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Epigenetic Priming in Cancer Initiation
Opinion
Epigenetic Priming in Cancer Initiation Carolina Vicente-Dueñas,1,5 Julia Hauer,2,5 César Cobaleda,3,6,* Arndt Borkhardt,2,6,* and Isidro Sánchez-García1,4,6,* Recent evidence from hematopoietic and epithelial tumors revealed that the contribution of oncogenes to cancer development is mediated mainly through epigenetic priming of cancer-initiating cells, suggesting that genetic lesions that initiate the cancer process might be dispensable for the posterior tumor progression and maintenance. Epigenetic priming may remain latent until it is later triggered by endogenous or environmental stimuli. This Opinion article addresses the impact of epigenetic priming in cancer development and in the design of new therapeutic approaches. Epigenetic Priming in Cancer Development Despite the enormous amount of data that we have gathered in the past four decades on the biology of tumor cells, our capacity for controlling the development of cancer unfortunately remains limited. We do not know yet how to prevent the conversion of a precancer cell (see Glossary) into a tumor, mainly due to the fact that the early events triggering the commitment to a new cancer lineage remain largely unknown [1]. A crucial point in the history of a tumor is the transition of a normal cell to a malignant state. Therefore, an unmet need in cancer research is to understand how to prevent the conversion of a precancer cell into a full-blown tumor. The mechanisms that establish and regulate cellular identity allow the emergence of aberrant cells. In this Opinion article, we discuss the importance of oncogenic hits in establishing the cell fate of the cancer-initiating cell and how tumor-primed cells evolve. Better understanding of largely neglected role of oncogenes may help in developing ways to attack precancer cellular states.
Interplay between the Oncogenic Hit and the Cancer-Initiating Cell Oncogene expression is necessary at the earliest stages of cancer development and for progression [2]. This seems to suggest that oncogenic hits play a similar molecular role throughout the origin, progression, and expansion of the tumor [2]. This fits with the concept of the clonal nature of human cancers, in which all tumor cells carry the same initiating oncogenic lesions. However, full-blown cancers exhibit a high degree of cellular heterogeneity, suggesting that the role of oncogenes might not be equal in all tumor cells, a fact that may explain why therapies targeting oncogenic hits have different efficacies against cells at different stages of evolution [3,4]. This could be better explained by a new model of cancer based on tumor epigenetic priming, in which an oncogenic hit ‘resets’ the epigenome and/or transcriptome of a normal cell (the cancer cell of origin) and gives rise to a pathological differentiation program that ultimately leads to tumor development (Figure 1). This concept implies a different role for an oncogenic hit in the cancer cell of origin and suggests that oncogenic alterations essential for tumor initiation and at the origin of clonal disease may not be required during tumor progression, once the cells have been primed towards malignancy [5,6] (Figure 2). Importantly, stem or progenitor cells (PCs) have a specific epigenetic program that renders them more susceptible to genetic alterations, and hence one could argue that specific developmental stages with their associated epigenetic states may be more susceptible to oncogenes and progression to cancer. This view of cancer is also supported by the fact that 408
Trends in Cancer, June 2018, Vol. 4, No. 6 © 2018 Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.trecan.2018.04.007
Highlights During cancer development, a new tumor cell identity is established and maintained mainly through epigenetic priming of cancer-initiating cells. This priming to cancer is associated with major epigenetic changes and its outcome is influenced by the initial susceptibility of the cancer cell of origin. Epigenetic priming may remain latent until it is triggered by either endogenous or environmental stimuli. Targeting this epigenetic network to block the transformation of latent precancerous cellular states in disease appears to be a novel, promising strategy for the prevention of cancer formation in susceptibility carriers.
1 Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain 2 Department of Pediatric Oncology, Hematology, and Clinical Immunology, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany 3 Department of Cell Biology and Immunology, Centro de Biologia Molecular Severo Ochoa (CBMSO), CSIC/UAM, Madrid 28049, Spain 4 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Campus M. de Unamuno s/n, 37007 Salamanca, Spain 5 Equal first author 6 Equal senior author
*Correspondence: [email protected] (C. Cobaleda), [email protected]. de (A. Borkhardt), [email protected] (I. Sánchez-García).
polycomb group target (PCGT) genes are preferentially methylated and silenced in all cancer stem cells (CSCs) [7–9]. Finally, the epigenetic priming model is supported by the tight association between a given chromosomal aberration and a specific tumor type, a clear indication of the fate-inducing nature of oncogenes [1]. The epigenetic priming model could also explain how, in the evolution of several human tumors, a precancer lesion can be stably maintained as the only proto-oncogenic alteration in an abnormal cell population that will give rise to a full-blown tumor only in response to secondary hits [10,11]. A good example of this apparent paradox is the effect of imatinib in chronic myelogenous leukemia (CML), where the drug fails to kill BCR-ABLp210+ CSCs because, somehow, it cannot block the capacity of the fusion oncogene to prime the epigenome towards malignancy in the stem cell compartment (see below) [6,12,13].
Epigenetic Priming as a Driver of Cancer: Restriction of Lineage Options during Transformation The epigenetic priming model implies that the initiating oncogenic hit may not be required for tumor progression. This fact cannot be experimentally demonstrated in human tumors, which, by their clonal nature, carry these initiating hits throughout their whole malignant cellular population [1]. Therefore, to test this model we would need a system that isolates the role of an oncogenic hit in the cancer cell of origin. Expression of the oncogenic hit would have to be restricted to the stem/ progenitor compartment and switched off afterwards to demonstrate that later oncogene expression is not necessary for tumor progression once epigenetic priming has already occurred (Box 1). Epigenomic studies such as genome-scale maps of DNA methylation from both stem cells and mature B cells in various hematopoietic animal cancer models [MafB in multiple myeloma (MM), Bcl6 in diffuse large B cell lymphoma (DLBCL), ETV6-RUNX1 in childhood B acute lymphoblastic leukemia (B-ALL)] [14–16] provided insights into the precise molecular mechanisms by which oncogenes reprogram stem cells into differentiated tumor cells. In all cases a substantial number of CpG islands and promoters were hyper- or hypomethylated in the stem cells of these transgenic models, setting a pattern that is inherited throughout B cell development and suggesting that molecular reprogramming is involved in tumor initiation. Therefore, according to evidence from animal experimental hematopoietic systems (Box 1), we postulate that cancer-initiating oncogenic hits epigenetically modify target genes that will remain in an epigenetically primed state in mature tumors even when the oncogene is no longer present (Figure 2). A growing number of epigenetic regulators essential for normal differentiation are being found to initiate oncogenic hits, such as DNMT3A and TET2 through deregulation of DNA methylation [17,18]. Dnmt3a loss in mice predisposes hematopoietic stem cells (HSCs) to malignant transformation [19] and these animals develop a spectrum of malignancies similar to those seen in patients with DNMT3A mutations [20–25]. Loss-offunction mutations in TET2 often occur in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), or acute myelogenous leukemia (AML) and are associated with a DNA hypermethylation phenotype [26,27] and with an increased mutagenesis rate in HSCs [28,29]. Also, TET1 acts as a tumor suppressor in hematopoietic malignancies [30], and restoration of TET2 activity prevents leukemia progression [26]. Similarly, in gliomas, mutations in the protooncogenes IDH1 and IDH2 induce genome-wide chromatin changes that lead to the appearance of a precancer population with a proliferative advantage and stem-like transcriptional features [31]. Mutations in IDH1 and IDH2 are frequent and mutually exclusive in de novo AML and result in a hypermethylation phenotype and are mutually exclusive with mutations in TET2, suggesting that these lesions may be biologically redundant by fullfilling similar functions in tumor development, which can even occur at different stages of tumoral evolution [32].
Glossary Cancer cell of origin (or cancerinitiating cell): the initially healthy cell that will be primed by an oncogenic hit and that will give rise to a (pre)cancer cell. Cancer stem cell (CSC) (or cancer-maintaining cell): the CSC theory postulates that tumors are stem cell-based tissues like any other tissue in the organism. This implies the existence of a hierarchical structure in the tumor where not all of the cancer cells are equally competent in regenerating the tumor. The CSCs are the only cells that regenerate and maintain the whole tumor. Epigenetic memory: in the context of tumor origin and development, epigenetic memory refers to the retention of transcriptional properties or chromatin or DNA methylation marks imposed by an oncogenic hit in the starting cancer cell of origin that are afterwards maintained from precancer to tumoral differentiation. Oncogenic hit: any molecular alteration that leads to the establishment of a precancer or cancer cellular state. It may be the activation of an oncogene (by mutation, gene fusion, gene amplification), the inactivation of a tumor suppressor, or an epigenetic alteration. Precancer cell: a cell that has already been primed towards malignancy but retains normal developmental and physiological behavior. Depending on susceptibility and environmental exposure, a precancer cell may or may not give rise to a full-blown tumor. Susceptibility: increased sensitivity of a cell (or an individual) to suffer negative consequences (e.g., precancer or cancer development) from exposure to a given agent or condition. It can be due to genetic (mono- or polygenic) or epigenetic causes and these can be congenital or appear de novo by exposure to related or unrelated agents or conditions.
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Figure 1. Gene–Environment Interactions in Cancer Development. Tumoral epigenetic priming can occur at any point in life and can remain dormant in the cancer cell of origin unless other posterior events trigger further tumor progression. This priming will be preserved through tumor evolution even if the initiating hit is no longer present or expressed. (A) Sometimes the second event can arise randomly. (B) However, a given epigenetic priming may be particularly sensitive to a certain environmental factor (‘gene–environment interaction’) so that exposure to this factor will favor the emergence of a second hit that will in turn drive cancer development. (C) In other cases, exposure to a certain environmental factor causes epigenetic reprogramming that increases the cancer susceptibility of the primed cell.
Malignant epigenetic rewiring in stem cells is not restricted to the hematopoietic system, but rather represents a common tumoral mechanism that is also at work in non-hematopoietic tumors. For example, the EWS-FLI-1 fusion gene, associated with many Ewing sarcoma tumors, triggers the expression of the embryonic stem cell genes OCT4, SOX2, and NANOG 410
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Figure 2. Cancer Epigenetic Priming. (A) In the current view of the initiation and progression of cancer, an initiating hit is required to immortalize a target cell. These cells are then predisposed to acquire additional genetic hits over time. The acquisition of additional hits further deregulates cell behavior leading to subclonal genetic heterogeneity. Specific subclones may contribute to the initiation of cancer, resistance to therapy, or relapse. In human cancers and in most animal models of cancer, oncogenic alterations are expressed in all malignant cells, from target cells to terminal differentiated cells. (B) Epigenetic priming model. The cancer-initiating effect of the oncogenic hit occurs in the stem/progenitor compartment, where the oncogene imposes an epigenetic regulatory state that does not interfere with normal differentiation but may become active at later developmental stages in response to external or internal factors, leading to the appearance of tumor-differentiated cells. (C) Can epigenetic priming be the sole driver of cancer? A key open question is how the normal epigenetic mechanisms that regulate physiological development and cellular identity are destabilized in such a way that oncogenic priming can persist and be propagated.
Box 1. Modeling Epigenetic Priming In Vivo In animal models of hematopoietic tumors, various genetic lesions associated with different hematopoietic cancers can epigenetically reprogram the stem cells and create a differentiation state from which tumor cells with differing oncogenespecific properties emerge [6,14–16,70,71]. CML is a stem cell tumor characterized by the presence of the chimeric BCR-ABLp210 oncogene. Accordingly, when a mouse model was generated in which the expression of BCR-ABL was restricted to HSCs/PCs (using the locus control region of the Sca1+ gene), mice developed CML [6]. In this experimental model the oncogene expression is switched off in the tumor-differentiated cells that form the main mass of the tumor, although leukemia initiation has occurred in the stem cell/progenitor population. The fact that CML arises under these circumstances indicates that sustained BCR-ABL expression is not required for the generation of differentiated tumor cells. These results strongly support the existence of an epigenetic priming-like mechanism in cancer development. A challenging disease to test this hypothesis would be a tumor where the main cell type is a mature differentiated cell, as in the case of MM or mature B cell lymphoma. The phenotypes and biology of MM (induced by the MafB oncogene) and of B cell lymphoma (induced either by the MALT1 or the BCL6 oncogene) can be accurately mimicked with the Sca1mediated stem cell targeting system described previously [12,14,16,71–73]. These results imply that the oncogene establishes an epigenetic program in the stem cells that in some way persists during differentiation and that will finally lead to a mature tumoral phenotype [12,14,16,52,64,71–73].
when present in human pediatric mesenchymal stem cells but not in adult cells and reprograms mesenchymal cells to Ewing sarcoma CSCs [33]. Similarly, the synovial sarcoma-associated oncogene SYT-SSX2 can reprogram mesenchymal stem cells by promoting differentiation towards a proneural lineage, which most likely constitutes the primary tumorigenic event in synovial sarcomas [34]. A similar event was recently described in chondroblastomas [35]. The driving of cancer by malignant epigenetic rewiring of stem cells also occurs in epithelial tumors such as bladder cancers [36], skin, ovarian, pancreatic, and prostate [37–39] and in brain
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tumors [40–42]. Some tumors, like ependymomas, are almost purely epigenetic with minimal genetic alterations [43]. A ‘hit-and-run’ epigenetic priming model, in which the oncogene is required only in the first reprogramming moment (the ‘hit’) and dispensable afterwards (it may ‘run away’), is not exclusive to cancer development. During normal hematopoietic development, cytokines, for example, are often required to trigger specific differentiation programs but are not needed once the programs are established. During reprogramming to pluripotency, Yamanaka factors are also not necessary to maintain pluripotency [5,44]. If cancers develop through an epigenetic priming mechanism, then, the oncogenes necessary to initiate the tumor might be dispensable for the ensuing tumor survival and/or progression.
Lineage Decisions in Hematopoiesis and Leukemias The traditional view of HSC differentiation is as a series of dichotomic branching steps diverting into mutually exclusive stable progenitor states. However, recent work suggests that hematopoiesis occurs through a mechanism of continuous lineage priming [45] and therefore the architecture of the system is much less compartmentalized than was previously considered, and more versatile in terms of lineage plasticity. ‘Defined’ progenitor populations can be mixtures of cells with several differentiation capabilities [46,47], in such a way that developing hematopoietic cells are gradually biased towards their final fates without sudden black-or-white developmental steps. This new point of view widens our understanding of preleukemic stem cells (pre-LSCs). It was shown in leukemias that the pre-LSCs possess multilineage potential, as in the case of CML, where preleukemic BCR-ABLp210+ stem cells can give rise to all different blood cell types [48]. In MDS a multipotent malignant stem cell gives rise to refractory anemias [18]. Preleukemic stem cells with multilineage differentiation potential suggest that initiating mutations arise in normal HSCs and the acquisition of additional mutations triggered by secondary events leads to subclones of lineage-restricted malignant blasts. This two-hit model of leukemogenesis (not to be confused with Knudson’s model of sequential inactivation of recessive tumor suppressors) relies on the stepwise acquisition and collaboration of two main groups of mutations: (i) those affecting genes of transcriptional or epigenetic regulators that can modify or restrict lineage options (e.g., the generation of chimeric oncogenes such as RUNX1-RUNX1T1 or PML-RARA, BCR-ABLp190, ETV6-RUNX1, or mutations in CEBPA, PAX5, or NPM1); and (ii) those activating signal-transduction pathways that confer survival or proliferative advantages (e.g., mutations in FLT3, RAS, or KIT). Therefore, the preleukemic oncogenic lesion is stably maintained as a single alteration in an abnormal cell population and will progress to open leukemia only when a secondary hit occurs [10,11] (Figure 3). The order of these alterations can be reversed, particularly in hematopoietic tumors (Figure 1); that is, epigenetic regulators are often the founding mutation (e.g., translocations affecting MLL genes in infant leukemias) and epigenetic priming precedes the acquisition of additional oncogenic drivers. The same gene can also behave differently at different stages of tumor evolution in different cancer types; for example, PAX5 can be either a susceptibility first-hit gene [49,50] or a mutated second hit in ALLs [51,52]. The requirement of multiple hits for full tumor development relates to the fact that changes that take cells away from their fate are inherently evolutionarily disfavored, and biological barriers are in place to minimize the risk of malignant transformation [5]. How easily each one of these differentiation intermediates can be primed towards a tumoral fate is a property that has been recently named cellular pliancy [53,54]. 412
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Figure 3. Molecular Underpinnings of Tumor Epigenetic Priming. In the initial stages of cancer development, a normal cell (A), the cancer cell of origin, which initially exhibits normal chromatin modifications according to its identity and developmental stage, is going to become a precancer cell (B) by the action of a given oncogenic hit [e.g., ETV6-RUNX1 chimeric fusion gene in B acute lymphoblastic leukemia (B-ALL), IDH1 mutation in acute myelogenous leukemia (AML), Pax5 haploinsufficiency in familial cases of B-ALL, BRCA1 mutations in breast cancer] or by exposure to an environmental factor (tobacco smoke in lung adenocarcinoma); both can ‘reset’ the epigenetic and/or transcriptome status and reprogram the epigenome to give rise to a precancer cell. Once its role in oncogenic reprogramming is performed, the initiating hit is no longer necessary for tumor progression. (C) The malignant epigenetic priming will remain silent until specific second events trigger the appearance of cancer. These second hits can occur randomly or be triggered by environmental exposures (infections in childhood B-ALL, K-RAS mutations in lung adenocarcinoma). Blue cylinders represent nucleosomes, red and white circles symbolize changes in DNA methylation, and green and orange curved lines denote histone modifications.
Epigenetic Priming and Environmental Signals Epigenomic studies in various animal models of hematologic cancers [14–16] and solid tumors such as chondroblastomas [35], ovarian, pancreatic, and prostate carcinomas [38,39,42], and brain tumors [40,41] provided insight into the precise molecular mechanisms by which the first (epi)genetic hit induces the epigenetic reprogramming of stem cells that culminates in the generation of tumor-differentiated cells (Figure 2). An important remaining question is how can the oncogenic epigenetically primed patterns escape the action of the normal epigenetic regulatory mechanisms? The clinical implication is important, since the frequency of healthy people carrying precancer cells is higher than was initially thought [55].
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During cancer development, the tumor-cell identity is highly specific and mostly unique for each different oncogenic-hit–tumor-type association, especially in hematopoietic tumors (ETV6RUNX1 with childhood B-ALL, BCR-ABLp210 with CML, PML-RARa with promyelocytic leukemia). By analogy with the reprogramming to pluripotency (iPSC) process, tumor epigenetic priming can possibly occur through different stages and mechanisms [56–59]. The persistence of epigenetic memory and the capacity to erase it (or not) is an important topic in the context of tumor priming, because this lingering memory can modify the behavior of tumor epigenetically primed cells and have important consequences for the modeling and prevention of cancer. Tumor-differentiated cells have epigenetic memory as a consequence of the reprogramming that occurred at the stem cell level [14,16,38]. Therefore, there is an epigenetic mark that is inherited throughout tumor development. As in the case of the generation of iPSCs, tumor generation involves at first epigenetic rather than genetic modifications. This opens a new window of opportunity for cancer prevention therapies in precancerous carriers, since epigenetic modifications, unlike genetic mutations, can be pharmacologically modified.
Epigenetic Priming and Gene–Environment Interaction Precancer lesions can exist in a cell population that will progress to full-blown cancer only as a result of secondary hits [10,11]. This malignant epigenetic priming can occur early in life. Sometimes these second hits might arise as a consequence of ‘bad luck’ according to the model of Vogelstein (i.e., in a random, unpredictable, and largely unpreventable manner) [60] (Figure 3). We currently know that epigenetic priming is highly plastic and responsive to environmental factors, which led to the proposal of a more complex model explaining the role of the gene–environment interaction in cancer development [61–63]. ‘Gene–environment interaction’ refers to the increased sensitivity of individuals carrying a specific germline or acquired alteration to certain environmental exposures (Figure 3). The best example of this is childhood B-ALL, in which the first oncogenic hit, which occurs through stem cell tumor epigenetic reprogramming, gives rise to a preleukemic clone that remains harmless until the child is exposed to other events, such as infections with common agents [15,64,65]. Similarly, inherited genetic factors (e.g., malignant reprogramming triggered by BRCA1 mutations) and environmental factors (e.g., exposure to exogenous hormones) contribute to the risk of developing breast cancer (Figure 3). In other cases the mechanism by which environmental exposures amplify the vulnerability of adults to cancer is by epigenetic priming rather than by direct induction of genetic mutations (Figure 3). In this case the effects of reprogramming should be detectable in the ‘healthy’ (but reprogrammed) cells before the development of cancer; for example, the way in which obesity drives epigenomic alterations in the colonic epithelium [66,67] or cigarette smoke causes epigenomic changes before the sensitization of bronchial cells to transformation by KRAS mutations [68]. Epidemiological studies also show a positive association between body weight at birth and the risk of developing childhood leukemia or adult breast cancer. This implies that, although some epigenetic reprogramming can be observed immediately after exposure to exogenous or endogenous agents, both aberrant epigenetic programming and altered disease susceptibility may manifest only later in life, long after the exposure occurred. This correlates well with the increase in epigenetic alterations of various types associated with aging, one of the most important cancer-driving forces [69]. Together these data lead us to propose that ‘cancer as a result of epigenetic priming’ is a type of gene–environment interaction that can cooperate with a genetic predisposition, not by inducing mutations, but by reprogramming the epigenome to modulate gene expression to promote cancer development (Box 2). 414
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Box 2. Cancer Therapy Directed at the Epigenome of Precancer Cells
Outstanding Questions
The epigenetic priming model posits that the main contribution of oncogenes to tumor development is reprogramming the epigenome of the cell of origin. It was recently shown that epigenetic reprogramming is the driving force behind intratumoral heterogeneity [74] and is the mechanism used by tumors to evade CD19 CAR immune therapy [75,76]. In addition, cancer cells have been reprogrammed to non-tumoral fates, losing their malignancy. For example, it is possible to produce mouse embryos from brain tumor-derived cells [77] and to reprogram embryonal carcinomas [78] or melanoma cells using nuclear transplantation [79]. Similarly, B-ALL cells have been reprogrammed to an alternativelineage cell fate without a malignant phenotype [80]. Recently, it was also shown that epigenetic reprogramming can be exploited in therapy to kill leukemia stem cells [81] or used to treat pediatric brain cancer [82,83]. The TET2 enzyme catalyzes the oxidation of 5-methylcytosine (5mC) ultimately leading to DNA demethylation. Loss-of-function mutations in TET2 occur frequently in acute leukemia and are associated with a DNA hypermethylation phenotype. In a Tet2deficient mouse model, treatment with vitamin C mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in HSCs/PCs, and it also suppresses human leukemic colony formation and leukemia progression in primary human leukemia patient-derived xenografts [26], indicating that rewiring the epigenetic programming of tumor cells is a viable therapeutic prospect. As for any other type of therapy, a detailed understanding of the epigenetic rewiring is a prerequisite for any potential intervention.
Can epigenetic priming be the sole driver of cancer? Can we learn to detect the priming before the development of a full-blown tumor? Can the epigenome be reverted to the one compatible with normal cellular development and function? Can we block specific epigenetic priming caused by different exposures?
Concluding Remarks As always occurs with theories offering new points of view, the epigenetic priming model opens new questions and possibilities (see Outstanding Questions). From the point of view of cancer prevention, we may identify specific exposures as triggers of epigenetic priming and, conversely, we may try to prevent subgroups of susceptible individuals from being exposed to environments that can trigger cancer (e.g., infections in ETV6-RUNX1carrying children). In terms of diagnosis and biomarkers, epigenetic marks are certainly more difficult to identify than classical genetic mutations. Hypothetically, epigenetic marks could be used to identify, for example, preleukemic cells. However, even with highly sensitive PCR approaches it remains technically challenging to identify carriers of preleukemic clones, like ETV6-RUNX1+ cells in pBALL [55]. Furthermore, at present we cannot genetically or epigenetically distinguish preleukemic stem cells from more differentiated preleukemic cellular descendant progenitors. With the development of new detection technologies, the identification of aberrant patterns of differentiation might become feasible in the future.
The decision to initiate cancer: does it occur at only a single time point during the tumoral differentiation process or does it comprise a series of consecutive decisions required to fully switch to a cancer-cell fate? Are all of the tumor-priming decisions cell autonomous? In other words, is a role played by the surrounding cells and extracellular matrix in the priming and/or evolution of the cancer cell of origin? What is the precise nature of the epigenetic pathways triggered by cancerinitiating gene defects?
From a therapeutic perspective, the existence of an epigenetically driven mechanism of tumor initiation clearly opens new possibilities to prevent or abolish cancer, since epigenetic modifications, unlike genetic changes, can be erased, manipulated, and reinitiated even before a precancerous cell might evolve into cancer. Acknowledgments This Opinion article is dedicated to the memory of the authors’ friend and colleague Ester Alonso-Escudero. They are indebted to all members of their groups for useful discussions and for their critical reading of the manuscript. Research in the C.V-D. group is partially supported by FEDER, a Miguel Servet grant (CP14/00082 – AES 2013–2016) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad), the Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III (PI17/00167), and the Lady Tata International Award for Research in Leukaemia 2016–2017. J.H. has been supported by the German Children’s Cancer Foundation, DJCLS 02R/2016, KKS A2016/07, and the Forschungskommission of the Medical Faculty of Heinrich Heine University. Research at C.C.’s laboratory was partially supported by FEDER, the Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III (PI14/00025), MINECO (SAF2017-83061), and the Fundación Ramón Areces. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged. A.B. has been supported by the German Children’s Cancer Foundation and the Federal Ministry of Education and Research, Bonn, Germany. Research in I.S-G.’s group is partially supported by FEDER and by MINECO (SAF2012-32810, SAF2015-64420-R, and Red de Excelencia Consolider OncoBIOSAF2014-57791-REDC),
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the Instituto de Salud Carlos III (PIE14/00066), ISCIII – Plan de Ayudas IBSAL 2015 Proyectos Integrados (IBY15/00003), Junta de Castilla y León (BIO/SA51/15, CSI001U14, UIC-017, and CSI001U16), and the Fundacion Inocente Inocente. I. S-G.’s laboratory is a member of the EuroSyStem and the DECIDE Network funded by the EU under the FP7 Programme. A.B. and I.S-G. have been supported by the German Carreras Foundation (DJCLS R13/26).
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Epigenetic Priming in Cancer Initiation Carolina Vicente-Dueñas,1,5 Julia Hauer,2,5 César Cobaleda,3,6,* Arndt Borkhardt,2,6,* and Isidro Sánchez-García1,4,6,* Recent evidence from hematopoietic and epithelial tumors revealed that the contribution of oncogenes to cancer development is mediated mainly through epigenetic priming of cancer-initiating cells, suggesting that genetic lesions that initiate the cancer process might be dispensable for the posterior tumor progression and maintenance. Epigenetic priming may remain latent until it is later triggered by endogenous or environmental stimuli. This Opinion article addresses the impact of epigenetic priming in cancer development and in the design of new therapeutic approaches. Epigenetic Priming in Cancer Development Despite the enormous amount of data that we have gathered in the past four decades on the biology of tumor cells, our capacity for controlling the development of cancer unfortunately remains limited. We do not know yet how to prevent the conversion of a precancer cell (see Glossary) into a tumor, mainly due to the fact that the early events triggering the commitment to a new cancer lineage remain largely unknown [1]. A crucial point in the history of a tumor is the transition of a normal cell to a malignant state. Therefore, an unmet need in cancer research is to understand how to prevent the conversion of a precancer cell into a full-blown tumor. The mechanisms that establish and regulate cellular identity allow the emergence of aberrant cells. In this Opinion article, we discuss the importance of oncogenic hits in establishing the cell fate of the cancer-initiating cell and how tumor-primed cells evolve. Better understanding of largely neglected role of oncogenes may help in developing ways to attack precancer cellular states.
Interplay between the Oncogenic Hit and the Cancer-Initiating Cell Oncogene expression is necessary at the earliest stages of cancer development and for progression [2]. This seems to suggest that oncogenic hits play a similar molecular role throughout the origin, progression, and expansion of the tumor [2]. This fits with the concept of the clonal nature of human cancers, in which all tumor cells carry the same initiating oncogenic lesions. However, full-blown cancers exhibit a high degree of cellular heterogeneity, suggesting that the role of oncogenes might not be equal in all tumor cells, a fact that may explain why therapies targeting oncogenic hits have different efficacies against cells at different stages of evolution [3,4]. This could be better explained by a new model of cancer based on tumor epigenetic priming, in which an oncogenic hit ‘resets’ the epigenome and/or transcriptome of a normal cell (the cancer cell of origin) and gives rise to a pathological differentiation program that ultimately leads to tumor development (Figure 1). This concept implies a different role for an oncogenic hit in the cancer cell of origin and suggests that oncogenic alterations essential for tumor initiation and at the origin of clonal disease may not be required during tumor progression, once the cells have been primed towards malignancy [5,6] (Figure 2). Importantly, stem or progenitor cells (PCs) have a specific epigenetic program that renders them more susceptible to genetic alterations, and hence one could argue that specific developmental stages with their associated epigenetic states may be more susceptible to oncogenes and progression to cancer. This view of cancer is also supported by the fact that 408
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https://doi.org/10.1016/j.trecan.2018.04.007
Highlights During cancer development, a new tumor cell identity is established and maintained mainly through epigenetic priming of cancer-initiating cells. This priming to cancer is associated with major epigenetic changes and its outcome is influenced by the initial susceptibility of the cancer cell of origin. Epigenetic priming may remain latent until it is triggered by either endogenous or environmental stimuli. Targeting this epigenetic network to block the transformation of latent precancerous cellular states in disease appears to be a novel, promising strategy for the prevention of cancer formation in susceptibility carriers.
1 Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain 2 Department of Pediatric Oncology, Hematology, and Clinical Immunology, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany 3 Department of Cell Biology and Immunology, Centro de Biologia Molecular Severo Ochoa (CBMSO), CSIC/UAM, Madrid 28049, Spain 4 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Campus M. de Unamuno s/n, 37007 Salamanca, Spain 5 Equal first author 6 Equal senior author
*Correspondence: [email protected] (C. Cobaleda), [email protected]. de (A. Borkhardt), [email protected] (I. Sánchez-García).
polycomb group target (PCGT) genes are preferentially methylated and silenced in all cancer stem cells (CSCs) [7–9]. Finally, the epigenetic priming model is supported by the tight association between a given chromosomal aberration and a specific tumor type, a clear indication of the fate-inducing nature of oncogenes [1]. The epigenetic priming model could also explain how, in the evolution of several human tumors, a precancer lesion can be stably maintained as the only proto-oncogenic alteration in an abnormal cell population that will give rise to a full-blown tumor only in response to secondary hits [10,11]. A good example of this apparent paradox is the effect of imatinib in chronic myelogenous leukemia (CML), where the drug fails to kill BCR-ABLp210+ CSCs because, somehow, it cannot block the capacity of the fusion oncogene to prime the epigenome towards malignancy in the stem cell compartment (see below) [6,12,13].
Epigenetic Priming as a Driver of Cancer: Restriction of Lineage Options during Transformation The epigenetic priming model implies that the initiating oncogenic hit may not be required for tumor progression. This fact cannot be experimentally demonstrated in human tumors, which, by their clonal nature, carry these initiating hits throughout their whole malignant cellular population [1]. Therefore, to test this model we would need a system that isolates the role of an oncogenic hit in the cancer cell of origin. Expression of the oncogenic hit would have to be restricted to the stem/ progenitor compartment and switched off afterwards to demonstrate that later oncogene expression is not necessary for tumor progression once epigenetic priming has already occurred (Box 1). Epigenomic studies such as genome-scale maps of DNA methylation from both stem cells and mature B cells in various hematopoietic animal cancer models [MafB in multiple myeloma (MM), Bcl6 in diffuse large B cell lymphoma (DLBCL), ETV6-RUNX1 in childhood B acute lymphoblastic leukemia (B-ALL)] [14–16] provided insights into the precise molecular mechanisms by which oncogenes reprogram stem cells into differentiated tumor cells. In all cases a substantial number of CpG islands and promoters were hyper- or hypomethylated in the stem cells of these transgenic models, setting a pattern that is inherited throughout B cell development and suggesting that molecular reprogramming is involved in tumor initiation. Therefore, according to evidence from animal experimental hematopoietic systems (Box 1), we postulate that cancer-initiating oncogenic hits epigenetically modify target genes that will remain in an epigenetically primed state in mature tumors even when the oncogene is no longer present (Figure 2). A growing number of epigenetic regulators essential for normal differentiation are being found to initiate oncogenic hits, such as DNMT3A and TET2 through deregulation of DNA methylation [17,18]. Dnmt3a loss in mice predisposes hematopoietic stem cells (HSCs) to malignant transformation [19] and these animals develop a spectrum of malignancies similar to those seen in patients with DNMT3A mutations [20–25]. Loss-offunction mutations in TET2 often occur in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), or acute myelogenous leukemia (AML) and are associated with a DNA hypermethylation phenotype [26,27] and with an increased mutagenesis rate in HSCs [28,29]. Also, TET1 acts as a tumor suppressor in hematopoietic malignancies [30], and restoration of TET2 activity prevents leukemia progression [26]. Similarly, in gliomas, mutations in the protooncogenes IDH1 and IDH2 induce genome-wide chromatin changes that lead to the appearance of a precancer population with a proliferative advantage and stem-like transcriptional features [31]. Mutations in IDH1 and IDH2 are frequent and mutually exclusive in de novo AML and result in a hypermethylation phenotype and are mutually exclusive with mutations in TET2, suggesting that these lesions may be biologically redundant by fullfilling similar functions in tumor development, which can even occur at different stages of tumoral evolution [32].
Glossary Cancer cell of origin (or cancerinitiating cell): the initially healthy cell that will be primed by an oncogenic hit and that will give rise to a (pre)cancer cell. Cancer stem cell (CSC) (or cancer-maintaining cell): the CSC theory postulates that tumors are stem cell-based tissues like any other tissue in the organism. This implies the existence of a hierarchical structure in the tumor where not all of the cancer cells are equally competent in regenerating the tumor. The CSCs are the only cells that regenerate and maintain the whole tumor. Epigenetic memory: in the context of tumor origin and development, epigenetic memory refers to the retention of transcriptional properties or chromatin or DNA methylation marks imposed by an oncogenic hit in the starting cancer cell of origin that are afterwards maintained from precancer to tumoral differentiation. Oncogenic hit: any molecular alteration that leads to the establishment of a precancer or cancer cellular state. It may be the activation of an oncogene (by mutation, gene fusion, gene amplification), the inactivation of a tumor suppressor, or an epigenetic alteration. Precancer cell: a cell that has already been primed towards malignancy but retains normal developmental and physiological behavior. Depending on susceptibility and environmental exposure, a precancer cell may or may not give rise to a full-blown tumor. Susceptibility: increased sensitivity of a cell (or an individual) to suffer negative consequences (e.g., precancer or cancer development) from exposure to a given agent or condition. It can be due to genetic (mono- or polygenic) or epigenetic causes and these can be congenital or appear de novo by exposure to related or unrelated agents or conditions.
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Figure 1. Gene–Environment Interactions in Cancer Development. Tumoral epigenetic priming can occur at any point in life and can remain dormant in the cancer cell of origin unless other posterior events trigger further tumor progression. This priming will be preserved through tumor evolution even if the initiating hit is no longer present or expressed. (A) Sometimes the second event can arise randomly. (B) However, a given epigenetic priming may be particularly sensitive to a certain environmental factor (‘gene–environment interaction’) so that exposure to this factor will favor the emergence of a second hit that will in turn drive cancer development. (C) In other cases, exposure to a certain environmental factor causes epigenetic reprogramming that increases the cancer susceptibility of the primed cell.
Malignant epigenetic rewiring in stem cells is not restricted to the hematopoietic system, but rather represents a common tumoral mechanism that is also at work in non-hematopoietic tumors. For example, the EWS-FLI-1 fusion gene, associated with many Ewing sarcoma tumors, triggers the expression of the embryonic stem cell genes OCT4, SOX2, and NANOG 410
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Figure 2. Cancer Epigenetic Priming. (A) In the current view of the initiation and progression of cancer, an initiating hit is required to immortalize a target cell. These cells are then predisposed to acquire additional genetic hits over time. The acquisition of additional hits further deregulates cell behavior leading to subclonal genetic heterogeneity. Specific subclones may contribute to the initiation of cancer, resistance to therapy, or relapse. In human cancers and in most animal models of cancer, oncogenic alterations are expressed in all malignant cells, from target cells to terminal differentiated cells. (B) Epigenetic priming model. The cancer-initiating effect of the oncogenic hit occurs in the stem/progenitor compartment, where the oncogene imposes an epigenetic regulatory state that does not interfere with normal differentiation but may become active at later developmental stages in response to external or internal factors, leading to the appearance of tumor-differentiated cells. (C) Can epigenetic priming be the sole driver of cancer? A key open question is how the normal epigenetic mechanisms that regulate physiological development and cellular identity are destabilized in such a way that oncogenic priming can persist and be propagated.
Box 1. Modeling Epigenetic Priming In Vivo In animal models of hematopoietic tumors, various genetic lesions associated with different hematopoietic cancers can epigenetically reprogram the stem cells and create a differentiation state from which tumor cells with differing oncogenespecific properties emerge [6,14–16,70,71]. CML is a stem cell tumor characterized by the presence of the chimeric BCR-ABLp210 oncogene. Accordingly, when a mouse model was generated in which the expression of BCR-ABL was restricted to HSCs/PCs (using the locus control region of the Sca1+ gene), mice developed CML [6]. In this experimental model the oncogene expression is switched off in the tumor-differentiated cells that form the main mass of the tumor, although leukemia initiation has occurred in the stem cell/progenitor population. The fact that CML arises under these circumstances indicates that sustained BCR-ABL expression is not required for the generation of differentiated tumor cells. These results strongly support the existence of an epigenetic priming-like mechanism in cancer development. A challenging disease to test this hypothesis would be a tumor where the main cell type is a mature differentiated cell, as in the case of MM or mature B cell lymphoma. The phenotypes and biology of MM (induced by the MafB oncogene) and of B cell lymphoma (induced either by the MALT1 or the BCL6 oncogene) can be accurately mimicked with the Sca1mediated stem cell targeting system described previously [12,14,16,71–73]. These results imply that the oncogene establishes an epigenetic program in the stem cells that in some way persists during differentiation and that will finally lead to a mature tumoral phenotype [12,14,16,52,64,71–73].
when present in human pediatric mesenchymal stem cells but not in adult cells and reprograms mesenchymal cells to Ewing sarcoma CSCs [33]. Similarly, the synovial sarcoma-associated oncogene SYT-SSX2 can reprogram mesenchymal stem cells by promoting differentiation towards a proneural lineage, which most likely constitutes the primary tumorigenic event in synovial sarcomas [34]. A similar event was recently described in chondroblastomas [35]. The driving of cancer by malignant epigenetic rewiring of stem cells also occurs in epithelial tumors such as bladder cancers [36], skin, ovarian, pancreatic, and prostate [37–39] and in brain
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tumors [40–42]. Some tumors, like ependymomas, are almost purely epigenetic with minimal genetic alterations [43]. A ‘hit-and-run’ epigenetic priming model, in which the oncogene is required only in the first reprogramming moment (the ‘hit’) and dispensable afterwards (it may ‘run away’), is not exclusive to cancer development. During normal hematopoietic development, cytokines, for example, are often required to trigger specific differentiation programs but are not needed once the programs are established. During reprogramming to pluripotency, Yamanaka factors are also not necessary to maintain pluripotency [5,44]. If cancers develop through an epigenetic priming mechanism, then, the oncogenes necessary to initiate the tumor might be dispensable for the ensuing tumor survival and/or progression.
Lineage Decisions in Hematopoiesis and Leukemias The traditional view of HSC differentiation is as a series of dichotomic branching steps diverting into mutually exclusive stable progenitor states. However, recent work suggests that hematopoiesis occurs through a mechanism of continuous lineage priming [45] and therefore the architecture of the system is much less compartmentalized than was previously considered, and more versatile in terms of lineage plasticity. ‘Defined’ progenitor populations can be mixtures of cells with several differentiation capabilities [46,47], in such a way that developing hematopoietic cells are gradually biased towards their final fates without sudden black-or-white developmental steps. This new point of view widens our understanding of preleukemic stem cells (pre-LSCs). It was shown in leukemias that the pre-LSCs possess multilineage potential, as in the case of CML, where preleukemic BCR-ABLp210+ stem cells can give rise to all different blood cell types [48]. In MDS a multipotent malignant stem cell gives rise to refractory anemias [18]. Preleukemic stem cells with multilineage differentiation potential suggest that initiating mutations arise in normal HSCs and the acquisition of additional mutations triggered by secondary events leads to subclones of lineage-restricted malignant blasts. This two-hit model of leukemogenesis (not to be confused with Knudson’s model of sequential inactivation of recessive tumor suppressors) relies on the stepwise acquisition and collaboration of two main groups of mutations: (i) those affecting genes of transcriptional or epigenetic regulators that can modify or restrict lineage options (e.g., the generation of chimeric oncogenes such as RUNX1-RUNX1T1 or PML-RARA, BCR-ABLp190, ETV6-RUNX1, or mutations in CEBPA, PAX5, or NPM1); and (ii) those activating signal-transduction pathways that confer survival or proliferative advantages (e.g., mutations in FLT3, RAS, or KIT). Therefore, the preleukemic oncogenic lesion is stably maintained as a single alteration in an abnormal cell population and will progress to open leukemia only when a secondary hit occurs [10,11] (Figure 3). The order of these alterations can be reversed, particularly in hematopoietic tumors (Figure 1); that is, epigenetic regulators are often the founding mutation (e.g., translocations affecting MLL genes in infant leukemias) and epigenetic priming precedes the acquisition of additional oncogenic drivers. The same gene can also behave differently at different stages of tumor evolution in different cancer types; for example, PAX5 can be either a susceptibility first-hit gene [49,50] or a mutated second hit in ALLs [51,52]. The requirement of multiple hits for full tumor development relates to the fact that changes that take cells away from their fate are inherently evolutionarily disfavored, and biological barriers are in place to minimize the risk of malignant transformation [5]. How easily each one of these differentiation intermediates can be primed towards a tumoral fate is a property that has been recently named cellular pliancy [53,54]. 412
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Figure 3. Molecular Underpinnings of Tumor Epigenetic Priming. In the initial stages of cancer development, a normal cell (A), the cancer cell of origin, which initially exhibits normal chromatin modifications according to its identity and developmental stage, is going to become a precancer cell (B) by the action of a given oncogenic hit [e.g., ETV6-RUNX1 chimeric fusion gene in B acute lymphoblastic leukemia (B-ALL), IDH1 mutation in acute myelogenous leukemia (AML), Pax5 haploinsufficiency in familial cases of B-ALL, BRCA1 mutations in breast cancer] or by exposure to an environmental factor (tobacco smoke in lung adenocarcinoma); both can ‘reset’ the epigenetic and/or transcriptome status and reprogram the epigenome to give rise to a precancer cell. Once its role in oncogenic reprogramming is performed, the initiating hit is no longer necessary for tumor progression. (C) The malignant epigenetic priming will remain silent until specific second events trigger the appearance of cancer. These second hits can occur randomly or be triggered by environmental exposures (infections in childhood B-ALL, K-RAS mutations in lung adenocarcinoma). Blue cylinders represent nucleosomes, red and white circles symbolize changes in DNA methylation, and green and orange curved lines denote histone modifications.
Epigenetic Priming and Environmental Signals Epigenomic studies in various animal models of hematologic cancers [14–16] and solid tumors such as chondroblastomas [35], ovarian, pancreatic, and prostate carcinomas [38,39,42], and brain tumors [40,41] provided insight into the precise molecular mechanisms by which the first (epi)genetic hit induces the epigenetic reprogramming of stem cells that culminates in the generation of tumor-differentiated cells (Figure 2). An important remaining question is how can the oncogenic epigenetically primed patterns escape the action of the normal epigenetic regulatory mechanisms? The clinical implication is important, since the frequency of healthy people carrying precancer cells is higher than was initially thought [55].
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During cancer development, the tumor-cell identity is highly specific and mostly unique for each different oncogenic-hit–tumor-type association, especially in hematopoietic tumors (ETV6RUNX1 with childhood B-ALL, BCR-ABLp210 with CML, PML-RARa with promyelocytic leukemia). By analogy with the reprogramming to pluripotency (iPSC) process, tumor epigenetic priming can possibly occur through different stages and mechanisms [56–59]. The persistence of epigenetic memory and the capacity to erase it (or not) is an important topic in the context of tumor priming, because this lingering memory can modify the behavior of tumor epigenetically primed cells and have important consequences for the modeling and prevention of cancer. Tumor-differentiated cells have epigenetic memory as a consequence of the reprogramming that occurred at the stem cell level [14,16,38]. Therefore, there is an epigenetic mark that is inherited throughout tumor development. As in the case of the generation of iPSCs, tumor generation involves at first epigenetic rather than genetic modifications. This opens a new window of opportunity for cancer prevention therapies in precancerous carriers, since epigenetic modifications, unlike genetic mutations, can be pharmacologically modified.
Epigenetic Priming and Gene–Environment Interaction Precancer lesions can exist in a cell population that will progress to full-blown cancer only as a result of secondary hits [10,11]. This malignant epigenetic priming can occur early in life. Sometimes these second hits might arise as a consequence of ‘bad luck’ according to the model of Vogelstein (i.e., in a random, unpredictable, and largely unpreventable manner) [60] (Figure 3). We currently know that epigenetic priming is highly plastic and responsive to environmental factors, which led to the proposal of a more complex model explaining the role of the gene–environment interaction in cancer development [61–63]. ‘Gene–environment interaction’ refers to the increased sensitivity of individuals carrying a specific germline or acquired alteration to certain environmental exposures (Figure 3). The best example of this is childhood B-ALL, in which the first oncogenic hit, which occurs through stem cell tumor epigenetic reprogramming, gives rise to a preleukemic clone that remains harmless until the child is exposed to other events, such as infections with common agents [15,64,65]. Similarly, inherited genetic factors (e.g., malignant reprogramming triggered by BRCA1 mutations) and environmental factors (e.g., exposure to exogenous hormones) contribute to the risk of developing breast cancer (Figure 3). In other cases the mechanism by which environmental exposures amplify the vulnerability of adults to cancer is by epigenetic priming rather than by direct induction of genetic mutations (Figure 3). In this case the effects of reprogramming should be detectable in the ‘healthy’ (but reprogrammed) cells before the development of cancer; for example, the way in which obesity drives epigenomic alterations in the colonic epithelium [66,67] or cigarette smoke causes epigenomic changes before the sensitization of bronchial cells to transformation by KRAS mutations [68]. Epidemiological studies also show a positive association between body weight at birth and the risk of developing childhood leukemia or adult breast cancer. This implies that, although some epigenetic reprogramming can be observed immediately after exposure to exogenous or endogenous agents, both aberrant epigenetic programming and altered disease susceptibility may manifest only later in life, long after the exposure occurred. This correlates well with the increase in epigenetic alterations of various types associated with aging, one of the most important cancer-driving forces [69]. Together these data lead us to propose that ‘cancer as a result of epigenetic priming’ is a type of gene–environment interaction that can cooperate with a genetic predisposition, not by inducing mutations, but by reprogramming the epigenome to modulate gene expression to promote cancer development (Box 2). 414
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Box 2. Cancer Therapy Directed at the Epigenome of Precancer Cells
Outstanding Questions
The epigenetic priming model posits that the main contribution of oncogenes to tumor development is reprogramming the epigenome of the cell of origin. It was recently shown that epigenetic reprogramming is the driving force behind intratumoral heterogeneity [74] and is the mechanism used by tumors to evade CD19 CAR immune therapy [75,76]. In addition, cancer cells have been reprogrammed to non-tumoral fates, losing their malignancy. For example, it is possible to produce mouse embryos from brain tumor-derived cells [77] and to reprogram embryonal carcinomas [78] or melanoma cells using nuclear transplantation [79]. Similarly, B-ALL cells have been reprogrammed to an alternativelineage cell fate without a malignant phenotype [80]. Recently, it was also shown that epigenetic reprogramming can be exploited in therapy to kill leukemia stem cells [81] or used to treat pediatric brain cancer [82,83]. The TET2 enzyme catalyzes the oxidation of 5-methylcytosine (5mC) ultimately leading to DNA demethylation. Loss-of-function mutations in TET2 occur frequently in acute leukemia and are associated with a DNA hypermethylation phenotype. In a Tet2deficient mouse model, treatment with vitamin C mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in HSCs/PCs, and it also suppresses human leukemic colony formation and leukemia progression in primary human leukemia patient-derived xenografts [26], indicating that rewiring the epigenetic programming of tumor cells is a viable therapeutic prospect. As for any other type of therapy, a detailed understanding of the epigenetic rewiring is a prerequisite for any potential intervention.
Can epigenetic priming be the sole driver of cancer? Can we learn to detect the priming before the development of a full-blown tumor? Can the epigenome be reverted to the one compatible with normal cellular development and function? Can we block specific epigenetic priming caused by different exposures?
Concluding Remarks As always occurs with theories offering new points of view, the epigenetic priming model opens new questions and possibilities (see Outstanding Questions). From the point of view of cancer prevention, we may identify specific exposures as triggers of epigenetic priming and, conversely, we may try to prevent subgroups of susceptible individuals from being exposed to environments that can trigger cancer (e.g., infections in ETV6-RUNX1carrying children). In terms of diagnosis and biomarkers, epigenetic marks are certainly more difficult to identify than classical genetic mutations. Hypothetically, epigenetic marks could be used to identify, for example, preleukemic cells. However, even with highly sensitive PCR approaches it remains technically challenging to identify carriers of preleukemic clones, like ETV6-RUNX1+ cells in pBALL [55]. Furthermore, at present we cannot genetically or epigenetically distinguish preleukemic stem cells from more differentiated preleukemic cellular descendant progenitors. With the development of new detection technologies, the identification of aberrant patterns of differentiation might become feasible in the future.
The decision to initiate cancer: does it occur at only a single time point during the tumoral differentiation process or does it comprise a series of consecutive decisions required to fully switch to a cancer-cell fate? Are all of the tumor-priming decisions cell autonomous? In other words, is a role played by the surrounding cells and extracellular matrix in the priming and/or evolution of the cancer cell of origin? What is the precise nature of the epigenetic pathways triggered by cancerinitiating gene defects?
From a therapeutic perspective, the existence of an epigenetically driven mechanism of tumor initiation clearly opens new possibilities to prevent or abolish cancer, since epigenetic modifications, unlike genetic changes, can be erased, manipulated, and reinitiated even before a precancerous cell might evolve into cancer. Acknowledgments This Opinion article is dedicated to the memory of the authors’ friend and colleague Ester Alonso-Escudero. They are indebted to all members of their groups for useful discussions and for their critical reading of the manuscript. Research in the C.V-D. group is partially supported by FEDER, a Miguel Servet grant (CP14/00082 – AES 2013–2016) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad), the Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III (PI17/00167), and the Lady Tata International Award for Research in Leukaemia 2016–2017. J.H. has been supported by the German Children’s Cancer Foundation, DJCLS 02R/2016, KKS A2016/07, and the Forschungskommission of the Medical Faculty of Heinrich Heine University. Research at C.C.’s laboratory was partially supported by FEDER, the Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III (PI14/00025), MINECO (SAF2017-83061), and the Fundación Ramón Areces. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged. A.B. has been supported by the German Children’s Cancer Foundation and the Federal Ministry of Education and Research, Bonn, Germany. Research in I.S-G.’s group is partially supported by FEDER and by MINECO (SAF2012-32810, SAF2015-64420-R, and Red de Excelencia Consolider OncoBIOSAF2014-57791-REDC),
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the Instituto de Salud Carlos III (PIE14/00066), ISCIII – Plan de Ayudas IBSAL 2015 Proyectos Integrados (IBY15/00003), Junta de Castilla y León (BIO/SA51/15, CSI001U14, UIC-017, and CSI001U16), and the Fundacion Inocente Inocente. I. S-G.’s laboratory is a member of the EuroSyStem and the DECIDE Network funded by the EU under the FP7 Programme. A.B. and I.S-G. have been supported by the German Carreras Foundation (DJCLS R13/26).
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