Oncogene (2003) 22, 64846488

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Epigenetic targets for immune intervention in human malignancies

Michele Maio*,1, Sandra Coral1, Elisabetta Fratta1, Maresa Altomonte1 and Luca Sigalotti1
1

Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientico, Aviano 33081, Italy

Emerging evidences suggest that epigenetic events associated with tumor development and progression, such as deregulated methylation of CpG dinucleotides and aberrant histone acetylation, may impair the immunogenic potential of cancer cells. In fact, DNA hypermethylation and/or histone deacetylation contribute to the absent or downregulated expression of different components of the tumor recognition complex (i.e., HLA class I antigens, cancer/testis antigens and accessory/costimulatory molecules) in solid and hemopoietic human malignancies. However, pharmacologic agents that induce DNA hypomethylation or inhibit histone deacetylation can modify these epigenetic phenomena, restoring the defective expression of selected components of the tumor recognition complex in cancer cells. These antigenic modications positively modulate the immunogenicity and the immune recognition of cancer cells, making epigenetic drugs attractive agents to design new combined chemoimmunotherapeutic strategies for the treatment of cancer patients. Oncogene (2003) 22, 64846488. doi:10.1038/sj.onc.1206956 Keywords: DNA methylation; histone acetylation; cancer; immunotherapy; HLA antigens; tumor antigens

Introduction Epigenetic events, that is, heritable changes in gene expression that are not accompanied by changes in DNA sequences, play an important role in tumorigenesis (for a review, see Nephew and Huang, 2003). The main epigenetic modication of the human genome is methylation of cytosine residues within the context of the CpG dinucleotides (Worm and Guldberg, 2002). In cancer, aberrant hypermethylation of CpG islands, associated with transcriptional silencing of selected genes, can affect multiple cellular functions, including cell growth and differentiation, cell cycle control, and DNA repair, as well as angiogenesis and invasion (for a

*Correspondence: M Maio, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, I.R.C.C.S.-Centro di Riferimento Oncologico, Via Pedemontana Occ.le, 12, Aviano 33081, Italy; E-mail: mmaio@cro.it

review, see Santini et al., 2001). Furthermore, hypoacetylation of histones is frequently associated with CpG islands hypermethylation, and results in a compact structure of chromatin, which is repressive for transcription (Cameron et al., 1999). Upcoming evidences show that the deregulation of the DNA methylation apparatus that occurs during cancer development provides new therapeutic targets for cancer treatment. Indeed, the genome-wide hypomethylation that is frequently observed in cancer cells can lead to the reactivation of genes silenced by DNA methylation (De Smet et al., 1996). Among these are the cancer/testis antigen (CTA) genes (De Smet et al., 1996), which encode for proteins that are recognized by the hosts immune system and can, therefore, be exploited to implement tumor-specic immunotherapeutic approaches (Traversari, 1999; Zendman et al., 2003). The presence and levels of CTA expression on neoplastic cells, combined with other components of the tumor recognition complex, including HLA class I antigens and costimulatory/accessory molecules, are mandatory prerequisites for the successful implementation of T-cell-based and humoral therapeutic strategies in cancer patients, and for the immunogenicity of neoplastic cells. In fact, T-cell activation requires an antigenspecic signal, delivered through the T-cell receptor via the peptide/HLA class I complex, and a nonspecic signal, resulting from the binding of costimulatory/ accessory molecules, like CD80/B7-1, CD86/B7-2, intercellular adhesion molecule-1 (ICAM-1/CD54) and lymphocyte function-associated antigen 3 (LFA-3/ CD58) expressed on cancer cells, with their respective counter-receptors CD28, CTLA-4, LFA-1 and CD2, expressed on T cells (Schwartz, 1990; Altomonte et al., 1993; Boussiotis et al., 1996; Zuckerman et al., 1998; Kim et al., 1999). These activating signals are frequently impaired in cancer cells, contributing to tumor escape from immune surveillance. The absence of a specic tumor antigen and/or loss or downregulation of HLA class I molecules expression represent the most frequent mechanisms underlying lack of T-cell recognition of cancer cells (for a review, see Seliger et al., 2002). An additional major mechanism of immune escape involves defective expression of costimulatory/accessory molecules on target cancer cells (Maeda et al., 2000). In the light of these evidences, the identication of the molecular mechanisms regulating the expression of

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each component of the tumor recognition complex is a mandatory prerequisite to circumvent the immune escape of cancer cells, and to design eventually more effective immunotherapeutic approaches in cancer patients. The aim of this review is to highlight the immunologic implications of cancer epigenetics and to propose new clinical approaches to enhance the immunogenicity of neoplastic cells and the clinical effectiveness of cancer immunotherapy.

HLA class I antigens abnormalities Neoplastic cells can lose their ability to present effectively tumor antigens to hosts immune system as a result of different mechanisms that affect the biosynthesis of HLA class I molecules. In fact, malignant transformation can associate with changes in the expression and/or function of HLA class I antigens. These changes include: (i) total HLA class I antigens loss; (ii) total HLA class I antigens downregulation; and (iii) selective loss or downregulation of distinct HLA class I allospecicities (for a review, see Seliger et al., 2002). Molecular mechanisms that have been found to underlie these distinct HLA class I phenotypes are generally represented by regulatory and structural defects in the components involved in the biosynthesis of the peptide/b2-microglobulin/HLA class I heavy chain complex (for a review, see Seliger et al., 2002).

Heterogeneity of CTA Distribution CTA include MAGE, NY-ESO-1 and SSX gene families and GAGE/PAGE/XAGE superfamilies (Zendman et al., 2003). The main characteristic of CTA is that they can be expressed by neoplasms of different histological origin, while they are not expressed by normal tissues, except for the germ cells of testis (Zendman et al., 2003); this pattern of expression clearly makes CTA optimal targets for cancer immunotherapy. Furthermore, distinct CTA can encode for different antigenic peptides that are presented to the immune system in association with various HLA class I or HLA class II allospecicities (Traversari, 1999), eliciting both cytotoxic T lymphocytes (CTL) and humoral immune responses (Jager et al., 2000). Despite these promising characteristics, the expression of CTA is heterogeneous in neoplastic lesions, showing percentages of CTA-positive lesions ranging from 0 to 66%, depending on the specic CTA investigated and on the tumor histotype analysed (Table 1), leukemias showing only a sporadic expression (Chambost et al., 2001; Sigalotti et al., 2003). Furthermore, the intralesional expression of CTA is characteristically heterogeneous; in fact, immunohistochemical analyses revealed that the majority of CTA-positive lesions show staining for the CTA MAGE-A1, NY-ESO-1 and SSX in less than 50% of neoplastic cells (dos Santos et al., 2000; Jungbluth et al., 2000, 2001).

Epigenetic regulation of the components of the tumor recognition complex Epigenetic events appear to represent the unique mechanism regulating the expression of CTA in cancer cells, and DNA methylation seems to play the major role; in fact, a correlation between hypomethylated CpG dinucleotides in MAGE promoters and their expression has been found in neoplastic cell lines (De Smet et al., 1996, 1999) and tissues (Sigalotti et al., 2002a). Moreover, unmethylated MAGE promoters drive the transcription of reporter genes in CTA-negative neoplastic cells, suggesting that availability of transcriptional factors is not involved in the heterogeneity of CTA expression and that DNA methylation is the main restricting factor for CTA expression (De Smet et al., 1995). Consistently, in vitro methylation of these reporter constructs is sufcient to block their transcriptional activity in MAGE-positive cells (Sigalotti et al., 2002a). In agreement with these observations, methylation of distinct CpG dinucleotides seems to be

Table 1 RTPCR analysis of the expression of selected CTA in human tumors of different histologic origin Tumor type MAGE-1 Breast carcinoma Colorectal carcinoma Cutaneous melanoma Hepatocellular carcinoma Esophageal carcinoma Gastric carcinoma Head and neck carcinoma Lung carcinoma Medullary thyroid carcinoma Neuroblastoma
a

CTA MAGE-3 12 20 57 24 47 23 43 60 22 33 NY-ESO-1 10 2 21 0 24 8 7 21 65 36 SSX-2 4 12 35 9 0 3 36 15 4 NDb

References

20a 30 40 19 53 23 33 36 17 66

Brasseur et al. (1992); Gaugler et al. (1994); Mashino et al. (2001) Mori et al. (1996); Mashino et al. (2001); Tureci et al. (1998) Sigalotti et al. (2002a); Tureci et al. (1998) Luo et al. (2002) Quillien et al. (1997); Mashino et al. (2001) Li et al. (1996); Mashino et al. (2001) Eura et al. (1995); Kienstra et al. (2003); Tureci et al. (1998) Scanlan et al. (2000) Maio et al. (2003) Soling et al. (1999)

Percent of CTA-positive specimens. ND: not determined.

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the leading cause generating the intratumor heterogeneity of CTA expression observed in melanoma lesions (Sigalotti and Maio, manuscript in preparation). Unlike CTA, the role of epigenetic mechanisms in the transcriptional regulation of HLA class I antigens is not fully understood. Recent studies suggest that DNA hypermethylation might be a major mechanism for the transcriptional inactivation of HLA class I genes in human esophageal squamous cell carcinomas (Nie et al., 2001), although it is exceptionally responsible for the total loss of HLA class I antigens expression in melanomas (Serrano et al., 2001; Fonsatti et al., 2003). Conversely, DNA hypermethylation is responsible for the downregulated expression of HLA class I antigens and allospecicities in the large majority of melanoma samples (Coral et al., 1999). Pharmacologic approaches to restore the expression of the components of the tumor recognition complex Several studies have demonstrated that epigenetic events can be reversed through pharmacologic approaches. In particular, it is well established that the expression of different tumor suppressor genes, which have been inactivated by aberrant DNA methylation, can be restored by the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-20 -deoxycytidine (5-AZA-CdR), which leads to the synthesis of hypomethylated DNA (Momparler and Bovenzi, 2000). The unique role of DNA methylation in regulating CTA expression offers the possibility to modify their constitutively heterogeneous expression utilizing pharmacologic inhibitors of the DNMT1. Indeed, treatment of neoplastic cells of different histotypes with 5-AZACdR induces a de novo expression of CTA (Weber et al., 1994; De Smet et al., 1996; Li et al., 1996; dos Santos et al., 2000; Weiser et al., 2001a, b; Coral et al., 2002; Sigalotti et al., 2002a, b), and upregulates their constitutive levels of expression (Weiser et al., 2001a, b; Sigalotti et al., 2002a, b), both at the mRNA and protein levels. Additionally, 5-AZA-CdR reduces the intratumor heterogeneity of CTA expression, as demonstrated by the homogeneous levels of MAGE-A3 obtained following treatment of singlecell melanoma clones displaying high, intermediate or low expression of MAGE-A3 (Maio, manuscript in preparation). Furthermore, the ability of DNA methylation patterns to be inherited throughout cellular divisions (Razin and Riggs, 1980) makes changes of the antigenic prole induced by 5-AZA-CdR long lasting. In fact, 5AZA-CdR-induced expression of CTA can be maintained in cancer cells for up to 60 days after drug removal (Coral et al., 1999, 2002), and indenitely in single-cell sarcoma (De Smet et al., 1999) and melanoma (De Smet et al., 1999; Maio et al., manuscript in preparation) clones generated from 5-AZA-CdR-treated parental cell lines. Whether these ndings underlie a selective growth advantage of CTA-negative over CTApositive neoplastic cells remains to be investigated.
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Additional evidences suggest that treatment with DNA hypomethylating agents (DHA) may also inuence the expression of other components of the tumor recognition complex on cancer cells. In fact, the role of DHA in restoring the downregulated expression of HLA class I antigens and allospecicities frequently detected in melanoma cells is well established: exposure to 5-AZA-CdR induced a persistent upregulation of the constitutive expression of HLA class I antigens and of HLA-A1 and -A2 allospecicities in a large panel of melanoma cell lines (Coral et al., 1999). Conversely, the de novo expression of HLA class I antigens induced by 5-AZA-CdR seems to be a sporadic phenomenon, since it was observed only in one melanoma cell line (Serrano et al., 2001) and in a human esophageal cell carcinoma cell line (Nie et al., 2001), but not in a panel of HLA class I-negative or HLA-A2-negative melanoma cells (Fonsatti et al., 2003). Exposure to DHA may also affect the expression of molecules providing the nonspecic signals required for an efcient T-cell activation. In fact, 5-AZA-CdR induced a persistent upregulation of the constitutive levels of ICAM-1 and LFA-3 expression in a panel of melanoma cell lines (Coral et al., 1999). It is noteworthy that this phenomenon is shared by neoplastic cells of different histotypes since 5-AZA-CdR reverted the downregulated expression of ICAM-1 in different ovarian cancer cell lines (Arnold et al., 2001) and upregulated the constitutive levels of ICAM-1 in renal cell carcinoma cells (RCC) (Maio et al., unpublished observation). The recent identication of a synergistic mechanism for gene repression involving DNA methylation and histone deacetylation (Cameron et al., 1999) has opened new promising routes to achieve higher levels of gene reactivation (Cameron et al., 1999). In particular, the combined use of 5-AZA-CdR and of the histone deacetylase inhibitor (IHD) depsipeptide has shown a synergistic effect in the induction of NY-ESO-1 and MAGE-A3 expression in thoracic cancers (Weiser et al., 2001a, b). Furthermore, recent studies have shown that chromatin modications induced by treatment with IHD might affect the expression of costimulatory molecules. In particular, the IHD sodium butyrate (SB) was able to induce or upregulate the expression of CD86, and to upregulate the expression of ICAM-1 in a panel of acute myeloid leukemia (AML) cell lines and in 30 clinical AML samples (Maeda et al., 2000). Conversely, the expression of HLA class I antigens, or other molecules involved in the antigen processing and presentation machinery (i.e., TAP1, TAP2, LMP2 and LMP7) does not seem to be affected by IHD in lung cancer cells (Weiser et al., 2001a). Functional role of the epigenetic targeting of cancer cells Besides the phenotypic changes, the restoration of the defective expression of distinct components of the tumor recognition complex through epigenetic

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targeting of cancer cells results in their efcient recognition and lysis by antigen-specic CTL. In fact, de novo expression of selected CTA induced by 5-AZACdR allowed specic CTL recognition of melanoma, lung cancer, esophageal cancer, mesothelioma and RCC cells (Weber et al., 1994; Weiser et al., 2001a; Coral et al., 2002). Furthermore, the upregulated expression of HLA class I antigens and allospecicities observed in melanoma cell lines after exposure to 5-AZA-CdR resulted in their increased recognition by a gp 100-specic HLAA2-restricted CTL clone (Fonsatti and Maio, manuscript in preparation). Despite the well-characterized functional role of phenotypic modications induced by 5-AZA-CdR, the immunologic role of IHD treatment requires further studies. In fact, an enhanced proliferation of allogeneic lymphocytes was mediated by myelomonocytic leukemia cells as a consequence of their upregulated expression of CD86 and ICAM-1 induced by SB (Maeda et al., 2000). Nevertheless, the upregulated expression of NY-ESO-1 obtained in neoplastic cells by combining depsipeptide and 5-AZA-CdR did not result in an increased CTA-specic CTL recognition, as compared to cells treated with 5-AZA-CdR alone (Weiser et al., 2001a).

patients. Furthermore, the ability of 5-AZA-CdR to induce concomitant and homogeneously high levels of expression of different CTA in a given population of neoplastic cells (Li et al., 1996; Fujie et al., 1997; Coral et al., 2002; Sigalotti et al., 2002a, b) suggests the possibility to pursue multitargeted immunotherapy for the treatment of cancer patients, with the aim to overcome the possible emergence of CTA-negative tumor clones. The potential clinical role of the immunobiologic features of DHA is further supported by the recent demonstration that a single cycle of 5-AZA-CdR induced a persistent de novo expression of different CTA in 11 patients affected by AML or myelodysplastic syndrome. These in vivo data further support the immunologic properties of 5-AZA-CdR, which may signicantly contribute to the therapeutic activity of the drug in combination with its well-dened cytotoxic/ cytostatic and differentiating potentials (Sigalotti et al., 2003).

Conclusion Epigenetic events have a crucial role in the expression of different components of the tumor recognition complex. Thus, systemic administration of epigenetic drugs (e.g., DHA, IHD) may represent a promising strategy to overcome the impaired immunogenicity and immune recognition of cancer cells, and to design new chemoimmunotherapeutic regimens to improve the therapeutic efcacy of CTA-based vaccination in cancer patients. This approach will be facilitated by the current evaluation of epigenetic agents as cytotoxic/cytostatic or differentiating agents in cancer patients (Wijermans et al., 2000; Daskalakis et al., 2002; Sandor et al., 2002; Silverman et al., 2002), and also by the recent identication of the new DHA zebularine, which is able to reactivate epigenetically silenced genes by oral administration with minimal toxicity (Cheng et al., 2003).
Acknowledgements We acknowledge Dr Anna DAmato for her skillful editorial assistance. This work was supported in part by the Associazione Italiana per la Ricerca sul Cancro, by the progetto Ricerca Finalizzata awarded by the Italian Ministry of Public Health and by the Compagnia di San Paolo, Turin, Italy.

Prospective immunotherapeutic applications of epigenetic targeting of cancer cells Based on the functional activities of the phenotypic modications induced by DHA and/or IHD in immune recognition of cancer cells, preclinical models have been developed to assess their clinical potential to overcome the reduced immunogenicity of neoplastic cells sustained by defects in the expression of distinct components of the tumor recognition complex. Preliminary results obtained by the molecular, biochemical and serological analyses of human melanoma xenografts explanted from nude mice treated with DHA demonstrated that treatment inuenced the constitutive pattern of expression of CTA and of HLA class I antigens (Maio et al., manuscript in preparation). These initial data suggest that systemic administration of 5-AZA-CdR to cancer patients may enhance the immunogenic potential of neoplastic cells in vivo, and may be utilized to overcome the heterogeneous expression of CTA in neoplastic tissues, concomitantly widening the biologic eligibility to CTA-specic vaccination to the large majority of cancer

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