The Future of Antisense Therapy: Combination with Anticancer Treatments

The future of antisense therapy: combination with anticancer treatments

Annamaria Biroccio1, Carlo Leonetti1 and Gabriella Zupi*,1

1Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy

The current direction in cancer research is rational drug et al., 2000). Although inhibition of gene expression design, which is based on the evidence that transformed seems to be the predominant biological mechanism of cells are characterized by alterations of genes devoted to ASOs, nonantisense effects, which may also contribute the regulation of both cell proliferation and apoptosis. A to the overall response, have been described. Nonanti- variety of approaches have been carried out to develop sense effects include potential immune stimulation by new agents selective for cancer cells. Among these, sequences such as CpG motifs or G quarter (Wooldridge antisense oligonucleotides (ASOs) are one of such class et al., 1997). of new agents able to inhibit specifically the synthesis of a The use of ASOs for inhibition of gene expression particular cancer-associated protein by binding to protein- raises several problems, including their possible degra- encoding RNA, thereby preventing RNA function. In the dation by nucleolytic enzymes and the difficulty of past decade, several ASOs have been developed and tested ASOs to cross cell membrane. To avoid problems in preclinical and clinical studies. Many have shown resulting from sensitivity to nuclease, various modifica- convincing in vitro reduction in target gene expression and tions to the oligonucleotide backbone have been made. promising activity against a wide variety of tumors. One of the earliest modifications brought to the ASOs However, because of the multigenic alterations of tumors, chain was the replacement of one of the nonbridging the use of ASOs as single agents does not seem to be oxygen atoms at each phosphorus by a methyl group, effective in the treatment of malignancies. Antisense thus producing a methylphosphonate (Miller and Ts’o, therapy that interferes with signaling pathways involved 1987; Miller, 1992). This substitution renders the ASO in cell proliferation and apoptosis are particularly molecules resistant to nuclease, but unfortunately it also promising in combination with conventional anticancer produces additional changes to the chemical nature of treatment. An overview of the progress of ASOs used in the molecule that might reduce its antisense activity. combination therapy is provided. Another important backbone modification was the Oncogene (2003) 22, 6579–6588. doi:10.1038/sj.onc.1206812 substitution of a sulfur atom for oxygen atoms at each phosphorus, generating a phosphorothioate (PS ASOs). Keywords: antisense; molecular targets; proliferation; This kind of substitution renders the ASO molecules apoptosis; combination therapy negatively charged compounds, highly soluble and relatively resistant to nuclease degradation (Stec et al., 1984). However, preclinical and clinical studies showed Antisense therapy: rationale and limitations that PS ASOs may have nonspecific effects, either due to nonsequence specific binding or activation of nonspe- The use of antisense oligonucleotides (ASOs) as cific mechanisms. In fact, it has been demonstrated that therapeutic agents in oncology has been proposed for PS ASOs are able to bind to heparin-binding proteins, longer than 10 years. ASOs are short single-stranded such as bFGF, PDGF, VEGF and their receptors DNA molecules, complementary to an RNA strand, (Fennewald and Rando, 1995; Guvakova et al., 1995). capable of specifically targeting and inhibiting the Under certain circumstances, PS ASOs are able to mRNA of a chosen gene. Once bound to the RNA activate SP1 transcription factor and their degradation target, its inhibition occurs through various mechan- products can affect cell proliferation and differentiation isms, including modulation of splicing and inhibition of (Perez et al., 1994). PS ASOs can also enhance NK cell protein translation. However, antisense-mediated inhi- activity, leading to biological effects combining bition of gene expression is mainly due to the recruit- sequence and nonsequence specific mechanisms (Boggs ment of endogenous RNase H, an ubiquitous et al., 1997). Notwithstanding the existence of endonuclease that recognizes the mRNA–oligos duplex nonspecific interaction, PS ASOs have been successfully and cleaves the RNA strand, leaving intact the used in a large number of experiments designed to antisense, which can bind other molecules of the RNA downregulate gene expression, and they have been and target (Baker and Monia, 1999; Crooke, 1999; Koller are currently used in several clinical therapeutic trials. A different class of modified oligos employed in clinical *Correspondence: G Zupi, Experimental Chemotherapy Laboratory, use is represented by the mixed backbone (MBO), Regina Elena Cancer Institute, Via delle Messi d’Oro 156, which contains phophorothioates and segments of Rome 00158, Italy; E-mail: [email protected] modified oligodeoxynucleotides or oligoribonucleotides. Antisense in combination therapy A Biroccio et al 6580 In particular, the incorporation of 20-O-methyloligor- targeting cancer-related genes that interfere with both ibonucleosides at the 50 and 30 ends of PS ASOs of these pathways are also shown in the figure. improves duplex affinity and also increases stability to This review summarizes the main promising results nuclease degradation (Agrawal and Zhao, 1998). These achieved by ASOs treatment used in combination with chemical modifications reduce toxic effects, while the conventional anticancer drugs. In fact, considering the antitumoral efficacy is maintained if compared to PS multigenic defects of human tumors, the combination of ASOs. ‘agents’, targeting specific genes involved in tumor The efficacy of ASOs in inhibiting gene expression progression, with antineoplastic drugs represents a also depends on their ability to penetrate targeted cells. promising strategy for improving cancer treatment. Considering the polyanionic features of ASOs, the Table 1 reports some preclinical studies of ASOs used difficulty of these compounds to cross the plasma- in combination. membrane passively can be understood. The uptake of ASOs occurs through active transport, which, in turn, depends on the temperature (Yakubov et al., 1989), the Antisense oligonucleotides against prosurvival-associated structure and the concentration of oligos (Vlassov et al., targets in combination therapy 1994). Several approaches have been explored to enhance cellular uptake and permeability of ASOs in Bcl-2 family order to increase their therapeutic efficacy, including conjugation to polycations, targeting cell surface recep- Bcl-2 and Bcl-XL proteins are inhibitors of the tors, coupling to cholesterol, encapsulation into lipid mitochondrial apoptosis pathway and exert their action particles such as microspheres and liposomes. The use of by blocking their proapoptotic counterparts, including vectors in antisense drug delivery in vivo seems to Bid and Bax, thereby preventing the release of improve their biological effects. cytochrome c and activation of caspases. Enthusiasm for antisense approach derives from the The bcl-2 gene has been the first negative regulator of identification of several cancer-associated proteins, apoptosis to be identified. It derives from 14–18 which can represent potential targets for a selective chromosomal translocation that places the bcl-2 gene anticancer therapy with less toxic side effects than under the control of immunoglobulin heavy chain, conventional chemotherapy. The preclinical develop- leading to its deregulated expression (Tsujimoto et al., ment of several antisense compounds targeting cancer- 1985; Cleary et al., 1986). Increased expression of Bcl-2 related genes has proceeded very rapidly. Figure 1 shows is found in a variety of human tumors, including the key signal pathways that regulate the proliferation lymphoma, melanoma, lung, colorectal and breast and the life/death decision in cancer cells. ASOs carcinomas (Reed, 1995). Several results demonstrated

Figure 1 Schematic representation of the signal pathways regulating the proliferation and life/death decision in cancer cells. The orange arrows indicate some of the most promising ASO targeted key proteins in pathways

Oncogene Antisense in combination therapy A Biroccio et al 6581 Table 1 Preclinical studies of antisense oligonucleotides in combination therapy on human tumors Target gene Biological end point Tumor type Combination with Combination with inhibitors of conventional therapy molecular targets bcl-2 Apoptosis Lymphoma, melanoma, DTIC, paclitaxel, DOX, MAPK inhibitor, PKA-I and prostate, gastric and cyclophosphamide, CDDP PLK1 antisense breast cancers bcl-2/bcl-xL Apoptosis Breast and colon carcinomas DOX, paclitaxel, PKA-I antisense, IRESSA cyclophosphamide Clusterin Apoptosis Prostate, bladder and renal CDDP, paclitaxel, MTX, ND cancers radiation, androgen ablation c-myb Proliferation Colon cancer, leukemia CDDP ND PKA-I Proliferation Colon, breast, lung and Taxol, irinotecan IRESSA, bcl-2 and bcl-2/bcl-xL prostate cancers antisense, hydroxycamptothecin c-myc Proliferation/apoptosis Melanoma CDDP ND mdm2 Proliferation/apoptosis Prostate, breast and Irinotecan, 5-FU, ND colon cancers paclitaxel, CDDP, DOX

PKA, protein kinase type A; DTIC, dacarbazine; CDDP, cisplatin; DOX, doxorubicin; MTX, mitroxantrone, 5-FU, 5-fluorouracil; MAPK, mitogen-activated protein kinase; PLK1, proliferation-associated polo-like kinase 1; ND, not done that overexpression of Bcl-2 inhibits apoptosis induced marked inhibition of tumor growth when compared by antineoplastic drugs, radiation and other damaging both to untreated mice and to mice treated with agents (Reed, 1994; Chao and Korsmeyer, 1998). Based control sequences. The analysis of Bcl-2 protein on these observations, ASOs targeting bcl-2 have been expression and apoptosis in tumor sections demon- used. Several studies have been performed by using the strates that the efficacy of G3139is associated with bcl-2 ASOs produced by Genta (G3139) either as single the reduction of Bcl-2 protein and with an increased agent or in combination with chemotherapy. G3139is number of apoptotic cells. Moreover, G3139enhances an 18-base PS ASO complementary to the first six the efficacy of dacarbazine (DTIC). In fact, while codons of the bcl-2 mRNA. Preclinical experiments treatment with DTIC alone reduces the tumor mass performed in B-cell lymphoma, exhibiting the t(14;18) by two-third if compared to untreated mice, the translocation and overexpressing Bcl-2, demonstrated combination with G3139leads to a complete regression that the treatment with G3139produces a significant of tumor in 10 out of 13 treated animals. The reduction of cell growth in vitro, compared with chemosensitizing effect produced by G3139in the untreated, sense- or nonsense-treated controls. On the different tumor histotypes appears to be a bcl-2-specific contrary, G3139does not produce any effect on viability antisense effect that is independent of immune stimula- either in normal fibroblasts not expressing Bcl-2 or in tion, as G3139and its immune-silent counterpart cause lymphoblastoid cells expressing Bcl-2, but without the similar effects (Wacheck et al., 2002). On the basis of the t(14;18) translocation. The administration of G3139in above preclinical data, phase I/II clinical studies have mice bearing lymphoma produces an increase in animal been concluded (Jansen et al., 2000). The phase III survival (Cotter, 1997). The combination of antisense randomized trial already started in patients with with a low dose of cyclophosphamide is able to cure the advanced melanoma by using G3139followed by DTIC. majority of treated animals, while chemotherapy alone Additional multicenter trials are ongoing in multiple does not have any significant effect on mice survival myeloma, chronic leukemia and lung cancer (www. (Klasa et al., 2000). Increased sensitivity of cytotoxic genta.com). agents is also observed in other tumor histotypes such as In addition to G3139, other bcl-2 ASOs complemen- breast, prostate, lung and gastric tumors (Zangemeister- tary to the translation initiation site of bcl-2 mRNA or Wittke et al., 1998; Gleave et al., 1999; Chi et al., 2000). targeting the bcl-2 mid-coding region were tested in a In particular, complete tumor regression is observed variety of preclinical models. A synergistic antitumoral when G3139is combined with cisplatin, paclitaxel and effect of bcl-2 antisense therapy combined with some docetaxel. Very interesting results are obtained in anticancer agents has been demonstrated in leukemia melanoma tumors (Jansen et al., 1998), where Bcl-2 and lung cancer cells overexpressing Bcl-2 (Zangemeis- protein is often overexpressed and has been associated ter-Wittke et al., 1998; Konopleva et al., 2000; Miayake with resistance to chemotherapy (Grover and Wilson, et al., 2000a). 1996; Reed, 1999). In particular, the treatment of some Bcl-2 PS ASOs have also been combined with human melanoma lines in vitro with G3139reduces Bcl- molecular therapies aimed at targeting other specific 2 protein expression and induces apoptosis, while no pathways. Synergistic induction of apoptosis has been significant effects are observed after treatment with observed by simultaneous disruption of the Bcl-2 and different control oligos. The administration of G3139in MEK/MAPK pathways (Milella et al., 2002) or Bcl-2 SCID mice bearing melanoma tumors produces a and the proliferation-associated polo-like kinase I

Oncogene Antisense in combination therapy A Biroccio et al 6582 (Elez et al., 2003). These studies provide evidence that a activity in breast and colon carcinomas both in vitro and combination of ASOs against both proliferation and in vivo (Tortora et al., 2003). prosurvival associated targets may represent a promising antitumoral therapeutic approach. Moreover, the Clusterin/Trpm-2 simultaneous suppression of different pathways significantly enhances the cytotoxicity of conventional anti- Clusterin, also known as testosterone-repressed prostate tumoral drugs; therefore it can be used in the treatment message-2 (TRPM-2), is a glycoprotein secreted by a of resistant tumors (Liu and Gazitt, 2003; Pakunlu et al., number of cell types, implicated in a wide variety of 2003; Tanabe et al., 2003). physiological processes, including tissue remodeling, Recent findings obtained by independent research reproduction, lipid transport, complement regulation groups, including ours, demonstrate that Bcl-2 is also and apoptosis. Moreover, since clusterin expression is involved in the angiogenesis process through a mechan- increased in various benign and malignant tissues ism that, at least in part, appears to be independent of its undergoing apoptosis, it has been regarded as a marker antiapoptotic function (Biroccio et al., 2000; Fernandez for cell death. In prostate cancer, experimental and et al., 2001, Iervolino et al., 2002). Based on these clinical studies suggest that clusterin increases after observations, ASOs targeting bcl-2 might provide a therapeutic cell death signals, such as androgen ablation therapeutic approach combining proapoptotic and or chemotherapy, and has a protective role against antiangiogenic activity. apoptotic cell death. Therefore, clusterin PS ASOs were Alternative splicing of bcl-x pre-mRNA leads to two designed to reduce mRNA and protein expression, thus distinct mRNAs: the antiapoptotic bcl-xL and the delaying tumor progression and enhancing chemother- proapoptotic variant bcl-xs. Recently, bcl-x PS ASOs, apy-induced apoptosis. Systemic administration of targeting regions shared by both splicing variants, have clusterin PS ASOs in mice bearing Shionogi tumors been used in malignant cells. All these oligos induce after castration leads to an earlier onset of apoptosis and apoptosis in various cancer cells and sensitize tumors to to a complete tumor regression, as well as a significant chemotherapy (Lebedeva et al., 2000; Leech et al., 2000; delay of emergence of androgen-independent recurrent Simoes-Wust et al., 2000). However, since the cross- tumors compared to control treatment (Liu and Gazitt, specificity for the proapoptotic bcl-xs could be undesir- 2003). However, although treatment with single-agent able, bcl-x PS ASOs designed to shift the splicing clusterin PS ASOs has no effect on prostate, renal and pattern of bcl-x pre-mRNA from the antiapopotic to the urothelial tumor growth, clusterin PS ASOs synergisti- proapoptotic variant, or PS ASOs targeting a cally enhance the effect of cytotoxic chemotherapy. In sequence in the bcl-x that is specific for bcl-xL, were particular, combined treatment of mice bearing andro- tested. Antisense-mediated shift of bcl-x pre-mRNA gen-independent prostate tumors with clusterin PS splicing is effective in sensitizing cells to apoptosis ASOs and paclitaxel inhibits tumor growth if compared and in enhancing the efficacy of standard anticancer to treatment with both agents alone, indicating that regimens (Taylor et al., 1999; Mercatante et al., 2002). clusterin PS ASOs may be useful in enhancing the effects Similarly, specific downregulation of Bcl-xL (4259) in of cytotoxic chemotherapy in hormone-refractory pros- different tumor histotypes leads to activation of tate cancer (Miayake et al., 2000c, d). Synergistic apoptosis, decreases cellular proliferation rate and chemosensitization and inhibition of tumor growth are resistance to cytotoxic chemotherapeutic agents observed following treatment with clusterin PS ASOs (Leech et al., 2000; Simoes-Wust et al., 2000; used in combination either with radiation or chemother- Heere-Ress et al., 2002; Vilenchik et al., 2002). Based apy in prostate, bladder and renal cancer (Miayake et al., on some evidences supporting the distinct biologic roles 2001; Zellweger et al., 2001a, 2002). Characteristic of Bcl-2 and Bcl-xL proteins in protecting cells from features of apoptotic cell death are observed both in apoptosis, bcl-2 and bcl-XL PS ASOs have also vitro and in vivo after combined treatment of PS ASOs been used in combination, even though several problems with paclitaxel or cisplatin, but not with either agents related to antisense complementarity have been alone. observed (Miayake et al., 2000b). To overcome this Second-generation MBO ASOs, targeting the limitation, a bcl-2/bcl-xL bispecific oligonucleotide translation initiation site of the clusterin gene (4625), which hybridizes to a short region of high (OGX-011), are more effective than conventional PS homology shared by bcl-2 and bcl-xL mRNA, was ASOs in decreasing mRNA and protein expression, generated. These oligos simultaneously inhibit the and they have a significantly longer in vivo tissue expression of both proteins and induce apoptosis in half-life with no additional side effects (Zellweger et al., tumor cells of different histotypes both in vitro and in 2001b). Weekly administration of OGX-011 is equiva- vivo (Zangemeister-Wittke et al., 2000; Gautschi et al., lent to daily treatment with clusterin PS ASOs in 2001; Olie et al., 2002; Jiang et al., 2003). Strong enhancing in vivo paclitaxel efficacy, thus supporting synergistic interactions between oligonucleotide 4625 the use of OGX-011 over PS ASOs by increasing and anticancer drugs were observed in breast carcinoma potency and allowing longer dosing intervals in cells (Simoes-Wust et al., 2002) and the combined clinical trials (Zellweger et al., 2001b). On the basis of inhibition of Bcl-2/Bcl-xL, epidermal growth factor preclinical results, the phase I clinical trial of OGX-011 receptor (EGFR) and protein kinase A (PKA) type I started in patients with prostate cancer (www. causes potent antitumor, apoptotic and antiangiogenic oncogenex.ca).

Oncogene Antisense in combination therapy A Biroccio et al 6583 Antisense oligonucleotides against proliferation- PARP, activation of caspase-3 and, finally, apoptosis led associated targets in combination therapy to combine PKAI and bcl-2 ASOs (Itano et al., 1996; Srivastava et al., 1998, 1999; Yang et al., 1998; Protein kinase A Srivastava et al., 1999). The results show that GEM 231 and the G3139have a marked cooperative effect on PKA is the primary mediator of cAMP action in inducing apoptosis and inhibiting the growth of human mammalian cells. It is composed of two distinct ovarian, breast and colon cancer both in vitro and in vivo isoforms, type I (PKAI) and type II (PKAII), which (Tortora et al., 2001). More recently, the simultaneous differ only in their regulatory subunit, RI and RII, blockade of EGFR, PKAI and bcl-2/bcl-xL proteins by respectively. PKAI is required for G1-S cell cycle Iressa, GEM 231 and 4625 PS ASOs exhibits higher transition and the transduction of mitogenic signals antiapoptotic and antitumoral effect than either agent from different growth factors and hormones. Expression alone or in twin combination (Tortora et al., 2003). of PKAI is increased in most human cancers and its Phase I/II dose-escalation trials of GEM 231 are now overexpression correlates with worse prognosis and ongoing in combination with taxanes (www.hybridon. decreased survival of cancer patients. These observa- com). tions led to the development of various inhibitors of PKAI, including unmodified and PS ASOs that show c-Myb the ability to inhibit PKAI expression specifically. PS ASOs against PKAI cause growth inhibition and The c-myb oncogene encodes a DNA-binding transcrip- differentiation of various cancer cell lines and exhibit tion factor involved in the control of the G1-S antitumor activity in human tumor xenografts (Yoko- transition of the cell cycle. c-Myb protein was first zaki et al., 1993; Cho-Chung, 1997). An improvement of detected in hematopoietic cells, where it regulates cell these effects is observed with the MBO GEM 231. This proliferation and differentiation. c-Myb protein is oligo elicits a better in vivo pharmacokinetic and overexpressed in leukemia cells, colon carcinoma, toxicological profile than PS ASOs, and displays small-cell lung carcinoma, teratocarcinoma and neuro- antitumor activity in different human tumor xenografts blastoma (Alitalo et al., 1984; Griffin and Baylin, 1985; when given orally to mice (Wang et al., 1999). Janssen et al., 1986; Thiele et al., 1987). c-Myb is also GEM 231 is also able to synergize with different expressed in human and murine colonic mucosa and antineoplastic drugs. In particular, oral administration elevated expression occurs in premalignant adenoma- of GEM 231 in combination with taxol significantly tous polyps and carcinomas (Thompson et al., 1998). prolongs the survival of colon cancer xenografted mice We found that enforced expression of c-Myb in a colon when compared to either of the single agents (Tortora tumor line causes upregulation of Bcl-xL protein, et al., 2000a). Moreover, it has been reported that GEM increased the tumorigenic ability and decreased the 231 sensitizes human tumors of different histotypes to apoptotic capacity of the tumor cells (Biroccio et al., irinotecan (Agrawal et al., 2002; Wang et al., 2002a) and 2001a). We also demonstrated that c-Myb overxpression to the topoisomerase I inhibitor hydroxycamptothecin is a marker of poor prognosis in colorectal cancer, by exerting a synergistic effect on inducing apoptosis frequently associated with Bcl-xL overexpression and and reducing the growth of colon carcinoma and aneuploidy. All these biological properties legitimate androgen-insensitive prostate cancer cells (Cho and c-myb gene as an ideal target for antisense therapy in Cho-Chung, 2003). human neoplasia where c-Myb protein is deregulated. PKAI interacts with the ligand-activated EGFR and It has been reported that c-myb PS ASOs inhibit the cooperates in the transduction of mitogenic signals cell growth of leukemic cell lines as well as the growth of involved in cancer pathogenesis and progression cells derived from patient biopsies in vitro and increase (Tortora et al., 1997). On the basis of this molecular animal survival (Calabretta et al., 1991; Ratajczak et al., interaction, a therapeutic strategy based on the com- 1992). c-myb PS ASOs were also employed as bone bined use of GEM 231 and Iressa (ZD1839, the selective marrow purging agents for chronic myelogenous leuke- inhibitor of EGFR) has been developed. Moreover, mia patients. The results demonstrate that purging of since PKAI and EGFR inhibitors have been shown to CD34 þ cells with c-myb PS ASOs decreases c-Myb and potentiate separately the efficacy of taxanes (Tortora Bcr/abl expression and cytogenetic analysis shows a and Ciardiello, 2000; Tortora et al., 2000a), the complete/partial response in 45% of patients (Luger antitumor efficacy of triple combination has been et al., 2002). Similarly, the treatment of melanoma evaluated. The results obtained demonstrate a coopera- tumors with c-myb PS ASOs inhibits the in vitro growth tive effect of the three agents in inhibiting the growth of of cells and decreases the growth of tumors implanted in xenografted tumors of different histotype and in SCID mice (Hijiya et al., 1994). In colon cancer, our increasing the survival of the animals. A cooperative group demonstrated that an 18-mer c-myb PS ASO, antiapoptotic and antiangiogenic activity of the combi- corresponding to the translation region of the human c- nation seems to account for the potent antitumor myb mRNA, inhibits in vitro cell growth. This effect is efficacy observed (Tortora et al., 2001). mainly due to cell cycle perturbations induced by oligos. The demonstration of the specific PKA phosphoryla- In fact, PS ASOs treatment causes an accumulation in tion site on Bcl-2 protein and the ability of PS ASOs the G0/G1 phases of the cell cycle with a relative S phase PKAI to induce Bcl-2 phosphorylation, cleavage of depletion. Systemic administration of c-myb PS ASOs

Oncogene Antisense in combination therapy A Biroccio et al 6584 for 7 consecutive days in nude mice bearing colon 1998; Ebinuma et al., 2001). The mechanism of growth tumors causes a significant tumor weight inhibition. arrest induced by c-myc PS ASOs appears to be a However, the efficacy of c-myb PS ASOs is reversible consequence of cyclin D1 inhibition (Carroll et al., and, 4 days after tumor growth delay, the tumors’ 2002). Tumor cell proliferation control has also been regrowth is similar to that of untreated tumors. The achieved by treating cells with c-myc PS ASOs in ability of PS ASOs in enhancing the efficacy of cytotoxic combination with different antineoplastic agents. A drugs has also been reported by our group. In combination of c-myc antisense with tamoxifene particular, we demonstrated that the combination of in breast cancer antagonized the tamoxifene-induced cisplatin and c-myb PS ASOs produces an increased increase in c-myc mRNA and protein expression (Kang inhibition of cell proliferation, which is maintained et al., 1996). Moreover, treatment with c-myc PS ASOs during the growth days. Cisplatin exerts its antiproli- in combination with all-trans retinoic acid inhibits the ferative activity independently of a direct effect on proliferation of small-cell lung cancer, and is much more c-myb expression, as c-myb mRNA levels were not effective than both agents given alone (Akie et al., 2000). downregulated in cultures treated with cisplatin alone. The use of c-myc PS ASOs as potential therapeutic The in vitro results were validated by the in vivo data agents in human melanoma treatment has been exten- showing a significant increase of tumor mass inhibition sively investigated by our group. We demonstrated that following the combination treatment without toxic treatment of some human melanoma lines with a 15-mer effects (Del Bufalo et al., 1996). The ability of c-myb c-myc PS ASO, complementary to the translation PS ASOs to sensitize human colon cancer cells initiation region of c-myc RNA, reduces cell prolifera- to cisplatin was confirmed by a study performed by tion and induces apoptosis by specifically inhibiting Funato et al. (2001). The authors used tumor cells c-myc mRNA and protein expression. In vivo experi- resistant to cisplatin treatment and overexpressing ments performed in mice bearing human melanoma cells c-Myb if compared to the sensitive parental line. reveal that c-myc PS ASOs induce a marked inhibition Further efforts must be focused on evaluating the of tumor growth and increase mice survival (Leonetti antitumor efficacy of c-myb PS ASOs combined with et al., 1996). A combination of c-myc PS ASOs and molecules capable of blocking the expression of target cisplatin, a drug commonly used in the treatment of genes involved in cancer progression and metastasis melanomas, exerts a significant antiproliferative effect (Cox-2, bcl-2, bcl-xL,c-myc) and/or antitumoral drugs and activation of apoptosis greater than either agents (Ramsay et al., 2003). used alone. Antitumoral effect is more effective when cisplatin is followed by c-myc PS ASOs treatment if compared to the opposite sequence. The importance of the sequence administration of the two agents has also Antisense oligonucleotides against genes involved in both been observed by other authors in Lewis lung carcinoma proliferation and survival pathways in combination by using cisplatin or taxol followed by phosphorodia- therapy midate morpholino oligomer (Knapp et al., 2003). We c-Myc also evaluated the efficacy of c-myc PS ASOs and cisplatin combination in vivo. Mice bearing human c-myc is a member of the myc gene family, which also melanoma tumors and treated with cisplatin and c-myc includes N-myc, L-Myc and B-myc. The most investi- PS ASOs combination show a higher inhibition of gated myc gene, c-myc, is a key regulator of cell growth tumor growth and an increase in lifespan if compared to that has been found altered by amplification or point animals treated with either of the agents alone (Citro mutation in a large subset of tumors, including et al., 1998). These results are illustrated in Figure 2. leukemia, melanoma, prostate, breast and colon carci- Moreover, by using a human metastatic melanoma nomas (Dang, 1999). Based on these findings, ASOs inherently resistant to cisplatin, we also demonstrated against c-myc have been studied as potential therapeutic that the treatment with c-myc PS ASOs is able to agents. Specific c-Myc downregulation by PS ASOs overcome drug resistance by activating the apototic reduces cell proliferation and induces terminal differ- program (Leonetti et al., 1999). Recently, we used a 16- entiation in leukemia cells in vitro (Holt et al., 1988; mer c-myc PS ASO (INX-6295) encapsulated in lipid Wickstrom et al., 1988). Simultaneously targeting c-myc particles. The systemic administration of encapsulated and bcr-abl by antisense strategy produces a marked INX-6295 improves the pharmacokinetics of oligo when increase in the survival of mice bearing leukemia cells compared to the free one, leading to a significant (Skorski et al., 1995, 1996). Moreover, the delay of enhancement of tumor accumulation and distribution. tumor appearance or prevention of tumor formation Animals treated with INX-6295 exhibit a prolonged was obtained in an animal model of Burkitt’s lymphoma reduction of c-Myc expression, a reduced tumor growth treated with c-myc PS ASOs (Huang et al., 1995; Smith and an increased survival. When administered in and Wickstrom, 1998a, b). c-myc antisense therapy has combination with cisplatin, INX-6295 produces a been used for the treatment of several other tumor complete tumor regression in approximately 30% of histotypes. In particular, treatment with c-myc PS ASOs treated mice (Leonetti et al., 2001). The efficacy of c- reduces viability and in vitro proliferation of human Myc downregulation to inhibit cell proliferation and to prostate, breast and liver cancer cells (Watson et al., synergize with cisplatin in melanoma cells has been 1991; Kang et al., 1996; Balaji et al., 1997; Steiner et al., subsequently observed by other authors by using

Oncogene Antisense in combination therapy A Biroccio et al 6585 the same experimental model, we also demonstrated that telomerase dysfunction caused by c-Myc downregulation is involved in the c-Myc-dependent tumorigenicity and drug sensitivity (Biroccio et al., 2002b, 2003). These results suggest the use of c-myc PS ASOs in combination with GSH depletors and/or agents targeting telomerase function in the treatment of melanoma tumors.

MDM2 mdm2 is an oncogene that has been found overexpressed in a variety of human malignancies. It works as a negative regulator of the p53 tumor suppressor, which interferes with the control of cell proliferation and apoptosis. MDM2 not only interacts with p53 but also Figure 2 Effect of c-myc PS ASOs and cisplatin alone or in with p14ARF, which antagonizes its function as a combination on the growth of the NG melanoma tumor implanted suppressor of p53, Rb and E2F, thus contributing to the in nude mice. , untreated mice; ’, mice treated with c-myc PS development and maintenance of malignant phenotype. ASOs; J, mice treated with cisplatin; & mice treated with For all these reasons, MDM2 is considered to be a cisplatin/c-myc PS ASOs. Data points, means; bars, s.d. potentially relevant target for cancer therapy. PS ASOs against mdm2 mRNA have been used in a wide panel of psoralen- and acridine-modified ASOs (Stewart et al., tumor lines both in vitro and in vivo. Moreover, a novel 2001, 2002). A dose-escalation phase I clinical study in class of MBO containing centrally modified or combination with cisplatin has been completed on end-modified nucleosides has been synthesized, to over- patients with solid tumors and lymphomas (Gelmon come some limitations displayed by PS ASOs in vivo and et al., 2001). The results demonstrated that c-myc PS tested in prostate, colon and breast carcinomas (Tortora ASOs are well tolerated and do not increase the toxicity et al., 2000b; Wang et al., 2001, 2002b, 2003). Inhibition of cisplatin. Partial responses have also been observed. of MDM2 expression induces both p53 and p21/WAF1 Further clinical studies are mandatory to establish the expression, accumulation and growth inhibition. mdm2 efficacy of this antitumoral strategy. MBOs also show a cooperative growth-inhibitory effect Notwithstanding the well-established involvement of with several classes of cytotoxic drugs with different c-Myc protein in cisplatin sensitivity, the susceptibility mechanism of action. mdm2 MBOs also induce apop- to other antineoplastic drugs is still controversial. In tosis and markedly enhance the apoptotic activity of fact, modulation of c-Myc expression has been reported antineoplastic drugs. Moreover, mdm2 MBOs show an to enhance tumor cell sensitivity and to induce resistance antitumoral activity in vivo, in terms of inhibition of to antineoplastic agents. These controversial results tumor growth, tumor growth delay and increase of mice seem to be dependent, at least in part, on the mechanism survival. In vivo administration of mdm2 MBOs leads to of drug action. In fact, by using stable transfected a synergistically or additive therapeutic effect of the melanoma clones, we demonstrated that c-Myc down- cancer chemotherapeutic agents irotecan, 5-fluorouracil, regulation increases the sensitivity to cisplatin, without paclitaxel, cisplatin and doxorubicin. Inhibition of affecting the response to adriamycin and camptothecin tumor growth, induction of apoptosis, and the chemo- (Biroccio et al., 2001b). Moreover, in human sensitizing effect after treatment with mdm2 MBOs p53-mutant small-cell lung cancer cells, we found that occurs both in wild-type and mutant p53 tumors (Wang inhibition of c-Myc expression by PS ASOs almost et al., 2001, 2002b). These results suggest that mdm2 abolishes the apoptotic response induced by doxorubi- MBOs have a role in cancer development and progres- cin (Supino et al., 2001). These results suggest the use of sion through both p53-dependent and -independent c-myc PS ASOs in combination with some kinds of mechanisms. drugs rather than others. In our laboratory, we performed experiments aimed at identifying other molecular targets involved in Conclusions and perspectives melanoma tumorigenicity and drug resistance, in order to improve the treatment of resistant tumors. Macro- The preclinical development of several ASOs targeting array analysis, performed to compare the gene expres- cancer-relevant genes has proceeded very rapidly. Many sion profile of control and c-Myc low-expressing cells, ASOs have shown convincing in vitro reduction in target reveals that downregulation of c-Myc produces striking gene expression and promising activity against a variety changes in the expression of glutathione (GSH)-related of human malignancies. However, considering the metabolism genes, where the expressions of g-glutamyl- multitude of molecular entities and signaling pathways cysteine synthetase and GSSG reductase are found to be that regulate the proliferation and the life/death decision significantly reduced. This imbalance of GSH is in cancer cells, inhibition of a single target gene is not responsible for the activation of apoptosis induced by sufficient to suppress tumor growth. Several evidences c-Myc downregulation (Biroccio et al., 2002a). By using from cell cultures and animal models established that

Oncogene Antisense in combination therapy A Biroccio et al 6586 targeted inhibition of genes involved in controlling cell settings and lack of appropriate selection of patients proliferation and apoptosis combined with anticancer based on a deep knowledge of the function of the chosen treatments is a promising approach for cancer therapy. target. Recent technological advances in both genomic Moreover, clinical trials showed that ASOs are well and proteomic wide views of tumor biology might in tolerated and do not increase the toxicity of conven- the future help address more speciﬁc and adequate tional treatments. However, the following stages of therapies. clinical studies demonstrated that even thought the antisense treatment resulted effective in improving the Acknowledgements response of patients to conventional therapies, most We thank Mrs Adele Petricca for her helpful assistance in antisense/drug combined strategy did not lead to a typing the manuscript. We also thank Dr Barbara Benassi for signiﬁcant increase of survival. This can be due both to a critical reading of the manuscript. Work performed in the limitations in our understanding of the molecular Experimental Chemotherapy Laboratory was supported by basis of cancer and, mostly, to inadequate clinical AIRC (Italian Association for Cancer Research).

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