Synthetic lethality: A promising therapeutic strategy for hepatocellular carcinoma

Cancer Letters 476 (2020) 120–128

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

Synthetic lethality: A promising therapeutic strategy for hepatocellular carcinoma

T

Linsong Tanga,b,1, Ronggao Chena,b,1, Xiao Xua,b,∗ a b

Department of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, China NHFPC Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, 310003, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Hepatocellular carcinoma Synthetic lethality Screening technology Genetic interactions

Hepatocellular carcinoma (HCC), the main cause of liver cancer-related death, is one of the main cancers in terms of incidence and mortality. However, HCC is difficult to target and develops strong drug resistance. Therefore, a new treatment strategy is urgently needed. The clinical application of the concept of synthetic lethality in recent years provides a new therapeutic direction for the accurate treatment of HCC. Here, we introduce the concept of synthetic lethality, the screening used to study synthetic lethality, and the identified and potential genetic interactions that induce synthetic lethality in HCC. In addition, we propose opportunities and challenges for translating synthetic lethal interactions to the clinical treatment of HCC.

1. Introduction Hepatocellular carcinoma (HCC), as the dominant cause of death among liver cancer, is the 6th most common cancer in terms of incidence and has the 4th highest cancer-related mortality around the world [1]. As one of the countries with a high HCC burden, HCC in China accounts for 55% of all HCC cases worldwide each year [2]. Chronic hepatitis B virus or hepatitis C virus infection, aflatoxin B1 exposure, alcohol abuse, diabetes and obesity are major risk factors for HCC [3,4]. The 5-year survival rate for advanced HCC is as low as 2%, which is the lowest among all solid tumor species [5]. The therapeutic strategies for HCC in the early to intermediate stages are surgical resection, liver transplantation, percutaneous ablation, transarterial chemoembolization and radioembolization [6]. However, for advanced HCC, few therapeutic strategies can provide a significant prognosis. Unfortunately, most patients diagnosed with HCC are in the advanced stage. The discovery of sorafenib, a tyrosine kinase inhibitor (TKi), was very impactful for patients with advanced HCC, extending the median overall survival from 7.9 months to 10.7 months [7]. Sorafenib was the only first-line agent for advanced HCC for a decade, until a phase 3 clinical trial in 2018 prompted the approval of lenvatinib as a first-line agent and alternative to sorafenib [8]. In addition, regorafenib showed survival benefits in patients with HCC that progressed on sorafenib treatment [9]. Recently, exciting advances have been made in

immunotherapy, with two programmed cell death-1 (PD-1) inhibitors, nivolumab and pembrolizumab, that were suggested to be useful in the treatment of advanced HCC [10,11]. Despite all this progress, the prognosis of HCC treated with sorafenib, lenvatinib and regorafenib remains poor, and nivolumab and pembrolizumab are only effective in a subset of patients. Therefore, it is of great urgency to explore new therapeutic strategies and agents for HCC. As a highly heterogeneous tumor, HCC is essentially the result of the accumulation of various mutations. Due to the development of the nextgeneration sequencing (NGS), a growing number of mutations have been found to promote the proliferation and metabolism of HCC, and these mutations are called driver mutations [12]. Correspondingly, other mutations that do not provide growth advantages are called passenger mutations. Recent studies revealed a series of HCC driver mutations, including TERT, TP53, CTNNB1, AXIN1, ARID2, ARID1A, PTEN, MYC and JAK1 [13–15]. Some of them, such as TP53 and PTEN, have been proven to play important roles in the treatment of HCC [16,17]. However, even though these findings have encouraged progress regarding gene-related therapies for HCC, there are still intractable problems. First, as a highly heterogeneous tumor [18], a single targeted therapy is not enough to cope with the increasing treatment demand for HCC. Moreover, many oncogenes, such as RAS and MYC, have proven to be partially druggable or undruggable [19]. Second, the alteration of signaling networks via bypassing therapeutic

∗

Corresponding author. Department of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, China. E-mail addresses: [email protected] (L. Tang), [email protected] (R. Chen), [email protected] (X. Xu). 1 Linsong Tang and Ronggao Chen contributed equally to this review. https://doi.org/10.1016/j.canlet.2020.02.016 Received 16 December 2019; Received in revised form 11 February 2020; Accepted 12 February 2020 0304-3835/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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[34,35]. Their studies provided the first direct evidence that synthetic lethality could be a new strategy for clinical treatment.

targets often occurs in resistance to target therapy [20]. Therefore, synthetic lethality, as a novel therapeutic strategy with increasing survival benefit [21], has been attracting considerable interest. Conceptually, synthetic lethality occurs when the simultaneous mutation of two genes leads to lethality of a cell, but the mutation of one alone does not lead to lethality of a cell. Recently, genome-wide RNA interference (RNAi) screening was applied to identify the common vulnerability among cancer subtypes, which was revealed to be synthetic lethality [22]. In this review, we discuss the development and application of synthetic lethality, the methods for synthetic lethality screening, as well as the known and putative synthetic lethal interactions with respect to substantiated or potential targeted therapy for HCC. In addition, we review the opportunities and challenges for translating synthetic lethal interactions to clinical treatment of HCC.

3. Synthetic lethality screening Due to the heterogeneity of hepatocellular carcinoma and the lack of driver genes, the development of therapies targeting synthetic lethal interactions in hepatocellular carcinoma is more challenging than that in other cancers. However, the clinical burden of a high mutation rate requires high-throughput and rapid synthetic lethal screening methods to find more targets for HCC. In long-term research, screening has evolved from Drosophila as a model organism to human cells, and a large number of high-throughput screening methods, such as yeast systemic genetic analysis (SGA) screening, drug screening, RNAi screening and clustered regularly interspaced short palindromic repeats (CRISPR) technology, have been extended for these studies (Fig. 2). In addition, the use of computational approaches to identify synthetic lethal interactions is increasing, which makes the identification of synthetic lethal interactions more convenient. These screenings greatly expand the synthetic lethal interaction database and provide more opportunities for selective clinical treatment.

2. The concept of synthetic lethality A synthetic lethal interaction was first reported in 1922, when Bridge discovered that the coexistence of two mutant genes led to death of Drosophila [23]. However, the concept of synthetic lethality was first proposed by Dobzhansky in 1946 to explain the same phenomenon observed by him and Bridge [24]. Synthetic lethality reflects the relationship between two genes, in which cell death occurs when both genes are mutated simultaneously, whereas mutation of only one of them does not result in cell death (Fig. 1a). The most famous example of synthetic lethality is the relationship between BRCA1/2 and poly (ADPribose) polymerase (PARP) in the DNA repair pathway; the inhibition of PARP, which is encoded by PARP1 gene, leads to death of BRCA1/2mutant cells. The concept of synthetic lethality can be expanded to synthetic dosage lethality (SDL) [25], also known as gain-of-function (GOF)/loss-of-function (LOF), which means that the overexpression of one gene is harmless to a cell while deficiency of the other gene leads to lethality (Fig. 1b). The typical example of SDL is MYC, a transcription factor overexpressed in most human cancers [26]. However, MYC is currently undruggable [27]. Thus, identifying the synthetic lethal partner gene for MYC has attracted much attention. In 2013, Pourdehnad et al. discovered that activation of the MYC gene leads to an increase in the expression of 4E binding protein-1 (4EBP1), a substrate of mTOR in the downstream pathway, which in turn maintains the occurrence and growth of tumors [28]. Therefore, EIF4EBP1 can be considered a synthetic lethal gene partner of MYC, which provides a drug-targeted treatment for MYC gene-related tumors. Another concept extending from synthetic lethality is metabolic synthetic lethality (MSL) [29]. Following the mutation of oncogenes, certain tumor-specific metabolic pathways are activated or inactivated, resulting in increased or decreased cellular demand for metabolic substrates. Consequently, altering the metabolic pathways of the substrates with drugs leads to cell death. These different pathways can be used to specifically kill cancer cells without affecting normal cells [30]. As a gene that is highly correlated with human tumors, P53 has been shown to restrict metabolism by regulating the transcription or activation of metabolic enzymes in glycolysis [31], while MYC and RAS can enhance the metabolic pathway in glycolysis [32]. As one of the important features of HCC, the Warburg effect plays a considerable role in the anticancer treatment of HCC; thus, it is possible to foresee the potential for metabolic synthetic lethality in HCC treatment. To better understand the concept of synthetic lethality, the cell can be visualized as an orderly castle, heavily guarded but constantly under pressure from the internal and external environment (Fig. 1c). Although the study of synthetic lethal interactions started many years ago, the clinical application has been slow to develop. It was not until half a century after the discovery of synthetic lethality that it was leveraged for the discovery of anticancer drug [33]. In 2005, Farmer and Bryant confirmed that cells with BRCA1 or BRCA2 dysfunction were significantly more sensitive to PARP inhibitor than were wild-type cells

3.1. Identification of synthetic lethal interactions based on model organisms Early studies on synthetic lethality were carried out in Drosophila until 1996, when a more effective model organism, yeast, was developed [36]. Synthetic lethal interaction between myo3 and myo5 in yeast were screened and found to cause growth defects in yeast strains. The revolutionary event in yeast screening was the application of SGA technology (Fig. 2). The analysis of cytoskeletal organization, DNA repair and uncharacterized functions, yielded 291 interaction networks of 204 genes, greatly increasing the number of known synthetic lethal interactions [37]. From then on, yeast has been widely used for screening because of its simplicity and low cost. A recent study used SGA technology to identify the interactions between 5400 genes in yeast, screening hundreds of thousands of synthetic lethal interactions [38]. Unfortunately, this method is currently not available for other model organisms, and the synthetic lethal interactions in yeast cannot be fully mapped to the human genome [39]. However, attempts in yeast have laid the foundation for the discovery of synthetic lethal interactions in human cancer; and suggest that the development of new targeted drugs for clinical treatment by identifying synthetic lethal interactions is promising. 3.2. Identification of synthetic lethal interaction in human cells At the beginning of the 21st century, synthetic lethal interactions of genes were first discovered in human cells [40]. In human cells, a multitude of methods can be used for the identification of synthetic lethal interactions, such as drug screening, RNAi and CRISPR technology (Fig. 2). Among them, drug screening, was the first method used in human cells. Through drug testing on cell lines with mutant genes, drugs that can selectively kill cancer cells are screened and added into chemical libraries [41]. Recently, a study reported the use of drugs to screen drug sensitivity in thousands of cancer cell lines, suggesting that drug screening can be used as a high-throughput screening method to establish a large library of synthetic lethal interactions [42]. In HCC, it has also been reported that drug screening has made good progress [43]. Even though drug screening allows high-throughput screening and the drugs that are screened can be used more quickly in the clinic; this shortcut can only be used for drug-targeted cells, and the targets of the drugs are usually unknown so that the screenings are easily random, in other words, many of these drugs have no clear target protein, so this method may not as accurate as RNAi. Previous studies have utilized that two genome-related libraries whose drug screening results are very inconsistent [44]. However, the overlap between the libraries makes an 121

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Fig. 1. The concept of synthetic lethality. (a) The simultaneous mutation of two genes leads to cell death, but mutation of only one of them does not. (b) The overexpression of one gene is harmless to the cell, while deficiency of the other gene causes lethality. (c) Normal cells are under pressure from the internal and external environments, but they have the ability to resist pressure. When one gene is changed (“mutiny” or “death”), it weakens the cell's ability to resist stress. When another gene is also changed, the cell dies via self- or external environment-induced mechanisms.

telangiectasia mutated and RAD3-related (ATR) inhibition [54]. Collectively, applying CRISPR technology for synthetic lethality screening is still in its infancy.

important contribution to the establishment of a synthetic lethal interaction library. Another tool, RNAi, is an increasingly important method for genetic screening [45]. RNAi technology provides a new opportunity for synthetic lethality screening since it allows gene knockout at the human cell level. Moreover, it can be applied in vivo to which stimulate genetic changes in cancer cells. Profiling with the use of RNAi has achieved a series of satisfactory results [46,47]. In HCC, RNAi technology has established a synthetic lethal interaction between HGS and CTNNB1 [48], and it was recently reported that PMPCB is a synthetic lethal partner of EPCAM [49], a gene that is overexpressed in HCC. It is undeniable that RNAi, which can achieve knockout of RNA levels and is not restricted by chromosome structure modification, has the advantage for synthetic lethal high-throughput screening [50]. However, the cytotoxicity caused by RNAi knockout becomes a potential risk factor, and the off-target effects cannot be ignored [51]. Recently, CRISPR technology, especially the CRISPR/Cas9 system, has been used for genome-wide screening and has vastly expanded the global genetic network map [52]. Compared to RNAi technology, CRISPR/Cas9 technology can directly and completely edit nuclear genes, resulting in a significant increase in accuracy. However, CRISPR screens may generate false-positive hits in chromosomally amplified genomic regions [53]. The latest study with CRISPR/Cas9 technology revealed synthetic lethality between RNASEH2 deficiency and ataxia

3.3. Identification of synthetic lethal interactions with computational approaches Both model organism and human cell-based synthetic lethal identification methods have achieved good results, but they all require complex experimental design. Relatively speaking, computational approaches are much more convenient and faster. These approaches can identify potential synthetic lethal gene pairs only by using various databases (Fig. 2) through methods such as the DAISY, which uses a genome-wide cancer database to screen synthetic lethal interactions [55]. By using DAISY, thousands of synthetic lethal gene pairs can be identified, including synthetic lethal interactions between PARP1 and BRCA1/2 and between MSH2 and DHFR, two gene pairs that have clinically important applications. Recently, a computational approach identified several potential synthetic lethal gene pairs for CDK1, CDK2, PLK1 and WEE1 [56]. In summary, computational methods have become increasingly important means of high-throughput screening for generating synthetic lethal libraries. However, these methods are all from cancer cells, and only detect gene-based coexpression or silencing, 122

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Fig. 2. Synthetic lethality screening. The top of the figure shows the method of synthetic lethality screening in yeast, using a query strain to mate with the gene-deficient strains. The synthetic lethal interactions can be screened by measuring the colony size. The middle part of the figure shows the method of synthetic lethality screening in human cells by adding different perturbations; the synthetic lethal partners were determined by comparing the survival of normal and cancer cells. The bottom of the figure shows a computational method for synthetic lethal screening, mutually exclusive mutations were screened through databases of cancer patients to identify potential synthetic lethal gene pairs.

Table 1 Identified and putative synthetic lethal interactions in HCC. Gene

Synthetic lethal partners Identified

PARP1

BRCA1/2 HDAC9

CDC7 PIN1 CDK6 ATR CDK7 BCL2

CTNNB1

TTK APC HGS

ARID1A

PTEN

ARID1B EZH2 PIK3CA PARP1 MTOR MET/MTOR PNKP CHD1 NLK

RAS

CTNNB1 PARP1/MEK STK33 PLK1

MYC

NUAK1 CDK9 TP53/AURKA EIF4EBP1 TP53 U2AF1 EFTUD2 BUD31

CD26 POLD1

BCL2L1 ATR

Mechanism

Reference

Drug screening Drug screening Drug screening Drug screening RNAi screening RNAi and Drug screening RNAi screening RNAi screening Drug screening Drug screening Drug screening Drug screening RNAi screening RNAi screening RNAi screening RNAi and Drug screening Drug screening Drug screening Drug screening CRISPR screening RNAi screening RNAi and CRISPR screening RNAi screening RNAi screening Drug screening RNAi screening RNAi screening RNAi screening RNAi screening RNAi screening Drug screening RNAi screening Drug screening Drug screening Drug screening Drug screening Drug screening

DNA damage repair DNA damage repair DNA damage repair Autophagy Cell cycle Senescence Cell cycle Cell cycle DNA damage repair Transcription Apoptosis Mitosis Unclear Unclear Proliferation Unclear Unclear DNA damage repair AKT/mTOR pathway dependence Unclear DNA damage repair Transcription Unclear Wnt pathway DNA damage repair Mitochondrial metabolism Chromosomal instability Apoptosis Transcription Cell cycle Translation Unclear Transcription Transcription Transcription Cell cycle DNA damage repair

[62] [64] [63] [66] [67] [80] [78] [79] [75] [76] [77] [82] [79] [48] [88] [86] [87] [89] [93,94] [95] [91] [92] [90] [100] [65] [97] [46] [106] [107] [112] [28] [79] [105] [105] [105] [108] [110]

Putative

RAD51 ATG5 CDK5 TP53

Methods of screening

123

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Fig. 3. The personalized treatment of HCC by synthetic lethal interactions. To determine the specific mutation type in HCC (e.g., gene A), the blocking agents of gene B, a synthetic lethal partner of gene A, can be used to achieve specific killing of cancer cells, thus achieving precision treatment.

Recently, autophagy was reported as an important factor in DDR, and combining autophagy inhibitors with PARPi led to synthetic lethality in HCC [66]. Since the autophagy pathway has been studied extensively, mutations associated with the inhibition of the autophagy pathway, such as the ATG5 gene, may promote synthetic lethal effects with PARPi in HCC. In addition to its role in DDR, PARPi has also been found to produce synthetic lethal effects with some non-HR-related inhibitors. The cyclindependent kinase (CDK) family plays an important role in tumorigenesis. High-throughput RNAi screening revealed that the silencing of CDK5 led to tumor cell sensitivity to PARPi [67]; a possible cause is the important role of CDK5 in cell cycle checkpoints. A recent study showed that CDK5 is one of the chief causes of HCC growth and metastasis [68]. Thus, the combination of a CDK5 inhibitor and PARPi may be a synthetic lethal strategy for the treatment of HCC. PARPi is also involved in the metabolism of HCC. A previous study has shown that PARP14 upregulates and promotes the Warburg effect in HCC [69], and the inhibition of PARP14 via the inhibitor PJ34 can make HCC cells sensitive to anticancer drugs [70].

so further experimental verification is needed [57]. As synthetic lethality plays an increasingly important role in targeted therapy, more and more technologies, including the combination of bioinformatic methods to develop more gene crossover networks, will be used in the future. 4. Identified and potential synthetic lethal interactions in HCC HCC is a highly drug-resistant cancer, and currently, the first-line drugs only provide limited survival benefits. HCC patients are in urgent need of new drug therapy. Synthetic lethal therapy has been reported as an effective strategy for the treatment of breast cancer [58], but it has not been clinically applied in HCC. Here, we review the emerging and potential synthetic lethal interactions involved in HCC. 4.1. PARP-related synthetic lethality in HCC PARP is an enzyme that plays a critical role in DNA damage repair (DDR) and is mainly involved in repairing single-strand DNA break (SSB) [59]. Once a single-stranded DNA cannot be repaired, it is converted into a double-stranded DNA break (DSB). Once homologous recombination (HR), a precise repair mechanism for DSBs, becomes deficient, cells accumulate a large amount of unrepaired DNA and experience lethality, which had been identified as the Achilles heel of the tumor cells. Previous studies have confirmed that deletions of HRrelated genes, such as BRCA and RAD51, sensitizes tumor cells to PARP inhibitors (PARPi) [60]. In HCC, the mechanisms of DDR are closely related to tumorigenesis [61]. Although DDR-associated synthetic lethal mutations are not common in HCC, some of the results that emerged in other cancers may also be potential synthetic lethal interactions in HCC. Lin et al. discovered that BRCA mutations, which occur in approximately 4.8% of HCC, make HCC sensitive to PARPi, although mutation patterns need to be considered [62]. Corylin, a drug that inhibits the expression of RAD51 protein, can block the function of DDR in HCC cells, thereby increasing the sensitivity of HCC cells to other chemotherapeutic drugs [63], suggesting that PARPi combined with corylin may have a therapeutic effect in HCC. A previous study showed that the combination of PARPi and histone deacetylase inhibitor (HDACi) can lead to synthetic lethality in HCC [64]. HDAC is an important enzyme in HR that controls the expression of HR-related genes and proteins. Sun et al. found that the combination of PARPi and mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors can play a therapeutic role in RAS mutant tumors, partly due to reduced homologous recombination-DNA damage repair capacity [65].

4.2. TP53-related synthetic lethality in HCC TP53 mutations occur in almost all human tumors, with mutations rates ranging from 38% to 50% [71]. In HCC, the mutation rate of TP53 is 31%, making it the most commonly mutated gene in HCC [72]. However, the safety of treatment with P53 inhibitors has been questioned [73], and the synthetic lethal strategy provides a new therapeutic horizon for treating TP53-mutant tumors. In colorectal cancer, the genes CSNK1E and CTNNB1 are lethal to TP53, mainly mediated by the Wnt signaling pathway [74]. TP53 also plays an important role in DDR, and TP53 mutation leads to cell cycle checkpoint defects. Once ATR is inhibited, the accumulation of unrepaired DNA damage leads to cytotoxicity, suggesting a synthetic lethal interaction between ATR and TP53 [75]. By chemical screening, Cdk7, a component of CDK-activated kinase and transcription factor IIH, was determined to have a lethal effect on P53-activated tumor cells [76]. TP53 also has an effect on apoptosis, and a previous study showed that Bcl-2 inhibitors can overcome the resistance of P53-activated tumor cell apoptosis [77]. Despite such a high mutation rate, there are a few reports of synthetic lethal interactions with TP53 in HCC. There is evidence that the silencing of PIN1 by RNAi technology leads to a significant decrease in invasion and metastasis ability in HCC cell lines with TP53 mutation [78]. Similarly, Wang et al. screened out the potential synthetic lethal 124

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AKT/mTOR pathway [93,94]. A recent study has shown that in HCC, PTEN deletion combined with MET gene overexpression (PTEN-/ MET+) in HCC cells confers sensitivity to mTORC2 inhibitors, mainly because deletion of mTORC2 inhibits PTEN-/MET + combined induction of AKT1 activation [95]. These results suggest that the PI3K/AKT/ mTOR pathway may contain a pool of potential synthetic lethal targets in PTEN-deficient HCC, and the synthetic lethal effect of a combination of mTOR inhibitors is of particular interest.

relationship between CDK4/6 and TP53 through RNAi screening and then, by chemical screening, showed that CDK4/6 inhibitors had a strong inhibitory effect on the proliferation of HCC cell lines with TP53 mutation [79]. A recent study revealed that inhibition of DNA-replication kinase CDC7 induces the senescence of HCC cells with TP53 mutation, leading to a “one-two punch” approach of eliminating senescent cells to kill cancer cells [80]. Thus, genes involved in the induction of cell senescence in HCC may be associated with the presence of TP53 in a synthetic lethal relationship.

4.6. RAS-related synthetic lethality in HCC 4.3. CTNNB1-related synthetic lethality in HCC The RAS gene is a common oncogene family consisting of three members, HRAS, NRAS, and KRAS, that control the development and programmed death of cells by participating in the RAS/Raf/MAPK pathway [96]. However, RAS is virtually undruggable, so, new targeting strategies are urgently needed for RAS-mutated tumors. Using the RNAi approach, Luo et al. described that mitotic kinase PLK1 inhibitors can lead to the death of tumor cells bearing RAS mutations, which may be associated with chromosomal instability caused by RAS mutations [46]. Using the same technique, Scholl et al. found that cells with KRAS mutations were sensitive to inhibition of the serine/threonine kinase 33 (STK33), which inhibits mitochondrial apoptosis mainly by regulating selective inactivation of S6K1-induced death agonist BAD [97]. With the continuous improvement of RNAi and CRISPR technology, high throughput screening for synthetic lethality has been achieved. In recent years, hundreds of RAS synthetic lethal partners have emerged through screening [79,98,99], implying that the vulnerability of RAS-mutated tumors has been greatly exposed. In HCC, the RAS mutation type is mainly KRAS, and the RAS mutation rate is less than 3% in HCC [13]. A recent study showed that coexpression of βcatenin and KRAS promoted tumorigenesis in HCC cells, and further studies found that cell viability and proliferation were decreased by inhibiting β-catenin [100]. Activation of the RAS/RAF/MAPK pathway is a major feature of patients with advanced HCC [6]. The initial strategy was to treat RAS-mutated HCC with MEK inhibitors, but phase II clinical trials showed no significant effect [101]. Thus, for RAS-mutated HCC, the combination of MEK inhibitor with other inhibitors, such as the combination of sorafenib and refametinib, a MEK inhibitor, has shown encouraging results and significantly improved the overall survival of patients [102].

The major role of the CTNNB1 gene is to promote the development of HCC, mainly through encoding β-catenin regulatory proteins [81]. CTNNB1 is second most frequently mutated gene in HCC, with a mutation rate of 27% [72]. It has been reported that the sensitivity of CTNNB1-mutant tumor cells to the spindle assembly checkpoint kinase TTK inhibitor (TTKi) is five times greater than the sensitivity of the wild-type of CTNNB1 [82]. TTK is a major stable mitotic kinase; thus, the imbalance of β-catenin in mitosis makes cells particularly sensitive to TTKi. In addition, CTNNB1 and HGS [48], as well as CTNNB1 and APC [79], have been screened in HCC by RNAi screening technology as synthetic lethal gene partners, although further validation is needed. 4.4. ARID1A-related synthetic lethality in HCC The ARID1A gene plays a crucially important role in the occurrence and metastasis of HCC. Its overexpression promotes tumorigenesis by mediating oxidative stress, while a deletion mutation promotes tumor metastasis through inhibitory factors [83]. ARID1A mutation occurs in many cancers. In gynecologic cancer, ARID1A deletion causes cells to be sensitive to elesclomol, a reactive oxygen species (ROS)-inducing agent [84]. In ovarian cancer, dasatinib has a lethal effect on ARID1A mutant ovarian clear cell tumors [85], and the silence of EZH2 is also a way to kill ARID1A mutant ovarian cancer cells [86]. In endometrial and breast cancer, ARID1A mutations make sensitize cancer cells to the activating mutations of PIK3CA [87]. ARID1A is usually coexpressed with ARID1B, so the comutation of the two genes is also the synthetic lethal interaction [88]. There are also reports that ARID1A is associated with DNA damage checkpoints, so its mutations can cause sensitivity to PARPi [89]. To date, the synthetic lethality of this mutation has not been verified in HCC, although the ARID1A mutation rate is only approximately 7% in HCC, the above various synthetic lethal relationships provide a reference for HCC treatment.

4.7. MYC-related synthetic lethality in HCC The MYC gene is overexpressed in most human cancers and is thought to be primarily a regulator of the tumor microenvironment [103]. However, MYC is undruggable due to the lack of binding sites on the surface of cells [104]. Therefore, synthetic lethal mechanisms have become an additional important therapeutic strategy for MYC-related cancers. Core spliceosomes components play important roles in the proliferation and invasion of MYC-mutant tumor cells; consequently, inhibition of core spliceosome components such as U2AF1, EFTUD2 and BUD31 leads to lethality of MYC-driven tumors [105]. MYC can also increase protein expression by promoting transcription in cancer cells, thereby inhibiting cancer cell translation and indicating synthetic lethality. A previous study discovered that the activation of 4EBP1 is an important feature in the development and maintenance of MYC-driven tumors [28], and further drug screening confirmed that 4EBP1 is a synthetic lethal target for MYC-driven tumors. P53 inhibits MYC expression, and the TP53 mutation is found to be dependent on MYC upregulation by computational method, indicating that MYC and TP53 have a synthetic lethal relationship [79]. In HCC, MYC is involved in cellular metabolic regulation, thus inhibiting AMPK-related kinase 5 (ARK5; also known as NUAK1), which leads to inhibition of ATP synthesis subsequently leading to apoptosis [106]. In addition, the transcriptional elongation of MYC-driven HCC is dependent on the expression of cyclin-dependent kinase 9 (CDK9), whose inhibition causes

4.5. PTEN-related synthetic lethality in HCC The PTEN gene is a tumor suppressor gene, and the PTEN mutation rate is 7% in HCC [72]. The PTEN gene inhibits tumor proliferation mainly through negative regulation of the AKT/mTOR pathway while maintaining chromosomal stability and DNA repair in the nucleus. A previous study found that PTEN-deficient tumors were sensitive to Nemo-like kinase (NLK) inhibitors, but the specific lethal mechanisms between the two genes remain unclear [90]. Deletion mutations in PTEN lead to an increase in DSBs, making cells sensitive to DSB repair agents. Therefore, PTEN-deficient tumors are also sensitive to PARPi. By using RNAi screening, PTEN-deficient tumors were also found to increase sensitivity to polynucleotide kinase/phosphatase (PNKP) inhibitors, inhibitors of DSB repair proteins [91]. Another study in breast and prostate cancers showed that the deficiency of chromatin helicase DNA-binding factor (CHD1) is lethal to PTEN mutant tumor cells [92], suggesting a synthetic lethal relationship between them. Several studies have demonstrated that cancers with PTEN deficiency are more sensitive to mTOR inhibitors, primarily mTORC1 inhibitors, mainly because PTEN inhibits tumor proliferation and survival by inhibiting the PI3K/ 125

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Authors' contributions

the death of HCC cells [107]. Although the MYC mutation rate is not high in HCC, less than 1% [13], considering the heterogeneity of HCC, synthetic lethality provides a new therapeutic perspective for MYCdriven HCC.

LT collected documents and wrote the manuscript. RC helped to revise the manuscript. XX designed and edited the manuscript. All authors read and approved the final manuscript.

4.8. Other synthetic lethal interactions in HCC

Declaration of competing interest

In addition to the synthetic lethal interactions described above, a large number of synthetic lethal interactions in HCC are still under development. Membrane proteins may also be relevant to synthetic lethality in HCC. CD26 is a membrane protein overexpressed in HCC, and its inhibition plus Bcl-xL (encoded by the BCL2L1 gene) inhibition can effectively inhibit HCC, mainly by dysregulation the cell cycle [108]. POLD1 is a gene that accounts for 1.9% of mutations in HCC [109]. A study has shown that ATR inhibition is lethal to POLD1-deficient cancers [110]. In addition, apart from two-genes pairs, the synthetic lethality of three-gene sets has also been reported [111,112]. At the same time, the discovery of lethality via drug combinations has further expanded the applicability of synthetic lethality [113,114]. Even though so many identified and potential synthetic lethal interactions have been discovered (Table 1), they are still just the tip of the iceberg, with more lethal interactions to be discovered.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank a professional English editor (American Journal Experts) for critical reading of the manuscript. This work was supported by grants from the National Science and Technology Major Project (Grant number: 2017ZX10203205), National Natural Science Funds for Distinguished Young Scholar of China (Grant number: 81625003). Abbreviations HCC TKi PD-1 NGS RNAi SDL GOF LOF 4EBP1 MSL PARP PARPi SGA CRISPR ATR DDR SSB DSB HR HDACi MAPK MEK CDK TTKi ROS NLK PNKP CHD1 STK33 ARK5 CDK9

5. Summary Synthetic lethality has been a popular topic in cancer therapy since its emergence, and it has provided another perspective for targeted therapy. Numerous studies have demonstrated that many drugs produce better outcomes when combined with sorafenib. Therefore, studies on synthetic lethality in HCC can not only increase the efficacy of existing targeted drugs, to enable the development of new effective therapeutic treatment options and circumvent drug resistance but can also provide opportunities for those genes or pathways that cannot be targeted. With the development of RNAi and CRISPR technologies (especially CRISPR/Cas9 technology), it will greatly promote the discovery of synthetic lethal interactions in HCC. In addition, bioinformatics is a nonnegligible tool for rapid research on genetic interactions. Through the application of these technologies, large-scale screening can be achieved to establish a synthetic lethal gene network; thus, more vulnerabilities of HCC can be exposed. Here we also reviewed the types and screening techniques of synthetic lethality, as well as the synthetic lethal interactions and potential synthetic lethal interactions in HCC. Further studies on synthetic lethality are expected to open up new approaches for the personalized treatment of HCC (Fig. 3). The current direction of synthetic lethality in cancer is mainly concentrated in the DNA damage response, synthetic dosage lethality and metabolic synthetic lethality. Although a significant portion of synthetic lethal interactions has been found in various cancers, with the exception of the clinical application of BRCA and PARPi in breast cancer, no more synthetic lethal interactions have yet been applied in clinical practice. In HCC, there are several reasons for this. First, only a few synthetic lethal interactions have been found, let alone clinical applications, in HCC. Second, the heterogeneity of HCC and the lack of important driver genes are also major causes of treatment difficulties. Third, there are only a few intersections between synthetic lethal networks screened by various screening methods, which greatly increases the difficulty of application. Fourth, both RNAi screening and CRISPR screening currently have severe off-target effects, which results in errors in the screening of synthetic lethal gene pairs. Yet, CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, due to their relative higher gene knockout efficiency [53]. Finally, the lack of suitable targets for certain synthetic lethal genes is also a limitation of clinical application. In any case, synthetic lethality has become an emerging and promising therapeutic strategy for HCC in the future.

Hepatocellular carcinoma Tyrosine kinase inhibitor Programmed cell death-1 Next-generation sequencing RNA interference Synthetic dosage lethality Gain-of-function Loss-of-function 4E binding protein-1 Metabolic synthetic lethality Poly (ADP-ribose) polymerase PARP inhibitor Systemic genetic analysis Clustered regularly interspaced short palindromic repeats Ataxia telangiectasia mutated and RAD3-related DNA damage repair Single-strand DNA break Double-stranded DNA break Homologous recombination Histone deacetylase inhibitor Mitogen-activated protein kinase MAPK kinase Cyclin-dependent kinase TTK inhibitor Reactive oxygen species Nemo-Like Kinase Polynucleotide kinase/phosphatase DNA-binding factor serine/threonine kinase 33 AMPK-related kinase 5 Cyclin-dependent kinase 9

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