The first MYC gene was identified in the late 1970s in the avian acute leukemia virus MC29. This virus was known to cause a range of malignancies and the sequence in the viral genome responsible for the transforming capacity was named v-myc (short for viral myelocytomatosis, a leukemia caused by the virus). In 1979, a cellular homologue was identified in several species and was subsequently called c-MYC, where “c” denotes cellular. In contrast to other oncogenes known at the time, MYC did not seem to be activated by point mutations in the coding sequence. Instead studies in the early 1980s led to the identification of three novel mechanisms of oncogene activation: insertional mutagenesis (virus integration into the host genome at or near proto-oncogenes resulting in high levels of expression driven by the viral promoter), chromosomal translocation, and gene amplification (see also “MYC in Cancer” below). Following the discovery of c-MYC, it was realized that it was a member of a larger family of oncogenes. In 1983, a new MYC gene was found to be amplified in neuroblastoma and was named MYCN, where “N” denotes neuroblastoma. Amplification of MYCN still remains one of the most important prognostic factors in neuroblastoma and is associated with aggressive disease and very poor prognosis. Two years later, yet another MYC family gene, MYCL, was identified in gene amplifications in small cell lung cancer. Since their discovery, studies of the MYC proteins have unveiled their fundamental roles during development and in normal function of various tissues and importantly, have led to novel understanding in cancer biology (Wasylishen and Penn 2010).
The MYC Transcription Factor
The MYC TAD contains two conserved regions, which are important for the transactivation and transforming activity of the protein referred to as MYC box (MB) I and MBII, respectively. MBII is mediating the interaction of MYC with several of its cofactors for transcriptional activation and repression, such as transformation/transcription domain-associated protein (TRRAP) and histone acetyl transferases (HATs). Located within MBI are residues that are important for regulation of MYC activity and turnover (see “Regulation of MYC Activity” below). Two additional conserved regions, MBIII and MBIV, are located in the central part of the MYC protein and are important for its full transforming activity (Cowling and Cole 2006).
To activate transcription, the MYC-MAX heterodimer binds to the conserved E-box sequence core motif 5′-CANNTG, where 5′-CACGTG is the preferred sequence (Fig. 1b). MYC then recruits different cofactors in order to drive transcription and can stimulate histone acetylation at promoters by recruiting TRRAP-containing complexes with either GCN5 or TIP60 HATs or the CBP/p300 acetyl transferase (Fig. 1b). Histone acetylation opens the chromatin and provides docking sites for additional proteins that promote transcription (Cowling and Cole 2006). In addition, MYC also stimulates transcriptional elongation through recruitment of P-TEFb and TFII-H, which in turn phosphorylate the C-terminal domain of RNA pol II, allowing its release from the promoter and subsequent elongation (Fig. 1b; Cole and Cowling 2008). Furthermore, MYC can repress the transcription of specific genes by binding to other transcription factors in core promoters and inhibiting their transactivation activity (Herkert and Eilers 2010). The most studied example is the POZ-domain containing protein MIZ-1, but other factors, including Sp1, can also be bound and inhibited by MYC. Several negative regulators of the cell cycle are among the transcriptional targets of MIZ-1 including the cyclin-dependent kinase inhibitors (CKIs) p21 and p15. By binding to MIZ-1, MYC can displace co-activators, such as CBP/p300, and instead recruit the DNA methylase Dnmt3, histone deacetylase 3 (HDAC3), and p14/ARF. This in turn leads to deacetylation of histones as well as methylation of DNA and histones promoting a repressed chromatin state (Fig. 1c; Herkert and Eilers 2010). It has also been recognized that regulation of specific microRNAs is another means whereby MYC indirectly regulates the expression of many genes (Bui and Mendell 2010). In addition to RNA pol II-mediated transcription of mRNA, MYC can also stimulate the transcription by RNA pol I and RNA pol III (Adhikary and Eilers 2005; van Riggelen et al. 2010). RNA pol I mediates the transcription of ribosomal RNA (rRNA) from rDNA. MYC promotes RNA pol I activity by binding E-boxes in rDNA promoter regions where it associates with the RNA pol I-specific promoter selectivity factor SL1. By binding to TFIIIB, a RNA pol III-specific transcription factor, MYC can also directly activate RNA pol III-mediated transcription of 5S rRNA and tRNA.
Several transcription-independent functions have been reported for MYC, including mRNA cap methylation and DNA replication (Cole and Cowling 2008). Cap methylation occurs during early stages of mRNA transcription, where the mRNA is first capped with an inverted guanosine, and this cap is subsequently methylated. Uncapped mRNA is rapidly degraded, and methylation of the cap is necessary for binding of translation factors. MYC stimulates the recruitment of enzymes responsible for both capping and methylation to specific mRNAs (Cole and Cowling 2008). In this way, the level of a protein can be increased by MYC, independent of any effects on transcription. A direct role for MYC at origins of replication has also been suggested (Herold et al. 2009). Defining the direct actions of MYC in regulating replication is not straightforward since MYC also stimulates DNA replication through transcription-dependent mechanisms. However, several components of the pre-replicative complex have been found to be direct interaction partners of MYC. Furthermore, MYC overexpression increased the number of origins of replication, even in the presence of transcription inhibitors, and could influence the rate of DNA synthesis in a Xenopus system, which supports cell cycle–regulated DNA replication in the absence of transcription and new protein synthesis (Herold et al. 2009). Recently, a cytosolic function of a cleavage product of the MYC protein was described (Conacci-Sorrell and Eisenman 2011). Calpain-mediated cleavage generates an N-terminal version of MYC, MYC-nick, that lacks nuclear localization signal and was shown to promote differentiation of muscle cells.
MYC in Cell Biology
Early on, MYC’s crucial role in driving cell proliferation was noted (Henriksson and Lüscher 1996; Wasylishen and Penn 2010). MYC was found to be an intermediate early response gene after serum stimulation and ectopic expression of MYC-promoted cell cycle entry of quiescent cells. The effect of MYC on the cell cycle is achieved through repression of cell cycle checkpoint proteins and CKIs, including p21, p15, p27, and GADD45. MYC also activates several proteins that are important for the progression of the cell cycle including cyclins D1, D2, E1, A2 as well as CDK4, CDC25A, E2F1, and E2F2 (Dang et al. 2006; Eilers and Eisenman 2008).
MYC drives cell division not only through regulation of cell cycle checkpoints but also by stimulating biosynthesis of macromolecules, allowing the doubling of cell mass required for every cell division. It is a direct regulator of ribosome biogenesis and thereby stimulates protein synthesis (van Riggelen et al. 2010). Through activation of the transcriptional activity of all three RNA polymerases, MYC stimulates synthesis of tRNAs, ribosomal RNAs (rRNA), and proteins, enzymes responsible for catalyzing the processing of rRNA, ribosome assembly, and export from the nucleus as well as translation initiation and elongation factors. In addition, MYC stimulates mitochondrial biogenesis and controls the activity of many metabolic pathways and the uptake of nutrients required to fuel these. Both glucose and glutamine transporters as well as several key enzymes in the glycolysis and glutaminolysis pathways are direct transcriptional targets (Dang et al. 2006, 2009). Furthermore, MYC has a broad role in driving biosynthetic reactions that are required to supply macromolecules to support cell growth and stimulates the production of amino acids, ribose sugars, and purine nucleotides.
Somewhat surprising when it first was discovered, MYC overexpression can also cause apoptosis (Wasylishen and Penn 2010). This is the result if MYC overexpression is present together with anti-proliferative signals and can be rescued by specific survival factors (Hoffman and Liebermann 2008). Abrogation of the apoptotic signaling by MYC strongly contributes to cancer development. MYC can promote apoptosis through p53-dependent and p53-independent functions (Hoffman and Liebermann 2008). Overexpression of MYC can be sensed by ARF, which in turn activates p53 through inhibition of HDM2. High expression of MYC also promotes DNA damage and genomic instability. This may be the reason for activation of the ATM and ATR kinases, which also contributes to p53 activation in response to MYC. There is evidence showing that MYC can alter the balance of pro- and anti-apoptotic members of the Bcl2 family, in parallel with or independent of p53 activation. For example, the expression of the anti-apoptotic proteins Bcl2 and Bclx can be repressed, while the pro-apoptotic protein Bim can be induced by overexpression of MYC (Hoffman and Liebermann 2008). In addition, multiple proteins involved in the death-receptor pathway can be regulated directly or indirectly by MYC, resulting in increased sensitivity to apoptosis induced by ligation of death receptors such as the Fas and TRAIL receptors.
Regulation of MYC Activity
MYC activity is tightly regulated by developmental and mitogenic signals. The transcript and protein have short half-lives, allowing rapid adjustments of levels and thereby MYC activity. Many signaling pathways are involved in the transcriptional regulation of the gene. Transcription of MYC is, for example, induced downstream of signaling by Notch, Wnt/β-catenin, and receptors tyrosine kinases (Fig. 2). Several of these pathways interact with each other, and there are multiple levels of regulation in each pathway. Thus MYC expression is controlled by a very complex network of intracellular signaling pathways allowing for tight regulation of gene activity. In addition, the cellular, developmental, and microenvironmental context will affect expression (see Wierstra and Alves 2008 for an extensive review).
Protein turnover is another means of regulating the expression level of MYC. Phosphorylation of two N-terminal sites, serine 62 (S62) and threonine 58 (T58) within the MBI, has been shown to have an important regulatory function for protein stability (Hann 2006; Vervoorts et al. 2006). Importantly, mutations of these residues have been found in Burkitt lymphomas and other lymphomas and are associated with stabilized MYC protein, increased transforming activity and impaired ability to induce apoptosis (Hann 2006). Phosphorylation of S62 and T58 is interdependent since T58 requires prior S62 phosphorylation (Henriksson et al. 1993). S62 phosphorylation stabilizes the MYC protein while phosphorylation of T58 promotes its degradation. Several kinases have been implicated in the phosphorylation of S62 including MEK/ERK, JNK, and CDK1 (Hann 2006). Once S62 is phosphorylated, MYC can be recognized by GSK3β, which phosphorylates T58 resulting in the targeting of MYC for proteasomal degradation. Upon T58 phosphorylation, S62 is dephosphorylated. T58-phosphorylated MYC can then be recognized by the F-box protein FBW7, which is a subunit of the SKP1-CUL1-F-box protein (SCF) complex that stimulates polyubiquitylation and subsequent proteasomal degradation of MYC (Hann 2006; Vervoorts et al. 2006). Ras signaling is believed to stabilize MYC through activation of Raf/ MEK/ERK leading to S62 phosphorylation and at the same time inhibiting T58 phosphorylation through activation of PI3K signaling, which in turn inhibits the activity of GSK3β (Hann 2006). The E3 ubiquitin ligase FBXO32 has been recently described to target MYC for proteasomal degradation. Interestingly, MYC activates FBXO32 transcription, suggesting a negative feedback regulatory loop (Mei et al. 2015). Another ubiquitin ligase, SKP2, can when recruited to MYC-regulated promoters mediate the ubiquitylation of MYC. This results first in transcriptional activation followed by proteasomal degradation of MYC. The HectH9 ubiquitin ligase regulates Myc transcriptional activity by forming an ubiquitin chain that, in this case, does not induce protein degradation but may be involved in a switch from inactive to active MYC protein (Adhikary et al. 2005). Furthermore, MYC can be phosphorylated at multiple additional sites and is also subject to acetylation (Vervoorts et al. 2006) and SUMOylation, which precedes its ubiquitination and proteasomal degradation (González-Prieto et al. 2015).
Development, Differentiation, and Stemness
The importance of MYC activity during development has been demonstrated by gene targeting in mice, where loss of either c-myc or mycn is embryonic lethal. c-myc −/− embryos die at embryonic day 10.5 due to severe hematopoietic as well as placental defects, while mycn −/− embryos succumb to neuroectodermal and heart abnormalities between embryonic day 10.5 and 11.5. Mice lacking mycl on the other hand appear normal: they are completely viable and fertile, and there are not any histological differences on tissues known to highly express mycl during development (Hatton et al. 1996). Intriguingly, myc +/− mice, although smaller than the wild-type counterparts, show increased life- and health span, that seem to be achieved through changes in metabolism, especially that of lipids, and in the immune system (Hofmann et al. 2015). It is likely that c-myc and mycn can at least partly compensate for the absence of each other during development since max −/− mice die already at embryonic day 6.5 (Laurenti et al. 2009).
It was early recognized that MYC expression negatively correlated with terminal differentiation of tissues and that MYC expression could prevent terminal differentiation. The role of MYC in differentiation now appears more complex, and the presence of MYC is important for many steps in differentiation in various tissues, including the hematopoietic system and the skin (Eilers and Eisenman 2008; Laurenti et al. 2009).
Furthermore, MYC function seems to be required for embryonic stem (ES) cell self-renewal. MYC was also one of the factors in the first described four-factor reprogramming of somatic cells to induced pluripotent stem (iPS) cells. While not strictly necessary for iPS generation, MYC has been found to enhance the reprogramming of cells. This has been linked to its capabilities to stimulate proliferation and to inhibit differentiation as well as to its ability to modify epigenetic patterns. Taken together, MYC can promote an open chromatin state and an ES cell-like chromatin landscape (Laurenti et al. 2009). In this sense, MYC is essential for the maintenance of proliferative capacity, but not for pluripotency, in ground-state ESCs. Similarly, MYC downregulation is sufficient and necessary for the induction of reversible diapause in mouse blastocysts through reduction of proliferation and protein synthesis, without effects on pluripotency (Scognamiglio et al. 2016).
MYC in Cancer
Deregulated expression of one of the MYC family genes is detected in a wide range of human cancers and has in many instances been associated with aggressive, poorly differentiated tumors. Activation of MYC in cancers can be achieved through different means and is in most cases not the consequence of activating point mutations, but rather increased expression achieved through gene amplification, copy number gain of super-enhancers (Zhang et al. 2016), translocation, overexpression, enhanced translation, or increased protein stability. Translocation of MYC to one of the immunoglobulin loci is the hallmark of Burkitt lymphoma, but also occurs at variable frequency in other lymphomas and leukemias (Vita and Henriksson 2006). Amplification of one of the MYC genes is found in a range of solid tumors, including breast cancer, prostate cancer, neuroblastoma, and lung cancer (Vita and Henriksson 2006; Albihn et al. 2010). Overexpression of MYC can also be achieved through deregulation of upstream signaling pathways, which regulate MYC transcription and/or MYC protein stability. Furthermore, mutations leading to enhanced translation, or affecting specific residues in MYC, such as T58, which are involved in the regulation of MYC protein turnover, can be the cause of enhanced MYC protein levels in tumors. In fact overexpression of MYC is one of the most common events coupled with tumorigenesis (Pelengaris and Khan 2003; Albihn et al. 2010).
Early in vitro experiments defining the oncogenic properties of MYC revealed that its constitutive overexpression in rat embryo fibroblasts (REFs) lead to immortalization and prevented exit from cell cycle. MYC’s contribution to tumorigenesis has been attributed mainly to its ability to promote tumor cell proliferation without the requirements of exogenous mitogenic signals resulting in uncontrolled proliferation. Overexpression of MYC can also promote tumorigenesis through its effects on metabolism, cell growth, and metastasis as well as on the tumor microenvironment, for example, through the regulation of angiogenic factors (Pelengaris and Khan 2003; Sodir and Evan 2009). Interestingly, oncogenic MYC is able to disrupt the cell circadian clock, unleashing glucose and glutamine metabolisms from their circadian control to favor biosynthesis and cell growth (Altman et al. 2015). The tumor-promoting effect of MYC was also verified in vivo in transgenic mice, first in the Eμ-myc model where MYC is overexpressed in B cells resulting in lymphoma, and then in many other tissues (Pelengaris and Khan 2003).
High levels of MYC expression promote apoptosis. While contradictory to its tumor-promoting function, this has come to be viewed as an intrinsic tumor suppressor function to guard against uncontrolled proliferation. The same holds true for other oncogenes, such as E2F. Cooperation in transformation is seen between MYC and other oncoproteins, for example, Ras and Bcl2 or loss of tumor suppressor proteins such as p53, and ARF that counteracts the apoptosis signaling induced by MYC. Another tumor suppressor function that can be affected by MYC is senescence. MYC can suppress senescence induced by oncoproteins such as Ras and BRAF. However, under certain circumstances, MYC can also induce senescence (Larsson and Henriksson 2010).
Conditional transgenic models where MYC overexpression can be turned on and off have shown that inactivation of MYC can lead to tumor regression (Felsher 2010). Some tumors are dependent on the sustained expression of MYC, so-called oncogene addiction, which implicate MYC as a therapeutic target at least in some tumor types. MYC inactivation in tumors can lead to proliferative arrest, differentiation, senescence, and/or apoptosis. In addition, inhibition of MYC can also affect the tissue microenvironment and tumor-induced angiogenesis. Studies in a collection of conditional transgenic models of MYC-driven tumors of various tissue origins have shown that the efficiency and reversibility of the effects of MYC inhibition depends on the cellular, genetic, and epigenetic contexts (Felsher 2010).
Approaches to target MYC include strategies that interfere with MYC expression such as antisense and RNA interference, inhibition of downstream MYC target genes, and small molecules that disrupt MYC-MAX interaction (Vita and Henriksson 2006; Larsson and Henriksson 2010; Prochownik and Vogt 2010). MYC has for several reasons been regarded as an impossible target for therapy, and development of small targeting molecules has lagged behind. Interfering with protein-protein interactions instead of the classical targeting strategy of gain-of-function mutants in cancer-associated kinases has been deemed difficult. Recent development of other protein interaction-targeting small molecule compounds, including nutlins, which disrupt the p53-HDM2 interaction, and ABT-737 that targets the interaction between specific Bcl2 family members, shows however that this strategy indeed is possible and can be successful (Prochownik and Vogt 2010).
Concerns have also been raised regarding the effect of inhibition of MYC in normal tissues. One study of systemic MYC inhibition in mice with the conditional expression of the MYC inhibitor Omomyc indicates, however, that the effects on normal tissues are mild and can be tolerated (Sodir and Evan 2009). During the last years, several effective strategies to intervene with MYC function both in vitro and in vivo have been developed. These approaches include direct inhibition of dimerization with MAX, such as 10058-F4 and 10074-G5, and indirect inhibitors targeting MYC-driven transcription through the inhibition of bromodomain and extraterminal domain (BET) proteins. BET proteins mediate the recognition of acetylated lysine residues in histones and recruit the machinery necessary for transcription elongation. Their inhibition abrogates both MYC-dependent transcription and MYC expression itself. Additionally, MYC overexpression shows synthetic lethality with inhibitors targeting several kinases (GSK3B, CDK1, Aurora kinase) as well as SUMOylation (Fletcher and Prochownik 2015). Some small molecule inhibitors of MYC are already in clinical trials, including the Aurora kinase A inhibitor alisertib, and bromodomain inhibitors including I-BET 762 and OTX015.
Taking into account the variety of cancer-promoting and cancer-supporting phenotypes elicited by oncogenic MYC, it is crucial to understand the mechanisms by which these processes occur and how they can be pharmacologically short circuited. The recent advances in MYC-driven tumor biology and in the development of small molecule inhibitors of MYC bring new hopes and open novel paths in the field, bringing the long-pursued objective of targeting MYC as an effective anticancer strategy closer to reality.
The MYC protein is a transcription factor, which appears to act as a global regulator of gene transcription, that controls many important normal as well as neoplastic cellular processes. The studies of MYC have in fact greatly contributed to our understanding of fundamental mechanisms of cell growth, cell death, and development and will likely continue to do so. Furthermore, the oncogenic role of MYC in tumor development is well established, and deregulated MYC is involved in several different if not all human cancers. This has stimulated the idea that inhibition of MYC could be an attractive strategy for development of novel cancer therapies. This notion is further encouraged by recent progress in the development of direct as well as indirect MYC-targeting small molecules.
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