COMMENTARY Epigenetic therapies for neuroblastoma: immunogenicity awakens nez1 Carlos Jime , Lucas Moreno2,3 and Miguel F. Segura2 1 Human Technopole, Milan, Italy noma de Barcelona (UAB), 2 Group of Childhood Cancer and Blood Disorders, Vall d’Hebron Research Institute (VHIR), Universitat Auto Spain 3 Paediatric Oncology and Haematology Department, Vall d’Hebron University Hospital, Barcelona, Spain Keywords The development of immunotherapies for neuroblastoma remains challeng- histone deacetylase inhibitors; ing owing to the low immunogenicity of neuroblastoma cells, as reflected immunotherapy; major histocompatibility by the low expression of one of the main triggers of immune recognition, complex class I; neuroblastoma the major histocompatibility complex class I (MHC-I). Cornel et al. Correspondence showed that epigenetic modulation of neuroblastoma cells with a histone M. F. Segura, Group of Childhood Cancer deacetylase inhibitor can boost the expression of major histocompatibility and Blood Disorders, Vall d’Hebron complex class I, among other immune receptors, priming their recognition Research Institute (VHIR), Universitat by T- and natural killer cells. By leveraging the developmentally related noma de Barcelona (UAB), Passeig Vall Auto aberrant epigenetic landscapes of neuroblastoma, these discoveries pave the Hebron 119-129, Collserola Building, way to overcome a major limitation in the field of neuroblastoma Laboratory 207-8, 08035 Barcelona, Spain immunotherapy. E-mail: miguel.segura@vhir.org (Received 14 February 2023, accepted 24 February 2023, available online 8 March 2023) doi:10.1002/1878-0261.13404 High-risk neuroblastoma remains a clinical challenge is the absence or low expression of MHC-I in neuroblas- worldwide, as this group of patients, representing close toma cells [4]. to half of all cases of neuroblastoma, still presents 5- MHC-I is expressed in all nucleated cells of post- year survival rates below 50% [1]. Although the survival embryonic tissues, and its expression is a key require- of patients with these peripheral nervous system neo- ment for the recognition of neoplasms and surveillance plasms has partially benefited from certain immunother- by the immune system. Loss of expression of this apies (i.e. anti-GD2 therapy), some immunotherapies antigen-presenting membrane receptor is a recurrent with promising results in adults such as anti-PD1 or immune-evading survival strategy of cancer cells, anti-CTLA4 do not have the expected results in children through mutations or gene silencing [5,6]. However, [2]. Thus, these above-mentioned immunotherapies are tumours of embryonal origin such as neuroblastoma limited by the low immunogenicity of neuroblastoma originate from early embryonal cell populations not cells [3]. A paradigmatic example reflecting this feature expressing MHC-I. In this case, the lack of expression Abbreviations CD137, cluster of differentiation 137; CTLA4, cytotoxic T lymphocyte-associated antigen 4; HDACi, histone deacetylase inhibitors; HLA-E, human leucocyte antigen E; IAPi, inhibitors of apoptosis proteins; IFN-a, interferon alpha; IFN-c, interferon gamma; MHC-I, histocompatibility complex class I; MIBG, metaiodobenzylguanidine; MICA/B, MHC class I chain-related protein A and B; N4BP1, NEDD4 binding protein 1; NFjB, nuclear factor kappa light chain enhancer of activated B cells; NK, natural killer; PD-1, programmed cell death protein 1; PRAME, preferentially expressed antigen in melanoma; RNAseq, RNA sequencing; TNFa, tumour necrosis factor alpha. 718 Molecular Oncology 17 (2023) 718–721 Ó 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. nez et al. C. Jime Epigenetic therapies for neuroblastoma: immunogenicity awakens is not due to a clonal selection process leading to its tested in children with relapsed or refractory malignan- loss, but to the specific gene expression programming cies in combination with nivolumab (NCT03838042). of the cell of origin, most usually through epigenetic Contrary to what was seen with IAPi, entinostat was mechanisms [7]. able to induce MHC-I expression in a large variety of Neuroblastoma, as a paediatric cancer, harbours low standard neuroblastoma cell lines as well as in patient- mutational rates. However, increasing evidence empha- derived organoids, across the diverse spectrum of sises the importance of the aberrant epigenomes of neu- genomic aberrations associated with neuroblastoma. roblastoma cells due to their oncogenic properties. Increase of MHC-I was also observed with other Neuroblastoma cells carry “frozen-in-time” epigenetic HDACi and was further enhanced by combinatorial landscapes that reflect their embryonal origins and treatment using cytokines known to regulate MHC-I. resemble those of the neural crest progenitors from The functional consequences of entinostat treatment which they were originated [8]. Therefore, understand- were analysed measuring the activation (via CD137 ing neuroblastoma epigenetics allows hacking these expression as well as TNFa and IFN-c secretion) and expression programs and reverting relevant oncogenic cytotoxic capacity of T-cells. Pre-treatment with enti- functions. In the case of immune evasion, the develop- nostat alone was sufficient to increase the expression mental inherited and reversible epigenetic silencing of of CD137, and this effect was further enhanced in the MHC-I, contrary to genetic loss in non-embryonal presence of other T-cell activation cytokines such as tumours, opens a promising opportunity for re- IFN-a and IFN-c. The consequences of T-cell activa- expressing MHC-I to induce neuroblastoma responsive- tion were analysed in a cytotoxic assay, which showed ness to the immune system. that entinostat pre-treatment resulted in a similar To revert one of the main mechanisms of immune increase in the cytotoxic capacity of T-cells as pre- evasion of neuroblastoma cells, Cornel et al. [9] sought treatment with other MHC-I-enhancing cytokines. The to screen for compounds that would restore the expres- fact that experiments were performed in the presence sion of MHC-I. The authors used two drug repurpos- of HDACi suggests that entinostat did not affect the ing screening libraries, testing approximately 3900 viability and cytotoxic capacity of T-cells. compounds in a range of nanomolar to low micromo- Because the effects of HDAC inhibition may alter lar concentrations. To dissect the mechanism of the many cellular processes, the authors further investigated drug tested, all these compounds were probed in a neu- whether the immunogenic effects of HDACi could be roblastoma cell line containing an NFjB gene reporter attributed to other factors. A whole transcriptomic system, as the authors and others had shown before analysis (by RNAseq) was performed to analyse differ- that this pathway mediates the upregulation of the entially expressed genes upon entinostat treatment. MHC-I gene induced by cytokines [10,11]. Among all Interestingly, the authors found a significant modula- drugs tested, two families of compounds stood out: his- tion of genes not only involved in MHC-I antigen pre- tone deacetylase inhibitors (HDACi) and inhibitors of sentation but also genes encoding natural killer (NK) Inhibitors of apoptosis proteins (IAPi), both of which signalling molecules. A fraction of these genes was also enhanced MHC-I expression in an NFjB-independent demonstrated to be upregulated and present on the sur- and NFjB-dependent manner, respectively. The func- face of the plasma membrane. Co-culture cytotoxicity tional consequences of this drug-mediated induction of experiments showed that entinostat pre-treatment of MHC-I were tested in a co-culture system consisting of neuroblastoma cells increased the cytotoxic capacity of neuroblastoma cell lines and T-cells directed against healthy-donor NK cells. In summary, these findings the neuroblastoma-specific Preferentially Expressed indicate that the immunogenic effects of entinostat Antigen in Melanoma (PRAME) antigen. The IAPi treatment are beyond the upregulation of MHC-I. AZD-5582 was able to induce the expression of MHC- Intriguingly, Cornel et al. also observed that entino- I and T-cell-mediated toxicity in a few neuroblastoma stat promoted a switch towards the mesenchymal line- cell lines, owing to the strong expression of negative age of neuroblastoma cells at the transcriptional level, modulators of the NFjB pathway, a common feature accompanying the increase in genes related to immune of neuroblastoma tumours [11]. The authors elegantly activation. Indeed, recent studies have revealed that showed that silencing of N4BP1, a well-known NFjB the immunogenic status of neuroblastoma is related to inhibitor, allowed AZD-5582-mediated induction of the adrenergic (less immunogenic) and mesenchymal MHC-I. (more immunogenic) cell lineages widely studied Because the effects of HDACi on MHC-I were not recently in these tumours [12]. The biological reason mediated by NFjB, the authors further focused on the for the higher immunogenicity of the more invasive HDACi entinostat, a drug that is currently being and therapy-resistant mesenchymal phenotype remains Molecular Oncology 17 (2023) 718–721 Ó 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of 719 Federation of European Biochemical Societies. Epigenetic therapies for neuroblastoma: immunogenicity awakens nez et al. C. Jime Fig. 1. Immunogenic activation of neuroblastoma cells through epigenetic therapy. Cornel et al. showed in their recent work that the expression of multiple immune system receptors, epigenetically silenced in neuroblastoma cells as a result of its developmental origins, can be restored with the histone deacetylase (HDAC) inhibitor entinostat. This epigenetic modulation boosts the expression of major histocompatibility complex class I (MHC-I), key for T-cell recognition, and human leukocyte antigen E (HLA-E) and MHC class I chain-related proteins A and B (MICA/B), receptors of natural killer (NK) cells, thereby triggering a cytotoxic immune response against neuroblastoma cells. elusive. However, this discovery revealed a key vulner- European Regional Development Fund/European ability of this subpopulation of aggressive neuroblas- Social Fund; “A way to make Europe”/“Investing in toma cells that could be exploited with entinostat your future”). We want to thank the parent’s founda- treatment, as proposed by the authors. tions that support the development of epigenetic thera- In summary, although T-cell dysfunction may be pies in neuroblastoma such as the NEN association; caused by multiple factors, the present study demon- Joan Petit foundation; Pulseras Candela foundation; strated a clear effect of HDACi on the immunogenic and Fundaci on Neuroblastoma. We also thank all mem- capacity of neuroblastoma cells by releasing the devel- bers of the Childhood Cancer and Blood Disorders opmental epigenetic brake that blocks the expression group (VHIR) for their support. of immune-related genes (Fig. 1). Validation in animal models showing the enhanced efficacy of immunother- apies in combination with HDACi will strength the Conflict of interest conclusions of this study. The authors declare no conflict of interest. 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