses effective
Drug Discovery Today: Technologies | Drug resistance
against at least some malignancies such as soft tissue sarcomas and ovarian cancer. Since to the best of our knowledge trabectedin is the only MGB that specifically alkylates N2 of guanine that is approved for clinical use, so we will overview the literature on this compound, although some other compounds will be mentioned too. Exposing Igrov-1 human ovarian cancer cells to increasing concentrations of trabectedin for short or long times, Erba et al. [13] obtained Igrov-1 sublines very resistant to trabectedin (i.e. approximately 50-fold), which overexpress P-gp; there was no increase in other multi-drug resistance-related proteins, such as MRP or LRP. Beumer et al. [14] have unambiguously shown that trabectedin is a P-gp substrate by investigating the vectorial transport in LLC-PK1 pig kidney, Madine-Darby canine kidney (MDCK) subclones and in the murine mdr1a and/or human MDR1 transfected subclones. The LLC-PK1 cells transfected with either mdr1a or MDR1 genes showed much higher trabectedin transport out of the cells than control untransfected cell lines. The same results were obtained by transfecting MDCK cells. In both models the P-gp transport system was blocked by LY335979 (a P-gp inhibitor) only in the MDR1 transfected cells, not in the control lines. The same authors examined a series of cell lines with high or moderate P-gp expression levels and found that trabectedin displayed the typical MDR phenotype only in highly P-gp expressing cells, but not in lines with moderate expression levels, more similar to clinical samples. In keeping with these observations were the reports by Scotlandi et al. [15,16] who found trabectedin was effective in a series of osteosarcoma and Ewing sarcoma cell lines resistant to doxorubicin, expressing moderate levels of P-gp. Since trabectedin is not a preferential substrate of P-gp, cell lines with a constitutive moderate expression of the MDR1 gene (sensitive to trabectedin) appeared resistant to anthracyclines or taxanes, the major substrates of P-gp. Since in some cases the MDR1 gene can be transcriptionally induced it could be interesting to investigate whether trabectedin inhibits this mechanism. Trabectedin can inhibit the transcription of some rapidly inducible genes such as heat shock proteins [17] or cell cycle genes [18]. Elegant in vitro experiments by Jin et al. [19] showed that trabectedin blocks the activation of transcription of the MDR1 gene promoter, induced by histone deacetylase inhibitors. In line with this observation Kanzaki et al. [20] reported that exposure to trabectedin caused downregulation of the MDR1 gene, enhancing the cytotoxicity and cellular accumulation of doxorubicin and vincristine in cell lines overexpressing P-gp. Duan et al. [21] investigated two chondrosarcoma cell lines made resistant to trabectedin or to Zalypsis1 – which has some structural analogies to trabectedin (Fig. 2) – exposing the cells to increasing concentrations. Analysis of differentially expressed genes showed that some zinc finger proteins, such www.drugdiscoverytoday.com
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HO NH
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Figure 2. MGBs that bind N2-guanine of DNA. The chemical structure of trabectedin and its derivatives Zalypsis1 and lurbinectedin.
as ZNF93 and ZNF43, were over-expressed in the resistant lines; silencing these genes reversed the resistance to trabectedin or Zalypsis1, suggesting their role in the resistance mechanisms. Marchini et al. [22] compared the gene expression profiles of trabectedin-resistant cell lines derived from an ovarian cancer or a chondrosarcoma cell line. Genes involved in the cytoskeleton dynamics such as g-actin, Rho E, Rac-b or other small GTPases were downregulated much more in the resistant than in the sensitive clones. In addition, in the two cell systems, the a-2 chain of collagen IV gene was down-regulated in the resistant line. These findings were consistent with data obtained in other cellular systems [23] suggesting extracellular matrix proteins are involved in the resistance to trabectedin. Takebayashi [24] isolated a HCT116 cell line resistant to trabectedin (HCT116/ER5); evidence that this line was also hypersensitive to UV radiation stimulated the investigation of the DNA repair pathways potentially involved. They found that functional XPG protein was not expressed in HCT116/ER5 cells because of a point mutation in the XPG gene, which resulted in a premature termination codon. These results are in keeping with reports [25–28] that cells deficient in NER, which are generally more sensitive to conventional alkylating agents and Pt complexes, are resistant to trabectedin. 76
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Tavecchio et al. [29] used an isogenic NER-proficient cellular system (CHO-AA8) and an NER-deficient one (CHO-UV-96) lacking functional ERCC-1, which are hypersensitive to UV light, and a UV-96 cell clone stably transfected with ERCC1(ERA-5) that shows the same sensitivity to UV light as AA8 cells. UV-96 cells were four times as resistant to trabectedin than AA8 or ERA-5 cells. Trabectedin different induced cell cycle perturbations in NER-deficient and NER-proficient UV-96 cells, even when treated with equitoxic drug concentrations (i.e. four times higher for NER-deficient cells). Biparametric flow cytometric methods indicated that trabectedin caused a block of cells in G2-M in NER-proficient AA8 or ERA5 cells and not in NER-deficient UV-96 cells. Applying a computer simulation method it was found that the dynamics of the cell cycle perturbations were complex, with blocks in both G1 and G2 and a delayed S phase [30]. In cells with functional NER, exit from G1 block was faster than in NER-deficient cells, then cells progressed slowly through S phase and were subsequently blocked irreversibly in G2-M phase, indicating they were unable to repair the damage [29]. These studies indicate that the resistance to trabectedin is associated with a different cell response from trabectedin-induced DNA damage, resulting in different cell cycle perturbations in NER-deficient or -proficient cells.
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Resistance mechanism to trabectedin related to the absence of NER has been observed in several cell lines with mutations of one of the proteins involved in NER, such as XPG or XPD or XPA [24]. Is this pattern of resistance unique to trabectedin? Another example has been reported for an anthracycline derivative that probably acts by a different mechanism from doxorubicin [31]. In a cell line made resistant to nemorubicin (30 -deamino-30 (2-(S)-methoxy-4-morpholinyl), a doxorubicin derivative that forms a metabolite, 30 -deamino-30 ,40 -anhidro-(20 (S)-methoxy-30 (R)-hydroxy-40 -morpholinyl) doxorubicin, which is 1000 times more potent than doxorubicin, the resistance appears to be related to the lack of XPG expression. Interestingly a cell line that did not express XPG because of hypermethylation of its promoter was cross-resistant with trabectedin, indicating that there may be an epigenetic mechanism of resistance to the drug [31]. Several laboratories have shown that cells made resistant to trabectedin by prolonged exposure often become hypersensitive to UV light, because they have a defect in the NER pathway, the main mechanism of repair of UV-induced DNA damage. Recently D’Incalci et al. (D’Incalci et al., abstract C93, AACRNCI-EORTC, Boston, October 2013) isolated a myxoid liposarcoma and an ovarian cancer cell line resistant to trabectedin, both lacking XPG expression. As expected, the resistant cells were hypersensitive to UV light. Since several reports had indicated that the sensitivity to cisplatin was increased in NERdeficient cell lines [26], the response to cisplatin was compared in the resistant and sensitive cell lines. Both trabectedin-resistant cell lines that do not express XPG were much more sensitive to cisplatin than the parental cell lines. These in vitro findings have been confirmed in vivo in immunodeficient mice. Xenografts from the trabectedinresistant ovarian cancer cell line were more sensitive to cisplatin, T/C 18% (where T and C are respectively the mean tumor weight of cisplatin-treated and control groups), than the parental cell line, T/C 64.2% (unpublished data obtained in our laboratory). These results provide the rationale for sequential use of trabectedin and Pt complexes in ovarian cancer (see the section ‘Clinical implications’). Although trabectedin was effective in several types of soft tissue sarcoma the most sensitive was certainly myxoid liposarcoma, in which the rate of objective responses as more than 50% in patients resistant to doxorubicin and ifosfamide [32,33]. The mechanism of sensitivity appears to be related to the ability of trabectedin to block the transactivating function of the fusion gene product FUS-CHOP, that is the pathogenic lesion of this type of liposarcoma, demonstrated both in vitro [34] and in vivo [35] in tumor biopsies from xenografts with myxoid liposarcomas. Uboldi et al. [36] isolated and characterised a trabectedin-resistant myxoid liposarcoma cell line, 402-91/ET, that appeared to be resistant to trabectedin by more than one mechanism. A mutation of XPG was one of the
Drug Discovery Today: Technologies | Drug resistance
mechanisms, also associated with increased sensitivity to UV radiation. However, when the 402-91/ET cell line was transfected with an expression vector encoding for XPG cDNA, the sensitivity to UV light was markedly reduced, with only partial reversion of the resistance to trabectedin, suggesting that other mechanisms play a role in the resistance. Chromatin-Immunoprecipitation (ChIP) assays showed that in the resistant cell line the FUS-CHOP was no longer able to transactivate its target promoters, suggesting that the mechanism of resistance was related to the change in the target of the drug action. This cell line showed cross-resistance to lurbinectedin and Zalypsis1 (Fig. 2) and collateral sensitivity to methylating agents, due to the lack of expression of the repair protein O6 methyl-guanineDNA methyl transferase (MGMT). In 402-91/ET cells, but not in the parental cell line, the MGMT gene promoter was highly methylated. The peculiar mechanism of resistance to trabectedin, involving regulation of transcription, prompted the authors to conduct a more detailed investigation of the behaviour of genes, microRNAs and proteins, in order to reconstruct in silico the regulatory networks leading to the resistance to trabectedin [37]. The combination of transcriptional, posttranscriptional and translational regulation made it possible to reconstruct putative gene–microRNA–protein circuits with altered trabectedin resistance. The transcriptome and proteome data agreed in recognizing anti-apoptosis and cell cycle regulation as the main biological processes distinguishing the sensitive and resistant sarcoma cell lines. This study illustrates how system biology approaches help clarify complex mechanisms of resistance to anticancer drugs. Although these studies provide interesting new molecular pharmacology data on cellular resistance to trabectedin, they require in vivo validation. This is particular so for trabectedin considering the recent findings by Germano et al. [38] who reported that a fibrosarcoma made resistant to trabectedin in vitro by prolonged drug exposure was no longer resistant when transplanted in mice. When the cells were regrown in vitro they were still resistant to trabectedin, suggesting that the in vivo antitumor activity was not due to a direct cytotoxic effect against cancer cells but was host-mediated. Recent evidence suggests in fact that trabectedin can reduce the number of tumor-associated macrophages (TAM) which have a pro-tumoral effect by secreting growth and angiogenic factors [38]. A decrease in the levels of some cytokines and chemokines was found in tumors of mice treated given therapeutic doses of trabectedin, suggesting that the modification of the tumor micro-environment is an important part of the mechanism of action of this drug.
Clinical implications There are many possible mechanisms of resistance to trabectedin, including overexpression of transport proteins, DNA repair, genetic and epigenetic mechanisms related to DNA www.drugdiscoverytoday.com
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Table 1. Cell lines made resistant to MGBs that bind N3-adenine or N2-guanine. Cell line
Drug
N3-Adenine LOVO/24517 L1210/245171 MCF-7/24517 (1)
TALLIMUSTINE TALLIMUSTINE TALLIMUSTINE
N2-Guanine 402-91/ET Igrov-1/25 ET HCT-116/ER5 SW480/ER0.5 Et-743 resistant CS-1 CS-1/ER CS-1/PR TC/ET 3nM TC/ET 6nM TC/ET 12nM
Fold resistance
Mechanisms involved in the resistance
References
56 48 10
P-gp overexpression P-gp overexpression GSH and unknown
[2] [3] [40]
TRABECTEDIN
10
[36]
TRABECTEDIN TRABECTEDIN TRABECTEDIN TRABECTEDIN TRABECTEDIN ZALYPSIS1 TRABECTEDIN TRABECTEDIN TRABECTEDIN
50 23 10 36 12 20 28 47 102
Defects in NER system (lack of XPG); change of FUS-CHOP promoter targeting P-gp overexpression Defects in NER system (lack of XPG) Defects in NER system (lack of XPG) Reorganization and repartitioning of actin cytoskeleton Overexpression ZNF93 Overexpression ZNF93 P-gp and IGF-1R overexpression P-gp and IGF-1R overexpression P-gp and IGF-1R overexpression
response pathways and transcription regulation. Although the very high level of expression of P-gp was reported to confer resistance to trabectedin, moderate overexpression, commonly observed in clinical tumor biopsies, does not cause significantly reduce the activity. The specific functional inhibition of a transcription factor by trabectedin for some sarcomas that express a fusion gene acting as an aberrant transcription factor explains why the mechanism of resistance of the drug seems to be related to changes in transcription regulation. Whether this also occurs for other tumors remains to be ascertained. The evidence that DNA repair, particularly TC-NER, is involved in the resistance mechanisms to trabectedin is quite strong in many preclinical systems [24,26,27,29], and confirmed in clinical studies [39]. The lack of expression of proteins involved in NER, for example, XPG, was related to mutations or epigenetic silencing [24,36]. A unique peculiar aspect of trabectedin molecular pharmacology is that cells deficient in NER are resistant to the drug, while they are hypersensitive to UV rays and other DNA damaging agents such as Pt complexes. Although the molecular mechanism of this paradoxical form of resistance has not been fully elucidated, it provides an opportunity to exploit collateral sensitivity to Pt complexes in patients treated with trabectedin who have become resistant. Trabectedin has been approved by EMA in Europe for the treatment of ovarian cancer patients who have relapsed six months after Pt-based therapies in combination with pegylated liposomal doxorubicin. After trabectedin treatment, these patients are eligible for other regimes containing Pt complexes that are potentially effective. Therefore the finding that the resistance to trabectedin, arising after prolonged treatment is often associated with a NER defect with a consequential increase in sensitivity to Pt derivatives, is of great clinical importance. Preliminary clinical data suggest that the sequence of trabectedin and carboplatin is in fact very effective, as indicated by 78
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[13] [24] [24] [23] [21] [21] [16] [16] [16]
patients’ overall survival (Callata H et al., abstract 144, 18th International Meeting, ESGO, Liverpool, 2013).
Conclusions Like for most active anticancer drugs, for MGBs too resistance is due to multiple mechanisms (Table 1). Transport and DNA repair pathways are the major ones. For trabectedin, so far the only MGB anticancer drug approved for clinical use, the resistance mechanism is related to transcriptional mechanisms and often to NER deficiency, so resistance might be related to collateral sensitivity to conventional alkylators such as Pt complexes. These data provide a rationale for clinical trials with trabectedin given before conventional DNA damaging agents.
Conflict of interest The authors have no conflict of interest to declare.
Acknowledgement Most of the experimental research performed on MGBs by this laboratory was supported by the Italian Association for Cancer Research (AIRC).
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