MAX and MYC: a Heritable Breakup

Published OnlineFirst June 15, 2012; DOI: 10.1158/0008-5472.CAN-11-3891

Cancer Review Research

MAX and MYC: A Heritable Breakup

Alberto Cascon 1,2 and Mercedes Robledo1,2

Abstract The overexpression of MYC, which occurs in many tumors, dramatically disrupts the equilibrium between activation and repression of the oncogenic MYC/MYC-associated protein X (MAX)/MAX dimerization protein 1 (MXD1) network, favoring MYC–MAX complexes and thereby impairing differentiation and promoting cell growth. Although for some time it has appeared that MAX is necessary for both the activation and repression of the axis, recent evidence shows that MYC retains considerable biologic function in the absence of MAX. The presence of germline MAX mutations in patients with hereditary pheochromocytoma supports the predominant role of MAX as a negative regulator of the network and suggests that MYC deregulation plays a role in hereditary cancer predisposition. This ﬁnding also conﬁrms the importance of impairment of the MYC/MAX/MXD1 axis in the development of aggressive neural tumors, because MYCN overexpression is an established genetic hallmark of malign neuroblastoma, and it is likely that MXI1 plays a relevant role in the development of medulloblastoma and glioblastoma. Finally, the likely malignant behavior of tumors with mutations in MAX points to MYC as a candidate therapeutic target in the treatment of metastatic pheochromocytoma. Cancer Res; 72(13); 1–6. 2012 AACR.

Introduction MAX probably led to the assumption that MAX integrity is MYC-associated protein X (MAX) is a ubiquitous, constitu- necessary not only for correct cell behavior but also for tumor tively expressed protein that plays a central role in controlling homeostasis, and that therefore it is unlikely that mutations MAX the MYC/MAX/MAX dimerization protein 1 (MXD1) axis, one affecting could occur in cancer. of the better-known cellular networks whose deregulation contributes to the genesis of many human cancers. MAX was MAX and Cancer the first-described bona fide MYC-interacting protein, and it MAX has gained special prominence in the field of human appears to be an essential dimerization partner for the other genetics since germline loss-of-function mutations in the MAX members of the axis to be active. Thus, whereas MYC activates gene were observed in patients with hereditary pheochromo- transcription binding to E-box DNA recognition sequences in cytoma (PCC; ref. 2), a rare neural crest cell tumor that is target gene promoters through heterodimerization with MAX, localized mainly within the adrenal medulla and rarely metas- heterodimers of MAX with MXD1 family members [MXD1, tasizes. Prior to this, 30% to 40% of patients with PCC were MAX interactor 1 (MXI1), MXD3, and MXD4], MAX-binding thought to be hereditary (3), with autosomal inheritance protein (MNT), and MAX gene–associated (MGA) antagonize caused by germline mutations affecting 1 of 9 susceptibility MYC-dependent cell transformation by transcriptional repres- genes (RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, NF1, and sion of the same E-box target DNA sequences (1). In addition, TMEM127). Mutations in MAX were identified by sequencing MAX is the only member of the network that homodimerizes the entire exome of 3 unrelated patients with a family history of efficiently, although the biologic role of these dimers remains PCC who did not have mutations in any of the known sus- unknown. The promiscuous interactions of MAX with MYC ceptibility genes (4). The patients were selected for the study and MYC repressors maintain a complex equilibrium that because their tumors were found to have very homogeneous dictates whether the transcription of thousands of target genes expression profiles in a previous transcriptional study. The will be activated or repressed. Recognition of this dual task of expression profile differentiated the 3 MAX tumors from all other PCCs, with or without germline alterations in the other PCC susceptibility genes, and suggested that they might have Authors' Affiliations: 1Hereditary Endocrine Cancer Group, Spanish the same genetic cause. The presence of germline mutations in National Cancer Research Centre; 2ISCIII Center for Biomedical Research the patients, as well as the loss of heterozygosity of the wild- on Rare Diseases, Madrid, Spain type allele and the absence of protein in the tumors, indicated Corresponding Authors: Alberto Cascon, Hereditary Endocrine Cancer that MAX is a tumor-suppressor gene that causes hereditary Group, Human Cancer Genetics Programme, Centro Nacional de Inves- tigaciones Oncologicas, Melchor Fernandez Almagro 3, Madrid 28029, PCC. Further analysis of 59 patients who were chosen because Spain. Phone: 34-91-224-6947; Fax: 34-91-224-6923; E-mail: they had bilateral PCC and/or age of onset of <30 years led to [email protected]; and Mercedes Robledo, [email protected] the detection of 5 additional cases with mutations in MAX. doi: 10.1158/0008-5472.CAN-11-3891 Most of the mutations affected highly conserved amino acids 2012 American Association for Cancer Research. within the basic helix-loop-helix leucine zipper (bHLHZip)

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domain of MAX. The bHLHZip domain is the common struc- is also noteworthy that the second hit observed for MAX- tural element among the members of this transcriptional related PCCs was the loss of heterozygosity, either by unipa- network and is responsible for the specificity and stability of rental disomy (UPD) or by loss of chromosome 14q, where homo- and heterodimer formation (HLHZip), and for DNA MAX is located (2). This is especially remarkable because loss recognition via interactions of the basic region and the major of chromosome 14q23-q32 has been described in 22% of groove (5). The presence of alterations affecting this critical primary neuroblastomas, and was inversely correlated with domain, which is involved in protein–protein interactions and MYCN amplification (11). In addition, 14q-UPD was recently DNA binding, could therefore destroy the ability of MAX to observed in neuroblastomas (12), and a significant downregu- antagonize MYC-dependent cell transformation, leading to lation of 2 microRNAs (miR-487b and miR-410) located on tumor development. In fact, 2 of the 3 MAX missense variants 14q32 was also associated with high-risk neuroblastoma (13). reported to date affect casein kinase II phosphorylation sites These findings suggest that MAX could be the target of this involved in MAX DNA binding (2). chromosomal loss, and that the deregulation of MYCN It has long been known that a homozygous chromosomal observed in neuroblastoma may occur as a result of either alteration involving the MAX gene leads to a protein that is direct overexpression of the protein or a lack of MAX-related incapable of homo- or heterodimerization and therefore is repression. It is noteworthy that no neuroblastomas were incapable of repressing transcription from E-box elements in found in MAX mutation carriers (2), although the small number PC12 cells (derived from rat adrenal PCC). Ribon and colleagues of patients studied meant that no definitive conclusion could (6) showed that the reintroduction of MAX in PC12 cells results be reached regarding the participation of MAX in neuro- in transcriptional repression and a reduction in growth rate. blastoma development. In addition, given the ubiquitous This finding both confirmed the role of MAX in the regulation of nature of MAX expression and the key role of MAX as a MYC-dependent transcriptional activation and revealed that regulator of this axis, one might expect patients with germline MYC may also function, at least in PC12 cells, in the absence mutations in this gene to develop other tumors as well. The of normally functioning MAX protein (6). The ability of MYC to analysis of a larger series of MAX mutation carriers will provide function independently of MAX was later shown in Drosophila further information about the overall involvement of MAX (reviewed in ref. 7), suggesting that the pivotal role of MAX in alterations in cancer predisposition. the MYC/MAX/MXD1 network is related more to repression Historically, most of the biologic properties of MYC have than to activation. The involvement of MAX in the genetic been attributed to its association with MAX. Moreover, susceptibility to PCC in humans also suggests that the loss of although it is known that MAX plays a pivotal role in the onco- MAX in PC12 cells contributes to the histogenesis of the primary genic function of MYC, it has been speculated that MAX may tumors rather than to the establishment of the cell line. also be required for the correct folding of MYC (14). Considering The presence of MAX mutations in patients with hereditary the essential roles of MYC, the fact that many of its biologic PCC highlights the importance of the MYC/MAX/MXD1 net- activities are independent of MAX could mean that cells need work in the development of neural crest tumors. Amplification protection mechanisms against MAX depletion. The mecha- and overexpression of MYCN are well-known genetic hall- nism by which MYC exerts its MAX-independent functions is marks in neuroblastoma (8), a neural crest cell pediatric tumor still unknown, but other known (or unknown) MYC-specific that originates primarily in the adrenal gland and is the second interacting proteins probably mediate these functions. most common type of solid tumor in children. MYCN amplification leading to mRNA and protein overexpression occurs in 20% to 25% of neuroblastomas and is strongly associated with MYC and Cancer advanced disease stages and rapid tumor progression (9). The Many, if not most, human tumors present with elevated involvement of MYCN expression in the biologic behavior of levels of MYC. Indeed, important aspects of tumor biology, these tumors remains unclear, but it was recently reported that such as proliferation, cell adhesion, and angiogenesis, are high-level expression of MYCN in neuroblastomas lacking affected by enhanced expression of MYC, which makes dereg- amplification of the MYCN locus results in a benign phenotype ulation of this oncogene a hallmark of cancer. Under normal (10). Thus, overexpression of MYCN leads to a more tumor- conditions, the stimulation of cells by internal and external igenic phenotype, but the degree of aggressiveness appears to signals leads to a rapid and transient overexpression of MYC depend directly on the MYCN copy number. Ablation of MAX's mRNA and protein that persists into the cell cycle and sub- transcriptional repression of MYC in PCC could lead to the sequently declines to low levels in quiescent cells (15, 16). In same oncogenic MYC deregulation that occurs in neuroblas- addition, MYC overexpression sensitizes primary cells to apo- toma. In this regard, the observation that 25% of patients with ptosis in response to growth-factor withdrawal, which may mutations in MAX develop metastasis suggests that a corre- provide protection against the potentially detrimental activity lation may exist between MAX loss of function and metastatic of MYC in terms of tumorigenesis (17). The oncogenic activity potential. If this is confirmed, the malignant behavior of MAX- of MYC proteins begins when their normal transcriptional related tumors would be congruous with what is known about regulation is disrupted by gene amplification and transloca- neuroblastomas, and would add weight to the idea that alteration, which then leads to abnormally increased levels of tions in the main regulator of MYC could lead to the same effect intracellular MYC. Overexpression of MYC promotes oncogen- in PCC that the amplification of MYCN provokes in neuro- ic transformation and tumorigenesis by on the one hand blastoma (i.e., highly malignant disease and poor prognosis). It activating the transcription of target genes that drive cell

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proliferation and stimulate angiogenesis, and on the other neuroblastoma. In addition, mice lacking MXI1 exhibited repressing cell differentiation (18). It is noteworthy that increased susceptibility to tumorigenesis [squamous cell car- although mutations within the open reading frame of MYC cinoma of the skin and malignant lymphoma (24)], and muta- occur infrequently, it is widely accepted that MYC deregulation tions in MXI1 were also observed in a small number of prostate is not solely restricted to translocations and amplifications at tumors (25). Despite the enhanced ability of MXI1-deficient the MYC locus. This suggests that the impact of MYC dereg- prostatic epithelium cells to proliferate, it seems that mutation ulation on cancer incidence in humans is greater than previ- of MXI1 is a minor event in prostate cancer and therefore of ously thought. Regarding cancer predisposition, inactivating unknown significance for prostate tumor development. germline mutations in MYCN have been found in patients with MNT is one of the most studied of the proposed transcrip- Feingold syndrome, and, as expected, carriers of heterozygous tional regulators of MYC because its chromosomal location MYCN alterations did not develop tumors (19). However, the (17p13.3) is frequently deleted in cancer and, like MAX, it is recently reported contribution of MAX germline mutations to ubiquitously expressed, with no fluctuations during the tran- cancer susceptibility highlights the importance of MYC regu- sition from G0-phase to S-phase. Although mice heterozygous lation in hereditary malignancies, and raises the possibility of for MNT mutations do not appear to be cancer prone, it was finding new alterations in other bHLHZip repressors of the shown that MNT-deficient mouse embryonic fibroblasts and network that are involved in tumorigenesis through their mammary glands exhibit many of the hallmark characteristics failure to repress MYC. of cells that overexpress MYC [i.e., prone to apoptosis, efficiently avoid senescence, and can be transformed with oncogenic RAS alone (26)]. Moreover, conditional deletion of MNT – MYC MAX Interacting Proteins and Cancer in T cells also leads to tumor formation (27). Nevertheless, The members of the MXD1 family (MXD1, MXI1, MXD3, and studies that focused on identifying MNT-inactivating muta- MXD4) have been shown in vivo to promote differentiation, tions in tumors with common 17p losses (e.g., medulloblas- block cellular growth and MYC-induced transformation, and toma) found no abnormalities (28). suppress the development of cancer (20). In addition, MNT is likely to be a key regulator of MYC activity, and MGA, whose biologic function remains unknown, appears to contribute to Therapeutic Strategies Targeting the the silencing of E2F- and MYC-responsive genes in quiescent MYC/MAX/MXD1 Pathway cells (21). The repression of MYC's transforming ability Site-directed mutagenesis experiments have consistently depends on the balance between the MYC–MAX and MXD/ revealed the critical role of various amino acids located within MNT/MGA–MAX complexes, and the regulating members of the bHLHZip domain of many components of the MYC/MAX/ the network exert their functions at different stages during the MXD1 network. Better knowledge of protein–protein and transition between proliferation and differentiation. Although protein–DNA interactions could help to focus future thera- distinct MYC-independent functions have been reported for peutic strategies on inhibitors of either the transforming some repressors, it seems that they could all behave in a very activity of the complex or the recognition of target genes. similar fashion. The exact significance of this putative func- Many compounds have already been tried because they inter- tional redundancy in MYC repressors is unclear, but it seems fere with MYC/MAX heterodimerization. However, given that that the differential timing of their induction ensures the MYC retains substantial MAX-independent activities, this may regulation of the axis both during MYC overexpression and not be the best strategy for all tumor types (7). On the other when MYC is subsequently downregulated. Taking into hand, it has been clearly shown that oncogene-induced tumor- account the detrimental effect of the failure to control MYC igenesis is reversible, and inactivation of a single oncogene expression (i.e., by MAX mutations), the presence of various within a primary tumor is sometimes sufficient to induce regulators is likely to be an essential cellular mechanism for tumor regression. Thus, specific inactivation of MYC was inhibiting the transforming capacities of MYC through dereg- shown to reverse the malignant properties of various tumors ulation of the network. (29, 30), and the combined inactivation of MYC and angiogen- The crucial role of MYC in cancer formed the basis of esis may be even more clinically effective (31). Another prom- numerous studies that attempted to elucidate the involvement ising therapeutic approach is to block MYC-induced neoplasia in tumorigenesis of the MXD1 family of transcriptional reg- by activating stress-signaling pathways (32). Phosphorylation ulators. MXI1 was proposed as a candidate tumor-suppressor of MYC by the protein kinase PAK2 leads to degradation of gene that accounts for the 10q deletion frequently found in MYC and therefore inhibition of proliferation and cell trans- brain tumors [i.e., 80% of glioblastoma multiforme and 15% of formation. Two recent studies (33, 34) showed that MYC primary medulloblastoma (22)]. Other findings that are con- suppression and subsequent deregulation of the MYC tran- sistent with this include the identification of a mutation scriptome can be achieved through pharmacologic inhibition affecting MXI1 in a medulloblastoma cell line, and the obser- against the BET bromodomain family of chromatin adaptors. vation that the reintroduction of MXI1 in a glioblastoma cell The finding that BET proteins act as regulatory factors for MYC line lacking endogenous MXI1 resulted in a decreased growth in various hematologic malignancies reveals a new thera- rate and an accumulation of cells in the G2–M phase (23). These peutic strategy for other tumors characterized by pathologic findings suggest that MXI1 may play a role in the development activation of MYC. As previously mentioned, aberrant MYC of other neural malignancies, as do MAX in PCC and MYCN in expression is usually due to induction caused by upstream

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Figure 1. The PIK3CA/AKT1/mTOR pathway mediates downstream activation of genes involved in multiple cell processes, such as regulation of cell growth, division, survival, and, when disrupted, tumorigenesis. In normal cells, upon binding to the GDNF family of ligands, the receptor tyrosine kinase RET triggers PIK3CA signaling via the recruitment of IRS proteins. PIK3CA subsequently activates (phosphorylates) AKT, and this activated form of the protein regulates up to 100 downstream effector proteins that are involved in cell proliferation. In addition, the AKT-mediated phosphorylation of TSC2 indirectly leads to the activation of mTOR, which regulates cell growth through phosphorylated S6K1. S6K1 also inhibits the tumor-suppressor function of MAD1 and thus leads to deregulation of MYC, cell growth, and activation of proliferation. Upstream, the pathway is negatively regulated by both PTEN, which directly antagonizes PIK3CA, and NF1 through RAS inhibition. TMEM127 is a negative regulator of mTOR, and MAX represses MYC-dependent transcriptional activation. GDNF, glial-cell-line–derived neurotrophic factor; IRS, insulin receptor substrate.

signals rather than by mutations in the gene. Therefore, the mutated PCCs (data not shown). This finding is especially inhibition of MYC would tend to target the consequence more relevant because deregulation of both the mTOR pathway and than the cause of oncogenesis (35). In addition, given that MYC the upstream PTEN/PIK3CA/AKT1 axis seems to be essential is essential for the development of a broad range of adult for the development of many PCCs [i.e., with mutations in RET, organs, blocking MYC function might systemically trigger NF1, TMEM127,orMAX (Fig. 1)]. Conditional PTEN knockout devastating and irreversible side effects. mice present with metastatic bilateral PCCs (37), and RET- Oncogenes are more attractive therapeutic targets than mediated cell-transforming capacity is critically dependent on tumor-suppressor genes because it is easier to inhibit excessive activation of the PIK3CA/AKT1 pathway (38). Furthermore, activity than to restore lost activity in tumor cells. Nevertheless, mTOR is constitutively activated in both NF1-deficient cells strategies to restore protein function are now considered and human tumors through the inactivation of tuberin by AKT viable anticancer therapeutic approaches (36), and thus may (39). Finally, the PCC susceptibility gene TMEM127 also func- be an option for MAX-related PCCs that have a potentially tions by negatively regulating the mTOR pathway (40). Con- malignant phenotype. It is noteworthy that the transcriptional sidering the transcriptional profile of MAX tumors, and the fact profile of MAX tumors shows a significant enrichment of that crosstalk between the PIK3CA/AKT1/mTOR and MYC/ mTOR pathway components when compared with other MAX/MXD1 pathways was recently proposed (41), it is likely

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that mutations in MAX not only deregulate the MYC neoplastic would be an effective therapy for patients with malignant PCC switch but also lead to impairment of the mTOR pathway and carrying germline MAX mutations. PCC development. It was reported that mTOR inhibitors (e.g., rapamycin) prevent the proliferation of human neuroblastoma Disclosure of Potential Conflicts of Interest fl cells (42), and also inhibit the development of PCC in PTEN No potential con icts of interest were disclosed. knockout mice (43). In addition, it seems that sunitinib (which Grant Support was recently shown to be a successful treatment for malignant Fondo de Investigaciones Sanitarias (PS09/00942 and P1080883 to A. Cascon PCCs) induces apoptosis in PC12 cells by inhibiting the and M. Robledo, respectively), and Mutua Madrilena~ (AP2775/2008 to M. VEGFR-2/AKT1/mTOR/S6K1 pathways. Because PC12 cells Robledo). fi constitute a speci c double-knockout model for MAX-related Received November 29, 2011; revised January 10, 2012; accepted January 20, PCC, it seems plausible that targeting the mTOR pathway 2012; published OnlineFirst June 15, 2012.

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OF6 Cancer Res; 72(13) July 1, 2012 Cancer Research

MAX and MYC: A Heritable Breakup

Alberto Cascón and Mercedes Robledo

Cancer Res Published OnlineFirst June 15, 2012.

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