PERSPECTIVE The long journey to bring a Myc inhibitor to the clinic Jonathan R. Whitfield1 and Laura Soucek1,2,3,4 The oncogene Myc is deregulated in the majority of human tumors and drives numerous hallmarks of cancer. Despite its indisputable role in cancer development and maintenance, Myc is still undrugged. Developing a clinical inhibitor for Myc has been particularly challenging owing to its intrinsically disordered nature and lack of a binding pocket, coupled with concerns regarding potentially deleterious side effects in normal proliferating tissues. However, major breakthroughs in the development of Myc inhibitors have arisen in the last couple of years. Notably, the direct Myc inhibitor that we developed has just entered clinical trials. Celebrating this milestone, with this Perspective, we pay homage to the different strategies developed so far against Myc and all of the researchers focused on developing treatments for a target long deemed undruggable. Introduction glimpse of the potential of these approaches for cancer patient 2021 started with great expectations and the responsibility of treatment. holding hope for the entire world: everybody is looking forward to seeing the Sars-CoV-2 pandemic finally coming to an end, Myc as master of all cancers thanks to the newly developed vaccines that are competing to Myc is a nuclear transcription factor that coordinates intracel- defeat “the beast” that has held us hostage for more than a year lular and extracellular programs that allow cells to divide in an now and still threatens to keep us away from our normal life for orderly manner. The Myc family consists of three functionally a while longer. From a personal point of view, though, 2021 also related genes: c-myc (MYC), l-myc (MYCL), and n-myc (MYCN; comes with another huge expectation and milestone: the clinical Massó-Vallés et al., 2020). MYC was the first gene to be dis- assessment of a new first-in-class Myc inhibitor, which we have covered in this family, almost four decades ago, due to homology been developing for more than two decades, is finally beginning, with the viral gene v-myc, carried by the avian virus myelocy- with the hope of making a difference in the treatment of cancer tomatosis (Beaulieu et al., 2020; Vennstrom et al., 1982). The patients. Indeed, in the last few decades, cancer survival has other two members of the family, MYCL and MYCN, were found increased significantly, especially for certain oncological in- expressed in lung cancer and brain development, respectively. dications. However, there are still too many cancer deaths that All three proteins (from now on, Myc) fall in the category of we are not able to prevent. This motivates all of us to keep intrinsically disordered proteins, lacking a well-defined 3D looking for new and effective therapeutic targets. Myc is one structure in solution (Dang et al., 2017). However, the Myc such target, infamous in the scientific community for a long time C-terminus is characterized by the presence of a basic-loop- as the oncogene underpinning most human cancers. Neverthe- helix-leucine zipper domain that allows it to dimerize with the less, no Myc inhibitor is yet approved for clinical use, and in- Myc-associated protein X (MAX) protein. This binding triggers a tense efforts around the world are underway to make this conformational change in Myc that enables binding to DNA, opportunity a reality for patients. We are just one of the many usually at sequences called enhancer boxes (E-boxes; Beaulieu research groups driving toward this goal, contributing to it with et al., 2020; Tansey, 2014) that characterize high-affinity target the design and development of Omomyc, the best characterized genes, and at noncanonical sequences, which are instead rec- direct Myc inhibitor known to date, which reached clinical trials ognized with lower affinity, especially in contexts where Myc is this year. overexpressed (Lorenzin et al., 2016). In physiological con- With this Perspective, we acknowledge all those people ditions, Myc is transiently expressed to allow cells to efficiently who have not given up on the search for Myc inhibitors, and progress through the cell cycle. However, in cancer, this regu- here briefly describe the multiple strategies that have been lation is lost. This alteration in Myc expression levels is rarely adopted so far to target Myc. Hopefully, 2021 will show a first due to direct mutation, in contrast to what happens for other ............................................................................................................................................................................. 1Vall d’Hebron Institute of Oncology, Edifici Cellex, Barcelona, Spain; 2Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain; 3Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Bellaterra, Spain; 4Peptomyc S.L., Barcelona, Spain. Correspondence to Laura Soucek: lsoucek@vhio.net. © 2021 Whitfield and Soucek. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1083/jcb.202103090 1 of 11 J. Cell Biol. 2021 Vol. 220 No. 8 e202103090 common oncogenes. Instead, it is usually the consequence of happens to have such an actionable structure (Yang and Hurley, upstream oncogenic signals, which all funnel through Myc in the 2006), and several studies have shown that some small-molecule nuclei, where it executes the transcriptional programs that ul- ligands (e.g., cationic porphyrins and quindolines), such as CX- timately lead to uncontrolled tumor growth (Dang, 2012). In this 33543 or quarfloxin, can stabilize G-quadruplexes in the Myc context, deregulated, tonic signaling through Myc can be as promoter, resulting in Myc down-regulation (Brooks et al., 2010; tumorigenic as elevated Myc levels (Murphy et al., 2008). Brown et al., 2011; Fig. 1 A). The phase III trials for quarfloxin Importantly, the role of Myc in cancer is not only related to were discontinued due to high albumin binding, but other cell division. In fact, Myc has been shown to contribute to es- G-quadruplexes are in the pipeline for further development sentially all hallmarks of cancer, promoting angiogenesis, coor- (Asamitsu et al., 2019). One of the most advanced currently is dinating cross-talk with the tumor microenvironment (Whitfield APTO-253, developed by Aptose Biosciences, currently in a and Soucek, 2012), blocking the antitumor immune response phase Ia/b trial in patients with relapsed or refractory acute (Casey et al., 2016), and even conferring resistance to many myeloid leukemia or high-risk myelodysplastic syndrome. standard-of-care therapies (Carabet et al., 2018). APTO-253 has been related to stabilization of G4 structures For all these reasons, Myc is considered a particularly ap- at least in telomeres, Myc, and KIT promoters (Local et al., pealing target for cancer treatment and is currently considered 2018). The initial clinical trial (in 2014) was temporarily dis- one of the “most wanted” targets in cancer therapy (Dang et al., continued because of formulation issues, but it was recently 2017). Nevertheless, no Myc inhibitor is available in the clinic resumed and is due to be completed in May 2022 (clinicaltrials.gov; yet, and the race is on to develop the first (Whitfield et al., 2017). NCT02267863). New G4 stabilizers appearing in the last year include (a) a Challenges and strategies in targeting Myc short peptide sequence (DM039; Minard et al., 2020); (b) a bi- Designing a clinically viable Myc inhibitor has been considered sacridine derivative called a9, which binds with nM affinity to extremely difficult, if not impossible, as Myc was deemed es- the Myc G4 and appears to be a dual G-quadruplex/i-motif sentially “undruggable” by most of the scientific community. binder effective in both oncogene replication and transcription This connotation is mainly due to technical reasons related to its (Kuang et al., 2020); (c) a new curcumin analogue (Pandya et al., intrinsically disordered nature, but also to the preconceived 2021); and even (d) a PARP-1 inhibitor derived from 7-azaindole- notion that inhibiting it would cause severe side effects in 1-carboxamide (Dallavalle et al., 2021). All of them need to be normal proliferative tissues. From a technical point of view, further characterized to properly assess their potential phar- standard molecule approaches have had limited success in macological application. For a comprehensive review of the tackling Myc with enough specificity. Such a task is complicated topic, refer to Wang et al. (2020). by the fact that Myc lacks a classic druggable enzymatic pocket and is harder to reach in the nuclear compartment, which many Indirect strategy: Bromodomain and extraterminal (BET) drugs do not enter efficiently (Beaulieu and Soucek, 2019). With domain inhibitors regard to the fear of catastrophic side effects in normal prolif- The BET domain inhibitors (BETis) have been found to be able to erating tissues, this mainly arose from the initial study of con- displace bromodomain chromatin regulators from gene super- stitutive Myc knockout mice, which present with embryonic enhancers. Their role is clearly not limited only to the Myc gene, lethality between embryonic day 9.5 and 10.5, with severe de- but the first one to demonstrate potential for its inhibition was fects in vasculogenesis and erythropoiesis (Baudino et al., 2002). JQ1 (Fig. 1 B), initially in multiple myeloma, followed by Burkitt’s However, whether such a dramatic phenotype would be ob- lymphoma and acute myeloid leukemia, where the Myc gene is served in adult animals was unclear until, many years later, we frequently amplified (Delmore et al., 2011; Lovén et al., 2013; finally managed to demonstrate it was not the case (Soucek et al., Mertz et al., 2011). JQ1 and other BETis, though, clearly extend 2008). their activity beyond Myc, to affect multiple genes within cancer Here we summarize some of the best characterized strategies cells that might also contribute to the tumorigenic phenotype developed to date to inhibit Myc in vitro and in vivo and some (Andrieu et al., 2016; Donato et al., 2017; Hogg et al., 2016). In new approaches that, in our view, hold promise for their future fact, sensitivity to BETi displayed by various types of cancers clinical application. For the various approaches listed herein, we does not show any correlation with Myc regulation. Neverthe- can distinguish between direct and indirect strategies that focus less, despite their often poor selectivity for Myc, many BETis are on tackling Myc itself or its regulators, respectively. In addition, currently in early phase clinical trials in various malignancies we discuss the parallel approach of synthetic lethality that tar- (for a review, see Alqahtani et al. [2019]) because of their generic gets entirely different proteins and pathways to generate le- antitumorigenic function. thality in cancer cells that overexpress or deregulate Myc. Blocking Myc translation Impairing Myc transcription Antisense oligonucleotides (ASOs) Direct strategy: G-quadruplex stabilizers One of the very early strategies used against Myc was ASOs, G-quadruplexes are four-stranded DNA structures formed in which, based on their sequence homology, can hybridize with guanine-rich regions. They can act as silencer elements, re- and induce the degradation of Myc mRNA (Prochownik et al., pressing transcription of proximal genes, or activators (e.g., 1988; Sklar et al., 1991; Fig. 2 A). This direct approach was ef- when acting instead on noncoding strands). The Myc promoter fective in multiple cell lines and was already tested in the clinic Whitfield and Soucek Journal of Cell Biology 2 of 11 A first Myc inhibitor in the clinic? https://doi.org/10.1083/jcb.202103090 Figure 1. Strategies to inhibit Myc transcription. (A) Mechanism of action of G-quadruplex stabilizers on the Myc promoter. G4 stabilizers, such as quarfloxin or APTO-253, in- terfere with transcription of Myc by RNApol I. (B) Mechanism of action of BETis on Myc transcription. BETis, such as JQ1, prevent protein–protein interaction between BET proteins and acety- lated histones, interfering with Myc gene transcription. Figure created with BioRender. two decades ago, where AVI BioPharma (now Sarepta) took AVI- prevent Myc translation, one possibility is to inhibit mTOR 4126 as far as phase II clinical trials, although for coronary artery or its upstream controllers (phosphatidylinositol 3-kinase; disease (Kipshidze et al., 2007). Not much information is phosphatase and tensin homolog; protein kinase B and Ras/Raf/ available regarding why it has not been pursued further. Re- Mitogen-activated protein kinase, ERK kinase; and extracellu- cently, the use of a Myc-specific ASO based on an oligonucleo- lar-signal-regulated kinase; Fig. 2 C). In this context, a small- tide phosphoramidate conjugated with lipid groups has been molecule inhibitor of eukaryotic initiation factor-4A (eIF4A), shown to display high target specificity and was proposed as silvestrol, showed efficacy in reducing Myc translation and in- therapy in mouse models of Myc-driven hepatocellular carci- hibiting tumor growth (Wiegering et al., 2015). The good news is noma and renal cell carcinoma, demonstrating that new in- that multiple mTOR and mTORC1/2 kinase inhibitors are cur- carnations of this strategy still hold promise for potential future rently approved for clinical use (Whitfield et al., 2017), although clinical application (Dhanasekaran et al., 2020). their specific impact on Myc is dubious, since they impinge on a general cellular process involving multiple targets. siRNA or shRNA Another small-molecule inhibitor called saracatinib was also RNA is also the tool for another therapeutic approach based on found to inhibit the ERK1/2-MNK1-eIF4E-mediated Cap-dependent the use of siRNA or shRNA. They are usually delivered in vivo translation of Myc (Jain et al., 2015), and more recently, rocaglates thanks to encapsulation in nanocarriers, and once inside the have shown to hold some promise in inhibiting Myc translation cells, they are both processed by Dicer and the RNA-induced initiation in Myc-driven lymphomas through stabilization of RNA- silencing complex to become suitable for targeting of Myc eIF4A interaction (Zhang et al., 2020). Again, Myc is only one of mRNA before it can be translated into a functional protein (Fig. 2 their targets, making it difficult to estimate how much of their ac- B). This direct approach recently reached clinical trials spon- tivity is actually due to Myc inhibition only. sored by Dicerna, where it was tested as Myc RNAi (DCR-MYC) encapsulated in lipid nanoparticles for the treatment of patients Inhibitors of Myc dimerization and DNA binding with solid tumors, multiple myeloma, or lymphoma (Miller Small molecules et al., 2020). Unfortunately, the trial did not meet expectations After antisense, the earliest attempts to inhibit Myc made use of of Myc knockdown or efficacy and was discontinued. For a re- small molecules to interfere with Myc/MAX interaction and/or cent review on the subject, please refer to Habib et al. (2020). prevent their binding to DNA (Prochownik and Vogt, 2010; Yin et al., 2003). Most small molecules, unfortunately, have been Interference with internal ribosome entry site (IRES)-dependent described (at least until recently) as frequently suffering from translation poor bioavailability (Fletcher and Prochownik, 2015; Prochownik Myc mRNA can be translated both by 59Cap-dependent and and Vogt, 2010). However, some small-molecule Myc inhibitors IRES-dependent mechanisms (Nanbru et al., 1997). Hence, to selected to interfere with Myc/MAX dimerization (Fig. 3 B) have Whitfield and Soucek Journal of Cell Biology 3 of 11 A first Myc inhibitor in the clinic? https://doi.org/10.1083/jcb.202103090 shown interesting in vivo capabilities in the last few years. Some are mentioned below. 3jc48-3. Small molecule 3jc48-3 is related to one of the ear- liest described compounds (10074-G5) and displayed im- proved activity as well as a 17-h intracellular half-life (Chauhan et al., 2014), although no further in vivo studies have been reported. Mycro3. Mycro3 showed efficacy in vivo upon oral gavage administration, increasing survival in mouse models of pan- creatic cancer (Stellas et al., 2014). KJ-Pyr-9. KJ-Pyr-9 is an inhibitor found in a Kröhnke pyri- dine library; it has a very low dissociation constant (Kd; 6.5 nM) and blocks the growth of Myc-amplified human cancer cell line xenografts, even crossing the blood–brain barrier (Hart et al., 2014). MYCMI-6. MYCMI-6 was shown to be able to inhibit tumor cell growth in a Myc-dependent manner, with half-maximal inhibitory concentrations (IC50) as low as 0.5 µM, and to de- crease proliferation and increase apoptosis in a Myc-driven tu- mor xenograft model (Castell et al., 2018). Novel computational techniques are also being used to virtually screen binding to different intrinsically disordered protein conformations, and compounds were identified with micromolar affinity for Myc (Yu et al., 2016). MYCi975. MYCi975 was recently discovered through leveraging a large in silico library followed by a rapid in vivo screen to streamline the process of identifying small molecules already possessing tolerability and efficacy in vivo (Truica et al., 2021). EN4. EN4 is possibly the most recent, identified by activity- based protein profiling; it is a functional covalent ligand that binds to disordered Myc domain, reduces its stability and transcriptional activity, and possesses in vivo activity, reducing tumor growth in a breast cancer xenograft model (Boike et al., 2021). KSI-3716 and MYRA-A. Among compounds that can instead disrupt binding to DNA, KSI-3716 was effective in mouse models of bladder cancer (Jeong et al., 2014; Seo et al., 2014), and MYRA-A inhibits DNA binding of Myc family proteins without inter- fering with c-Myc/Max dimerization (Mo and Henriksson, 2006; Fig. 3 B). MI1-PD. Given the number of effective small molecules out Figure 2. Approaches to block Myc translation. (A) Mechanism of action there that are hampered by in vivo delivery issues, it would also of Myc ASO. Myc ASO alters mRNA expression and translation through a be interesting to see more attempts at incorporating these variety of mechanisms, including decay of the pre-mRNA mediated by ribo- compounds into nanoparticles. To this end, MI1-PD is an nuclease H (represented by scissors in the figure), direct steric blockage, or integrin-targeted, lipid-encapsulated nanoparticle formulation modulation of splicing by binding of the pre-mRNA. (B) Mechanism of action of a Myc-MAX dimerization inhibitor that showed in vivo effi- of Myc shRNA. Schematic of mode of action of a Myc shRNA, administered in the form of liposomal nanoparticles. The shRNA is processed intracellularly cacy in a mouse model of multiple myeloma (Soodgupta et al., by Dicer and the RNA-induced silencing complex to become single-strand 2015). RNA capable of hybridizing with Myc mRNA and induce its degradation and/or KI-MS2-008. A complementary and maybe more indirect prevent its translation into a Myc protein. (C) Schematic of 59Cap-dependent approach is instead to stabilize Myc’s binding partner MAX, thus Myc mRNA translation inhibition. mTOR pathway inhibition offers the op- occupying DNA target sites with MAX homodimers and pre- portunity of impairing Cap-dependent translation, the most common mech- anism through which Myc protein is produced. Figure created with venting Myc transcriptional activity (Jiang et al., 2009). Re- BioRender. cently the compound KI-MS2-008 has been described as antagonizing Myc-dependent gene expression in cells and de- laying growth of Myc-driven tumors (Struntz et al., 2019; Fig. 3 C). Whitfield and Soucek Journal of Cell Biology 4 of 11 A first Myc inhibitor in the clinic? https://doi.org/10.1083/jcb.202103090 Figure 3. Different potential mechanisms of action of small-molecule and peptide inhibitors. (A) Schematic of the mechanism of action of a Myc/MAX dimer destabilizer. (B) An inhibitor of Myc/MAX binding to DNA. (C) A MAX/MAX dimer stabilizer. (D) Omomyc as Myc dominant-negative. Figure created with BioRender. Miniproteins or protein domains recombinantly produced Omomyc miniprotein has been shown to Several peptides and miniproteins based on domains from Myc have unexpected cell-penetrating properties and therapeutic ac- family members have been described as potential therapeutic tivity in non–small-cell lung cancer, both in vitro and in vivo. tools. The first and best characterized to date is Omomyc, a Upon intranasal or intravenous administration, Omomyc reaches mutant Myc basic-loop-helix-leucine zipper domain that we the tumor tissue and efficiently penetrates both cellular and nu- designed and have validated over the past 20 years, that func- clear membranes (Beaulieu et al., 2019). This miniprotein is being tions as a very efficient Myc dominant negative (Massó-Vallés developed by Peptomyc S.L. as a drug (OMO-103) to be tested in and Soucek, 2020; Soucek et al., 1998; Soucek et al., 2002). clinical trials in 2021 (Beaulieu et al., 2019; Beaulieu and Soucek, Omomyc works through several mechanisms, forming hetero- 2019). To us, this is an extremely exciting time and we are looking dimers with Myc unable to bind DNA, and also homodimers and forward to watching its performance in patients. heterodimers with MAX, that occupy E-boxes with transcrip- Some smaller peptides based on the Omomyc basic region tionally inactive complexes (Fig. 3 D). Hence, Omomyc displays have also been developed for interference with Myc/MAX two modes of action at once: it sequesters Myc away from DNA binding to the E-box. Such peptides recapitulated at least one of while also competing for DNA binding in the form of alternative Omomyc’s mechanisms of action and have shown promise dimers compared with Myc/MAX. It should be noted that in vitro but remain to be tested in vivo (Brown et al., 2020). Omomyc was initially used as a transgene in multiple mouse Omomyc and other Myc domains have also been fused to models of cancer to establish the proof of concept that Myc in- additional sequences for targeting and delivery. Omomyc was hibition is feasible and effective and lacks significant side ef- fused with a functional penetrating “phylomer” peptide (FPPa) fects, serving as a paradigm shift for the applicability of systemic as a therapeutic strategy to inhibit Myc in triple-negative breast Myc inhibition in vivo (Alimova et al., 2019; Annibali et al., 2014; cancer (FPPa-OmoMYC; Wang et al., 2019). A similar approach Duffy et al., 2021; Fiorentino et al., 2016; Sodir et al., 2011; was successful in delivering the Myc helix1 (H1) domain fused to Soucek et al., 2004; Soucek et al., 2008; Soucek et al., 2013). an elastin-like polypeptide (ELP, a thermally responsive bio- Recently though—and most relevant for clinical application—the polymer that forms aggregates above a characteristic transition Whitfield and Soucek Journal of Cell Biology 5 of 11 A first Myc inhibitor in the clinic? https://doi.org/10.1083/jcb.202103090 temperature) and a cell-penetrating sequence (Bac) to treat a rat have shown in vivo activity in multiple myeloma models glioma model and an orthotopic model of breast cancer (Bac- (Crawford et al., 2020; Kunz et al., 2020), although in one case, ELP-H1; Bidwell et al., 2013; Bidwell et al., 2012). In the past year, Myc-dependent gene regulation was not altered (Kunz et al., the same H1 was fused to Pseudomonas exotoxin, which was 2020), and thus the involvement of Myc is not always clearly employed as a peptide nuclear delivery device (PNDD) to inhibit demonstrated. Myc–dependent transcription at nanomolar concentrations in Alternatively, since the phosphorylation status of Myc is key different tumor cell lines (PNDD1; Ting et al., 2020). However, to for its physiological turnover, one strategy focuses on the tumor our knowledge, none of these approaches is close to clinical suppressor protein phosphatase 2A (PP2A), which destabilizes application yet. Myc by targeting its serine 62 (Sears, 2004). It has been shown Another miniprotein modulating the Myc network has been that cellular inhibitors of PP2A, the SE translocation (SET) on- derived from MXD1: Mad. Like Omomyc, Mad dimerizes with coprotein and CIP2A, are increased in human cancers and lead to MAX and binds the E-box, interfering with Myc-mediated Myc stabilization (Junttila and Westermarck, 2008). Hence, in- transcription in cell models (Demma et al., 2020). Further hibitors of SET (OP449) and CIP2A have been developed pre- studies will be needed to test its performance in vivo. Also, a clinically to reduce Myc levels and activity (Farrell et al., 2014; small minimalist hybrid protein called ME47 was designed to Janghorban et al., 2014). Once again, though, their specificity for disrupt MAX:E-box binding and block Myc transcriptional ac- Myc only is virtually impossible. tivity (Lustig et al., 2017). ME47 inhibited xenograft tumor As a more direct approach, a recent report showed that an growth upon induced expression but was not tested by direct Escherichia coli protease could degrade Myc, and treatment of protein delivery. mouse models reduced tumor growth, notably without side ef- fects or reduction of Myc levels in the intestine (Butler et al., Induction of Myc degradation 2021). In physiological conditions, Myc has a short half-life of ∼30 min The field of “degraders” in general is growing, especially in proliferating cells (Hann and Eisenman, 1984), mainly de- thanks to proteolysis-targeting chimeras (PROTACs) based on termined by its phosphorylation and consequent degradation by coupling a protein-targeting ligand with an E3 ubiquitin ligase the ubiquitin-proteosome pathway (Sears, 2004). Hence, a po- (Sun et al., 2019; Fig. 4 C). Given the short half-life of Myc and its tential strategy to decrease its activity in cells is to either increase intrinsically disordered nature (which makes its high-affinity the activity of ubiquitinases or interfere with deubiquitinases targeting more complicated), the applicability of this new ap- (Fig. 4 A). Below are some examples of these strategies, typically proach to Myc is still to be demonstrated. So far, a promising indirect approaches that rely on targeting molecules responsible result has been achieved by indirect targeting with pan-BETi for Myc ubiquitination or phosphorylation. PROTAC ARV-771, which reduced Myc expression and caused Oridonin has been associated with activation of FBXW7, xenograft tumor regression in prostate cancer mouse models which is an F-box protein that targets Myc for degradation (Raina et al., 2016). Similarly, the BRD4 protein degrader ARV- (Huang et al., 2012; Yada et al., 2004). Some of its derivatives 825 reduced MYCN expression and decreased growth of neu- reached clinical trials (e.g., HAO472), although its anti-cancer roblastoma xenografts (Li et al., 2020), although it affects activity has not necessarily been linked to Myc regulation. additional targets besides Myc (Saraswat et al., 2020). According MYCN has been specifically targeted because of its role in to their developer, Arvinas Inc., these PROTACs are at the ex- pediatric oncology and adult brain tumors, where it is often ploratory stage, and no clinical trials have yet been announced. highly amplified. These approaches are described more exten- sively in a number of recent reviews (Borgenvik et al., 2021; Liu Interference with Myc transcriptional cofactors et al., 2021; Wolpaw et al., 2021). In this context, the role of The N-terminal segment of Myc contains the transactivating Aurora-A kinase is particularly relevant, since it can complex domain and several highly conserved regions termed Myc boxes with MYCN, enabling its escape from proteasomal degradation (MBs; Beaulieu et al., 2020). MBII (aa 128–143), in particular, is (Otto et al., 2009). Hence Aurora-A inhibitors (i.e., MLN8054 involved in the assembly of transcriptional machinery through and MLN8237) have been developed to overcome this protec- interaction with a wide range of transcription factors (Conacci- tion. Unfortunately, MLN8054 was terminated by Millennium Sorrell et al., 2014). Although the MYC transactivating domain is in 2008 owing to side effects (Macarulla et al., 2010). A second- inherently disordered, MBII may acquire a defined structure generation inhibitor, MLN8237 (Alisertib), was evaluated in when complexed with these cofactors, offering an opportunity multiple phase II and III studies, and some positive results were for therapeutic intervention that could significantly impair Myc reported (Beltran et al., 2019; Falchook et al., 2019a; Falchook transcriptional activity. Just to mention some examples: WDR5 et al., 2019b), while others observed toxicity and disappointing plays a role in recruiting Myc to chromatin, and genetic disruption responses (Mossé et al., 2019). Current trials are mostly focused of WDR5/Myc interferes with tumor maintenance (Thomas et al., on combining Alisertib with other therapeutics. Additional tar- 2019), prompting the design of small-molecule inhibitors of this geting options are also being explored, such as by chemical interaction. These compounds, however, still need substantial degradation (Adhikari et al., 2020). optimization to allow for their application in cell and animal Small-molecule inhibitors against another ubiquitin ligase, studies (Chacón Simon et al., 2020; Thomas et al., 2020). The HUWE1, have also been shown to induce Myc and Miz1 degra- degrader approach has also been recently applied to WDR5 with dation (Peter et al., 2014). These inhibitors (BI8622 and BI8626) encouraging results (Dolle et al., 2021). Whitfield and Soucek Journal of Cell Biology 6 of 11 A first Myc inhibitor in the clinic? https://doi.org/10.1083/jcb.202103090 Figure 4. Targeting Myc for degradation. (A) Schematic of Myc degradation by modulation of the ubiquitin pathway. Myc is continuously subject to cycles of phosphorylation and dephosphorylation, strictly linked to its ubiquitination and degradation by the proteosome. This process can be enhanced by the use of ubiquitinase activators or inhibitors of deubiquitinases (DUB). (B) Interfering with Myc dephosphorylation to enhance degradation. (C) Targeting Myc for proteolytic degradation by PROTACs. Figure created with BioRender. Whitfield and Soucek Journal of Cell Biology 7 of 11 A first Myc inhibitor in the clinic? https://doi.org/10.1083/jcb.202103090 Another interaction that appears suitable for intervention is L. Soucek is co-founder and shareholder of Peptomyc, cur- that of Myc/TRRAP, which has been described to have a lower rently conducting the clinical trials of the Omomyc mini-protein free energy of association than the Myc/MAX interphase, and (OMO-103). J.R. Whitfield is a shareholder of Peptomyc. therefore could be a more desirable target for inhibition by small molecules (Feris et al., 2019). In this case too, development of Submitted: 15 March 2021 small-molecule inhibitors is still at its beginning, and hence Revised: 27 May 2021 these approaches appear quite far from clinical application. Accepted: 2 June 2021 Synthetic lethality In addition to the plethora of strategies described here, other References indirect approaches have also been employed to get around the Adhikari, B., J. Bozilovic, M. Diebold, J.D. Schwarz, J. Hofstetter, M. Schröder, issues with direct Myc targeting. These include synthetic lethal M. Wanior, A. Narain, M. Vogt, N. Dudvarski Stankovic, et al. 2020. PROTAC-mediated degradation reveals a non-catalytic function of approaches that could in theory target any specific dependence AURORA-A kinase. Nat. Chem. Biol. 16:1179–1188. https://doi.org/10 of Myc-driven tumor cells. Many diverse targets have already .1038/s41589-020-00652-y been identified, including SAE1/2 (Kessler et al., 2012), CDK2 Alimova, I., A. Pierce, E. Danis, A. Donson, D.K. Birks, A. Griesinger, N.K. Foreman, M. Santi, L. Soucek, S. Venkataraman, and R. Vibhakar. 2019. (Hydbring and Larsson, 2010), CDK9 (Huang et al., 2014), PIM Inhibition of MYC attenuates tumor cell self-renewal and promotes kinase (Horiuchi et al., 2016), and many more involved in me- senescence in SMARCB1-deficient Group 2 atypical teratoid rhabdoid tabolism (for a more complete discussion, see Thng et al. [2021]; tumors to suppress tumor growth in vivo. Int. J. Cancer. 144:1983–1995. https://doi.org/10.1002/ijc.31873 Whitfield et al. [2017]). One advantage for this group is that Alqahtani, A., K. Choucair, M. Ashraf, D.M. Hammouda, A. Alloghbi, T. Khan, novel synthetic lethal targets may already have a clinically ap- N. Senzer, and J. Nemunaitis. 2019. Bromodomain and extra-terminal proved inhibitor that could be quickly exploited for use in Myc- motif inhibitors: a review of preclinical and clinical advances in cancer therapy. Future Sci. OA. 5:FSO372. https://doi.org/10.4155/fsoa-2018 driven cancers. To date, there are few approved options, but -0115 many more are in current clinical trials (Thng et al., 2021). Andrieu, G., A.C. Belkina, and G.V. Denis. 2016. Clinical trials for BET in- hibitors run ahead of the science. Drug Discov. Today. Technol. 19:45–50. https://doi.org/10.1016/j.ddtec.2016.06.004 Conclusion Annibali, D., J.R. Whitfield, E. Favuzzi, T. Jauset, E. Serrano, I. Cuartas, S. We can all agree that this is an exciting time in the Myc inhi- Redondo-Campos, G. Folch, A. Gonzàlez-Juncà, N.M. Sodir, et al. bition world: many research groups (and now also biotechs and 2014. Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis. Nat. Commun. 5:4632. https://doi.org/10 Big Pharma) are involved in the hunt for the first and best Myc .1038/ncomms5632 inhibitor that can find its application in cancer patients. It has Asamitsu, S., S. Obata, Z. Yu, T. Bando, and H. 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