Targeting “undruggable” c-Myc protein by synthetic lethality

Abstract

Synthetic lethal screening, which exploits the combination of mutations that result in cell death, is a promising method for identifying novel drug targets. This method provides a new avenue for targeting “undruggable” proteins, such as c-Myc. Here, we revisit current methods used to target c-Myc and discuss the important functional nodes related to c-Myc in non-oncogene addicted network, whose inhibition may cause a catastrophe for tumor cell destiny but not for normal cells. We further discuss strategies to identify these functional nodes in the context of synthetic lethality. We review the progress and shortcomings of this research field and look forward to opportunities offered by synthetic lethal screening to treat tumors potently.

Keywords

synthetic lethality / undruggable / transcription factor / c-Myc

1 Introduction

More than 70% of human tumors overexpress either c-Myc or one of its two close paralogs, N-Myc and L-Myc [1,2]. MYC family members are broadly implicated in human cancers yet are presently considered “undruggable” due to its nuclear localization and lack of binding pocket and essential physiological functions to the maintenance of normal tissues [3]. Given the considerable relevance of c-Myc to human cancers and current “undruggablity,” researchers paid enormous attention to target it by inhibiting the transcription and translation of MYC and destroying the stability of c-Myc. However, these methods could cause severe side effects, especially on normal proliferative tissues [4–6]. Trumpp et al. reported that the Myc gene loss is more severe for proliferation cells, such as stem cells, than other cells. The differentiation of c-Myc-deficiency hematopoietic stem cells (HSCs) of a mouse model is impaired compared with that of normal HSCs [5]. Targeting c-Myc in mouse model and cell lines can incur unfavorable effects. Cleveland and colleagues [7] showed that Myc^–/– mice died as an embryo due to defects in growth and cardiac and neural development. Dominguez-Sola et al. clarified that transient Myc depletion by RNAi stalls DNA replication and hinders cell cycle [4].

Synthetic lethality could be used to identify pathways and functional nodes essential in the oncogenic c-Myc pathway [8,9]. This concept originates from studies in Drosophila model systems, in which a combinational mutation of two or more separate genes incurs death while either gene mutation alone could be buffered [10]. High-throughput screen technology propels the development of research on synthetic lethality. Based on high-throughput technology, synthetic lethal screening has identified over a hundred candidate genes that are potentially lethal to MYC [11]. Identifying critical functional nodes in synthetic lethal pathways related to undruggable oncoproteins holds great promise in cancer research area.

2 c-Myc and undruggability

c-Myc, encoded by MYC, is a master regulator of normal and cancer-associated processes [11]. As a key transcriptional factor, c-Myc regulates the expression of many genes with diverse biological functions, such as cell proliferation and apoptosis [12,13]. c-Myc also regulates the expression of mismatch repair genes by binding to the promoters of double-strand break repair genes during DNA damage repair [14,15]. Moreover, recent research depicts the critical roles of c-Myc in regulating metabolism, specifically glycolysis and glutaminolysis [16]. The MYC oncogene is a central driver in numerous cancers, such as breast cancer [17], liver tumor [6], colorectal carcinoma [18], and prostatic neoplasia [19]. This oncogene is dysregulated in>70% of human cancers, and the dysregulation is associated with poor prognosis and survival rates [20,21]. Excess c-Myc expression can be caused by chromosomal translocation [22] or amplification. Additionally, malfunctions in either the degradation process of c-Myc or the upstream pathways of c-Myc can increase c-Myc stability and oncogenic activity. The high frequency of MYC family dysregulation in human cancers suggests that a strategy to target Myc may benefit a broad population of patients; however, its simple protein structure (Fig. 1) and predominant nuclear location have impeded efforts to exploit it in drug discovery and development.

Potential molecular targets usually fall into two major categories, namely, druggable and undruggable. “Druggability” implies that the target molecule must have structures that should make it vulnerable to attack and inhibition by low-molecular-weight compounds. Moreover, a protein is considered druggable if it contains a cavity, usually a well-defined catalytic cleft. However, most transcription factors, including c-Myc, are widely thought to be undruggable due to the lack of catalytic clefts and the much-sought drug binding pockets. To date, targeting c-Myc in cancers with small molecular agents remains challenging. New strategies are urgently needed to regulate the stability or activity of c-Myc in cancer.

3 Current methods to target c-Myc

Clinical outcomes for cancer patients with high c-Myc activity remain dismal [23–25]. MYC-amplification is a relevant factor with poor outcomes in medulloblastoma [26], multiple myeloma [27], and diffuse large B cell lymphoma [24,25]. One contributing factor is the absence of effective therapeutics against c-Myc. As mentioned above, directly targeting c-Myc with small-molecule inhibitors is difficult [27–29]. Therefore, new agents that could indirectly regulate c-Myc stability or activity may shift the paradigm for treating c-Myc-dependent cancers (Fig. 2).

One method to indirectly target c-Myc is blocking the transcription of MYC and the corresponding target genes of the c-Myc protein. Bromodomain-containing 4 (BRD4) is involved in transcription elongation [30–32]. Based on multiple myeloma human cell line assays, a selective small-molecule bromodomain inhibitor, namely, JQ1, competes with BRD4 for binding to acetylated lysines and replaces BRD4 from super-enhancers within the MYC oncogene [33]; this phenomenon downregulates c-Myc transcription and leads to genome-wide downregulation of c-Myc target genes [33]. In vivo xenograft studies also showed the great potency of JQ1 [33]. JQ1 manifests powerful anti-tumor effects in multiple hematopoietic cancers and pancreatic ductal adenocarcinoma (PDAC) with c-Myc overexpression in vitro and in vivo [34–37]. Blocking c-Myc protein synthesis is an alternative method. Mammalian target of rapamycin complexe 1 (mTORC1) regulates protein synthesis [38]. The major regulators of protein synthesis downstream of mTORC1 are p70S6K1/2 and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4EBP1), which negatively regulates eIF4E, a key rate-limiting initiation factor for cap-dependent translation [38]. Pharmacological inhibition of the PI3K/AKT/mTOR pathway can suppress c-Myc protein level and may even manifest remarkable potential therapeutic efficacy in MYC-driven cancers [39,40]. MLN0128, a powerful mTOR active site inhibitor, inhibits 4EBP1 phosphorylation and achieves great efficacy in MYC-driven hematological cancer cell lines and mouse models [40]. Undermining the stability of c-Myc could be another method. The stability of c-Myc is under tight control by ubiquitin-proteasome system in normal tissues [41]. The stability of the c-Myc protein is regulated by phosphorylation at two sites: serine 62 phosphorylation (pS62) stabilizes c-Myc, while threonine 58 phosphorylation (pT58) promotes c-Myc degradation [42]. CAMKIIg, whose protein level correlates with c-Myc protein level in patients with T cell lymphomas (TCL), stabilizes c-Myc by phosphating it at Ser62 [43]. Pharmacologic inhibition of CAMKIIg with the specific inhibitor berbamine (BBM) could undermine the stability of c-Myc, suppress TCL development, and reduce the tumor burden [43,44].

During the course of existing cancer treatments, information may surface about the toxic side effects of drug on whole organisms. More importantly, the therapeutic index of an agent directs our attentions, that is, the efficiency with which it affects cancerous tissues compared with its toxic effects on normal tissues. Thus, an ideal agent should have high therapeutic index and wreak havoc on cancer cells while leaving normal tissues relatively untouched. All the methods mentioned above aim to reduce c-Myc expression. c-Myc, which is expressed in cancer cells, is also expressed by their normal counterparts, which is the fundamental obstacle to achieving high therapeutic index in cancer treatments.

4 Synthetic lethality in cancer

The aim of anti-cancer drug development is to direct drugs to specific molecular targets within cancer cells; if the aberrant biological state of cancer cells is derived from and depends on malfunctioning signaling nodes, then inhibiting or eliminating such nodes should result in a cytostatic response in these cells. Given that these signaling nodes function differently in normal and neoplastic cells, targeting them should yield substantial therapeutic indices, that is, selective killing of cancer cells and potentially reduce side-effect toxicity for cancer patients undergoing treatment.

Practicing synthetic lethality by destroying crucial malfunctioning signaling nodes within c-Myc oncogenic network will be a catastrophe for c-Myc-dependent tumor cells. Numerous redundancies developed in cellular signaling pathways to overcome the impediment of unexpected mutations [45] and maintain cell homeostasis. Intracellular redundant components also endow cancer cells with resistance to different treatments and unfavorable environment. Normal and neoplastic cells share similar protein components but have different ways of transmitting signals. Malfunctioning circuit nodes are critical for cancer cells, but similar pathway nodes may be dispensable or redundant in normal ones. Synthetic lethality can help screen malfunctioning circuit nodes to selectively kill cancer cells. Synthetic lethality originated from classical genetics and elaborated that synchronous mutation of two genes will lead to cell death, yet mutation of either gene alone is viable [46]. This exquisite principle provides a new insight into cancer treatment. After Ashworth and colleagues pointed out the potential of targeting DNA repair defect in BRCA mutant cells as a therapeutic strategy and successfully propelled poly (ADP-ribose) polymerase (PARP) inhibitors into clinics, more cancer researchers are embracing the concept of synthetic lethality [47–49]. Thus far, synthetic lethality has been expanded to heterotypic interactions across diverse cell types. LOX inhibition leads to synthetic lethality to PTEN null in glioblastoma multiforme (GBM) by markedly suppressing macrophage infiltration and tumor progression [50,51]. About 20 years ago, Hartwell and colleagues proposed that synthetic lethality could be used to identify new anticancer drug targets [52], especially those that are synthetically lethal to known cancer-causing mutations. When an oncogene mutation exists in certain tumor cells, we can exploit its synthetic lethal genes to induce cell death. Genes, which are synthetically lethal to other genes, do not need to be mutated if their functions are affected by environmental factors. As Robert G. Bristow and Ester M. Hammond reported [53], after severe acute hypoxia followed by reoxygenation or moderate chronic hypoxia treatments, replicating cells gain a homologous recombination defected phenotype, which is synthetically lethal with PARP1 inhibition.

Treatment methods based on synthetic lethality may outperform conventional medical options in the case of functional loss of a tumor suppressor gene (TSG) in a certain cancer regardless of deletion, inactivating mutation, or epigenetic silencing [54,55]. Restoring the TSG protein activity to fulfill its biochemical functions is difficult [56]. Chemotherapy causes indiscriminate harm to normal and cancerous cells, leading to a high risk of adverse events to patients [54]. Attractive prospects exist in targeting “undruggable” driver-mutations, such as MYC and KRAS, based on synthetic lethality [57,58].

Synthetic lethality may inspire and enrich drug-combinations as therapeutic methods to deal with drug resistance, a severe threat to overall patient survival [9] during cancer treatment. Conventional cancer treatments depend on standard protocols, where a given drug is designated as first-line therapy [9]. Drugs designated as second- and third-line therapies will be recommended in case drug resistance occurs. However, most of later-line therapies appear to be less effective than first-line therapy and do not overcome the resistance well. Drug resistance is a hindrance for conventional treatments but a weakness that can be exploited by synthetic lethality. Further investigation is needed on the drug resistance of cancer after first-line therapy and on how to use it to treat cancer instead. Following the principle of synthetic lethality, drug resistance might be exploited to target drug-resistant cancer cells potently.

Synthetic lethality has potential significance but also present issues that need to be answered. The urgent problem proposed here is how to identify critical functional nodes that are essential in MYC-driven tumor cells. These valuable nodes could be potential candidates of synthetic lethal partners with oncogenic c-Myc. Interfering with these critical functional nodes could tilt the relatively balanced cellular circumstance and trigger cell death [56,59,60].

5 Synthetic lethal screening

In general, synthetic lethal screening methods can be categorized into genetic screening and chemical screening based on platforms used.

5.1 Genetic screening

In isogenic human cell lines, large libraries of synthetic short interfering RNAs (siRNAs), libraries of short hairpin RNAs (shRNAs), and large collections of guide RNAs (gRNAs) for CRISPR/Cas9 genome editing, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) are applied to identify critical genes and their synthetic lethal partners [61–64]. Fig. 3 depicts a simplified cell-based synthetic lethal screening. The chimaera of the MYC gene fused with the hormone binding domain of the human estrogen receptor gene could be valuable for synthetic lethal screening. The activities of its protein product, c-Myc-ER, depend on hormone or its analog 4OH-tamoxifen and could be manipulated reversibly by removing the hormone. Cells with this chimera gene could be categorized into two groups: c-Myc-On and c-Myc-Off. Together with high-throughput screening mentioned, potential synthetic lethal interactions with c-Myc would surface out.

Cancer cell line panels with different MYC mutation status could be an alternative model for investigating its intracellular synthetic lethal partners [65,66]. In theory, only MYC mutation cells, rather than wild-type ones, would die when its synthetic lethal partners are genetically inhibited. The congenital weakness of this model is the enormous genetic complexity of most human cancer cell lines. This genetic background may obscure the results of synthetic lethal combination and even lead to false positive results.

Compared with CRISPR/Cas9 genome editing, shRNA-based gene knockdown incompletely inactivates genes. Moreover, shRNA-based gene knockdown efficiency varies. However, shRNA-based gene knockdown still has its position in synthetic lethal screening. The two aspects to consider are as follows. First, shRNA-mediated technology could mimic the effect of drugs closely. From this perspective alone, it outperforms the CRISPR/Cas9 genome editing technology. Second, a partial silencing of an essential gene may display the dosage-dependent synthetic lethal phenotype [9]. This process would be lethal itself in the case of complete inactivation. In a word, it can be a sharp sword depending on what we need.

CRISPR/Cas9 genome editing is not perfect all the time. Studies have shown that CRISPR targeting can be toxic in amplified genomic regions, especially in those highly aneuploidic ones [67,68]. CRISPRi or CRISPRa, which use a catalytically dead mutant of Cas9 fused to a transcriptional repressor or activator domain, could be an alternative choice. In contrast to genome-editing screening, CRISPRi and CRISPRa screenings are reversible and inducible, indicating the accuracy of the examination of the spatiotemporal dynamics of gene function [69,70].

In large-scale screening of model organisms, potent platforms have been introduced. For example, high-throughput mating methodologies in yeast, such as synthetic genetic array (SGA) analysis [71] and diploid synthetic lethality analysis with microarrays (dSLAM) [72], enable the large-scale construction of double mutants and the quantification of genetic interactions.

5.2 Chemical screening

This idea was born in screening for drug-like chemicals, which specifically kill yeast deletion mutations with defects in cell cycle checkpoints or DNA repair, as reported by Hartwell and Friend. The method is gradually extended to human cell lines. Kinzler and coworkers [73] cocultured KRAS-mutated colon cancer cells (engineered to produce blue fluorescent protein) with a subclone of which the mutant KRAS allele was removed by homologous recombination (and modified with yellow fluorescent protein), and the ratio of blue/yellow fluorescence was used to characterize and monitor differential cell viability. Wang and colleagues [74] discovered the increased sensitivity to the death receptor DR5 agonist tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) in vitro and in vivo in human cells, which overexpress Myc and link it to p53-independent induction of DR5 by Myc. Drug–drug interactions also come to the stage in recent years. Borisy and colleagues observed synergistic effects of drug–drug combination through their high-throughput cell-based assays [75]. Although, the underlying mechanism behind this should be further investigated.

6 Current challenges

Despite that numerous potential synthetic lethal interactions have been discovered, the lack of overlap in results from different screenings is an obstacle for further study and application to clinic trials [65]. First, wide-spreading high-throughput tools, including RNAi and CRISPR-based tools, could not exclude off-target effects completely [76,77]. Off-target effects may lead to false-positive results and contribute to the lack of overlap mentioned above. However, on-target efficiency could be improved by modifying the library of RNAi or guide RNA. Moreover, CAS9-induced DNA damage may lead to false-negative results and thus cover up the truth [78]. Second, screening based on cells with different genetic contexts or from different tissues might account for the lack of overlap [79]. The way that we culture cell lines cannot faithfully mimic the microenvironment around tumor cells in patients. Three-dimensional culture systems may also be helpful [65]. Considering that microenvironmental changes, such as hypoxia and high reactive oxygen species, may also influence synthetic lethality interactions, scholars have encountered difficulty in further in vivo studies of potential synthetic lethal interactions [53,79].

7 Applauding progresses

Selectively killing tumor cells rather than normal ones is important in cancer treatments and is a research direction that is worth pursuing. Synthetic lethality is a potent tool to fulfill this goal and help researchers achieve applauding progress. Genotype-selective synthetic lethality capitalizes on the notion that a mutation gained by cancer cells is also associated with new vulnerability that could be exploited therapeutically. Such vulnerability could be ascribed to stresses inside mutated cells. Thus, normal cells would not display increased sensitivity to synthetic lethality drug target, but cancer cells with specific mutation would. Cells lacking certain mutation can also phenocopy the similar clinical behavior as cells that do have that certain mutation [80,81]. BRCA mutation confers homologous recombination (HR) deficiency to cells. Mutations of alternative genes, whose protein products participate in HR process, may also result in HR defect [82]. A research reported hypoxia status as a microenvironmental change could lead to the phenotype of HR deficiency [53]. Thus, cancer cells with or without certain mutation but sharing similar clinical behavior can be selectively targeted. Developing effective drugs that are synthetic lethal to these genotypes is critical.

At present, PARP inhibitors are the only clinically proven drugs that can selectively target tumors in patients who carry germline mutations in BRCA1 or BRCA2 [49]. This finding is a milestone in synthetic lethal research and would constantly inspire future works. Based on this successful synthetic lethal interaction, clinic trials have been extended to “BRCAness” cancers [82,83], which share similar clinical behavior to the BRCA mutant one. A clinical trial (NCT01676753) related to synthetic lethality of Myc is ongoing at phase Ib stage. As reported [84], in Myc-overexpressing triple-negative breast cancer xenografts, CDK1 inhibition with dinaciclib results in synthetic lethality and attenuates distant metastasis. In syngeneic models, the combination of dinaciclib with anti-PD1 therapy has synergistic effects and increases immune cell tumor infiltration and activation. This clinical trial is still under further evaluation. Synthetic lethal drug combinations may show greater potency in clinical trials than genotype-selective monotherapy [9]. Moreover, synthetic lethal drug combinations have more realistic significance, when we take into consideration that FDA-approved drugs may exert far more potent efficiency on specific cancers after rationally-designed drug combining based on synthetic lethality. As reported by Liu and colleagues [85], the addition of cediranib to olaparib in a phase 2 study improved the median progression-free survival than olaparib alone in women with platinum-sensitive high-grade serous or endometrioid ovarian cancers. A joint research team reported [86] that the drug combination of olaparib (a PARP inhibitor of diagnosed advanced ovarian cancer with a BRCA mutation) and bevacizumab (a vascular endothelial growth factor receptor inhibitor, which may indirectly sensitize olaparib by acquisition of homologous-recombination defect [87]), significantly improved the progression-free survival in patients with advanced ovarian cancer. This finding was substantial in patients with homologous-recombination deficiency-positive tumors, including those without BRCA mutation. However, overwhelming work is needed in clinical application of synthetic lethal drug combinations. Undoubtedly, all these progresses have inspired us.

Despite limited clinic trials on synthetic lethality, many meaningful preclinical investigations are ongoing. Zhao et al. revealed the synthetic lethal interaction in patient-derived human breast cancer xenograft models with high Myc protein level between X-box binding protein 1 (XBP1) inhibition and c-Myc hyperactivation, which play important roles with inositol-requiring enzyme 1 (IRE1) in unfolded protein response (UPR) that is activated in multiple human cancers and involved in tumor initiation, progression, and therapy resistance [88]. More importantly, they also discovered a pharmacological inhibitor to mammalian IRE1, namely, fourth-generation salicylaldehyde class inhibitor (8866). 8866 could selectively restrain Myc-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models with efficacy comparable with that of the standard-of-care chemotherapy docetaxel. Hsu and collaborators identified BUD31 as a Myc-synthetic lethal gene in human mammary epithelial cells, and BUD31 is a component of the core spliceosome required for its assembly and catalytic activity [89].

Toyoshima and coworkers identified 102 potential synthetic lethal interactions with c-Myc overexpression in a collection of ~3300 druggable genes by applying high-throughput siRNA screening to an isogenic pair of primary cells (human foreskin fibroblasts); CAMK2G was determined to be one of the 102 potential genes involved in synthetic lethal interaction with c-Myc overexpression [57]. Further, we provided evidence that targeting Ca²⁺/calmodulin-dependent protein kinase II g (CAMKIIg), encoded by CAMK2G, can effectively inhibit T cell lymphoma (TCL) by destabilizing the c-Myc protein [43]. Moreover, pharmacologic inhibition of CAMKIIg with specific inhibitor berbamine suppressed TCL development and reduced the tumor burden [44]. Such findings highlight a potential therapeutic strategy whereby c-Myc-associated malignancies could be targeted by synthetic lethality.

Synthetic lethality will be an excellent alternative to drug “undruggable” oncogene. Pathways and functional nodes essential in the context of oncogene but not to normal cells will be identified through a functional genomics approach to reveal an undiscovered therapeutic space linked to a previously “undruggable” oncogene.

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