As mentioned above, tumour cells acquire mutations that allow them to grow rapidly over time. While these characteristics give cancerous cells advantages in proliferation and survival, they also become therapeutic targets. The differential regulation of genes between cancer cells and their corresponding normal cells allow researchers to identify those that can be targeted to induce synthetic lethality in a cancer-specific manner. The various types of interactions are described below.
Loss-of-function/Loss-of function
Loss-of-function (LOF) mutations in tumour suppressors are extremely common in human cancers. One such example is the genome guardian
p53, the most frequently mutated gene in human cancers, ranging from 25 to 50 % in various tumour types including ovarian, breast, colorectal, head and neck, and lung cancers [
36]. Although p53 mutations have been studied extensively, it has been a difficult target as it has no enzymatic activity and primarily functions through protein-protein interactions [
37]. One approach to target this “undruggable” target has focused on strategies to restore the wild-type function of mutated p53 [
38]. Another therapeutic approach exploits the vulnerabilities or genetic dependencies arising from loss of wild-type p53 functions [
39]. For example, based on differential gene expression analyses, Wang and Simon proposed a list of 98 candidate genes that, when suppressed, may induce synthetic lethality in p53-deficient cancers [
40]. We define such a strategy as LOF/LOF as it involves inhibiting a gene/pathway/function in the background of genetic or pharmacological inactivation of another gene to induce synthetic lethality.
To date, the biggest clinical success in synthetic lethal targeting is the use of PARP (poly (ADP-ribose) polymerase) inhibitors against tumours with BRCA mutations. BRCA1 and BRCA2 deficiencies cause defects in homologous recombination (HR) and have a significant role in hereditary breast and ovarian cancers [
41]. It is known that mice lacking PARP expression are viable but are defective in repairing single-stranded DNA breaks. Loss of PARP function results in the dependence on the use of homologous recombination (HR) to repair DNA damage [
42]. Based on these observations, Bryant et al. hypothesized and then demonstrated that BRCA-deficient cancers are hypersensitive to PARP inhibitors [
42]. The discovery of this truly synthetic lethal interaction (by definition) has revolutionized our approach to the treatment of BRCA-associated cancers and served as the paradigm for uncovering other synthetic lethal interactions.
PTEN, the second most frequently mutated tumour suppressor in cancer, is a similar therapeutic target [
43]. Inspired by the synthetic lethality induced by loss of BRCA2 and PARP functions, Mendes-Pereira et al. demonstrated that cancers with PTEN deficiency, which also cause HR defects, are sensitive to PARP inhibitors [
44]. This synthetic lethal interaction was validated in vitro and in vivo. As a result, PARP inhibitors are currently in Phase II clinical trials for treatment of PTEN-deficient cancers. Exploiting deficiencies in DNA repair proteins has since revealed additional synthetic lethal interactions with particular relevance to lung cancer. Similar to the case of PARP and BRCA, Riabinska
et. al. observed that cancers with ATM-deficiency are defective in HR and dependent on nonhomologous end joining (NHEJ) for DNA repair and cell survival [
45]. Through genetic and pharmacological methods, they subsequently demonstrated that inhibition of the catalytic subunit of an essential NHEJ protein, DNA-dependent protein kinase (PRKDC), induces synthetic lethality specifically in ATM-deficient cancers, but not normal cells or cancer cells with active ATM. Likewise, after developing a potent and selective inhibitor of the DNA damage response (DDR) kinase ATR, Reaper and colleagues found that it induced synthetic lethality specifically in ATM- or p53-deficient cancers in the context of genotoxic stress. Since ATM is mutated and inactivated in ~10 % of LACs, these findings provide a potential therapeutic strategy for a large subset of patients, one which is especially resistant to standard chemotherapeutics.
While the above examples were mainly identified in other cancer types, many of the described genes are also disrupted in lung cancer suggesting that similar synthetic lethal interactions may also exist in this context. However, screens for lung cancer specific LOF/LOF synthetic lethal interactions have also been performed. For example, through a chemical library screen, Shackelford and colleagues found that LKB1 (STK11) deficient lung cancer cell lines were acutely sensitive to phenformin, an inhibitor of mitochondrial function [
46]. LKB1 is mutated in ~20 % of NSCLC and this work, subsequently validated in Lkb1 mutant mouse models, suggests that metabolism based therapeutics may be effective in this subset of cancer patients. Using a loss-of-function whole-genome shRNA screen, inhibition of the canonical Wnt pathway, specifically the positive regulators tankyrase 1 and 2, was found to induce cell death in LAC cells only in the context of EGFR inhibition [
47]. Furthermore, computational approaches have revealed LOF/LOF synthetic lethality of MAX and BRG1 in SCLC and RB1 and CDKN2A in both SCLC and NSCLC [
48,
49].
Lastly, it is important to note a unique subcategory of LOF/LOF interactions that only occur in the presence of a gain-of-function (GOF) oncogene mutation. These interactions typically involve inhibiting the mutant oncogene with a small molecule inhibitor and another gene with pharmacological or genetic methods. Since the inhibitors only work – and hence synthetic lethality is only induced - in the context of the GOF mutation, we term this association GOF/LOF/LOF, even though only two genes are involved. Examples of this association include the use of EGFR kinase inhibitors in the background of mutant EGFR (mEGFR). For instance, using RNAi screening in the presence of the EGFR kinase inhibitor Erlotinib, Bivona and colleagues found that inhibiting FAS and other components of the NF-kB pathway specifically enhanced cell death in mEGFR LAC cell lines [
50,
51]. Likewise, Sudo et. al. found a similar association between NF-kB inhibition and Gefitinib (another EGFR kinase inhibitor) and also identified a novel gene candidate, PRKCSH, that induced cell death in Gefitnib-treated mEGFR LAC cells [
52]. Finally, a pooled shRNA screen in KRAS mutant cancer cells, which are dependent on MEK activation, found that inhibition of BCL-XL cooperated with MEK inhibitors to induce cell death [
53]. These findings were validated using the MEK inhibitor selumetinib and the BCL-XL inhibitor ABT-263 in a genetically engineered KRAS-driven mouse model of lung cancer; highlighting the potential clinical relevance of this interaction.
Gain-of-function/Loss-of function (synthetic dosage lethality)
While loss of tumour suppressor functions is a common characteristic in cancer cells, activation of various oncogenes through gain-of-function (GOF) mutations is another major contributor to tumour development. Like LOF mutations, GOF mutations rewire cancer cells, making them susceptible to additional changes in cellular functions that would cause little or no harm to normal cells. When lethality occurs as a result of one gene being genetically activated (GOF) and another being inactivated through genetics or drug targeting (LOF), the interaction is known as synthetic dosage lethality.
As an example, constitutive activation of RAS signaling, particularly KRAS, is known to be the oncogenic driver for approximately 20 % of all human cancers [
54]. Cancers of the pancreas, colon, and lung are known to have high frequencies of KRAS mutations [
55]. KRAS has long been considered an “undruggable” target due to the lack of suitable binding pockets for small molecule inhibitors [
56]. To target KRAS mutants, several groups have utilized high throughput RNAi screening to identify synthetic lethal partners of activated mutant KRAS. Luo et al. performed a genome-wide shRNA screen in colorectal cancer cells and found that KRAS mutants are hypersensitive to PLK1, APC/C, and proteasome inhibition relative to isogenic cells with wild-type KRAS [
57]. Other synthetic lethal partners that have been identified through RNAi screening within an activated KRAS background include STK33, TBK1, WT1, CDK1, CDK4, GATA2, and Snail2, studies conducted primarily using colon and lung cancer cells [
58‐
64]. These interactions have been reviewed elsewhere.
Hyperactivation of members of the MYC protein family is very common in a wide range of cancers.
c-MYC is amplified in about 10 % of LAC and c-MYC, MYCL or MYCN amplification/overexpression occurs in >20 % of SCLC. MYCs are transcription factors - and thus, difficult to inhibit directly – thus, synthetic lethal strategies are being used to define novel treatments for cancers driven by the activation of these proteins. Inhibition of mTOR, Aurora A/B, SAE2, or CDK1 has been shown to induce synthetic lethality in MYC-driven cancers [
65‐
68]. Synthetic lethal relationships between hyperactivated MYCN and suppression of BRD4 and CSNK1e have also been demonstrated in acute myeloid leukaemia and neuroblastoma, respectively [
69,
70]. Importantly these interactions are being tested clinically where Phase II clinical trials are assessing therapeutic benefit of inhibitors targeting CDK1 or BRD4 in patients with MYC-amplified tumours. Recently, through a pooled shRNA screen, inhibition PRKDC was found to specifically induce synthetic lethality in SCLC cell lines with amplification/overexpression of MYC family members but not in those without [
71]. This was attributed to the role of PRKDC in controlling MYC protein levels as well as facilitating repair of MYC-induced DNA damage. These results could be recapitulated with the PRKDC inhibitor, NU-7441, suggesting a possible therapeutic avenue for this aggressive lung cancer subtype.
Lastly, although EGFR kinase inhibitors have proven effective at improving outcomes of LAC patients with activating EGFR mutations, all patients treated with these inhibitors eventually develop resistance and relapse. Identifying the key factors necessary for mEGFR-mediated tumorigenesis may offer avenues for combination based therapies that can overcome EGFR kinase inhibitor resistance. Through charactering the LAC‐specific mEGFR interactome through global analysis of protein–protein interactions and phosphorylation, Li and collegues identified 8 key proteins necessary for survival mEGFR lung cancer cell lines in addition to EGFR itself: GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A [
72]. When inhibited through siRNAs, these genes induced cell death specifically in mEGFR, but not EGFR wild-type, LAC cells. This clearly suggested a synthetic lethal relationship. Indeed, ablation of ARHG5 in mEGFR LAC cells induced apoptosis and was shown to interact with downstream EGFR signalling proteins including SHC1 and GRB2, highlighting its potential as a target for combination based therapy.
Gain-of-function/Gain-of-function
With the tools currently available, it is relatively easy to identify synthetic lethal interactions using gene knockdown or knockout approaches. In contrast, there has yet to be a large-scale GOF screen in human cells to search for synthetic lethality in cancers driven by known oncogenes. It is, however, not impossible to define synthetic lethal interactions that are based on two gain-of-function mutations using information derived from studies assessing human tumour evolution. For instance, it has been known for years that oncogenic mutations in KRAS and EGFR are not only common, but also mutually exclusive in LAC [
73‐
75]. It was typically assumed that since the two oncogenes work through the activation of similar pathways, mutation in both genes was functionally redundant and thus, not positively selected during tumour development. However, in a recent study, Unni et al. discovered that these two mutations are mutually exclusive because activation of both genes in lung cells induces synthetic lethality. This explained why there is selectedion against cells expressing both mutations together during tumour evolution [
76]. Expressing both mutant oncogenes in a mouse model of lung cancer led to the selection of tumours expressing a single oncogene while forced expression of mEGFR or mKRAS in LAC cells with endogenous mutations in the reciprocal oncogene induced cell death through uncontrolled macropinocytosis and catastrophic cell vacuolization. The later events were likely controlled through increased MAPK signalling. From a clinical perspective, while it is challenging to increase expression of a gene product, this study suggests that there may be opportunities to activate combinations of pathways to induce lethality. The method through which this interaction was revealed and validated suggests that additional synthetic lethal interactions may be identified through exploring co-activation of mutually exclusive driver mutations and their respective pathways. Indeed, a subsequent study revealed a similar association between mutant BRAF and mKRAS in LAC, demonstrating the validity of this approach.
Although difficult to categorize, chemical compound screens have also revealed interactions suggestive of synthetic lethality induced by GOF/GOF associations. As an example, Guo et al. identified a small molecular weight compound (oncrasin-1) that selectively and effectively killed human lung cancer cells with mKRAS through induction of apoptosis [
77]. A search for a target of this novel compound revealed no changes to the activity of known RAS signalling pathway components. However, treatment with oncrasin-1 led to abnormal aggregation of PKCι in the nucleus of sensitive, but not in resistant cells. While oncrasin-1 did not change the activity of PKCι, it did induce a GOF change in terms of modifying PKCι subcellular localization. This was, in turn, associated with cell death in KRAS mutant cells. It is conceivable that compounds that stimulate or modify pathway signalling/function may also provide an opportunity to induce synthetic lethality in cancer cells in the context of GOF/GOF interactions.
Collateral lethality
A more recent concept of synthetic lethality has been termed “collateral lethality” [
78]. In the process of malignant transformation, some tumour suppressor genes may be inactivated through chromosomal deletions. Loss of heterozygosity or homozygous deletions results in complete inactivation of a tumour suppressor; which in itself enables cancer-specific targeting of synthetic lethal partners as described above. However, collateral lethality takes advantage of “passenger” or “neighboring” genes that are co-deleted “unintentionally” in this process [
78,
79]. These passenger genes may encode for housekeeping functions that are essential to cell viability but are masked by the presence of redundant genes encoded elsewhere to complement for the loss. Targeting homologues of these passengers forms the basis of collateral lethality. As an example,
ENO1 is a gene that is homozygously deleted in glioblastoma (GBM) as a result of deletion in the 1p36 tumour-suppressor locus [
79]. This gene encodes for enolase, an enzyme that is essential for glycolysis. Although ENO1 accounts for up to 90 % of the enolase activity in GBM, its loss is tolerated through the expression of
ENO2 which is exclusively expressed in neural cells [
55]. Loss of
ENO2 does not cause reduced viability in the presence of
ENO1 expression, but GBM cells with deletion in 1p36 are highly sensitive to ENO2 inhibition due to collateral lethality [
79]. Muller et al. have identified other homozygously deleted essential genes that have potential collateral lethal partners in GBM [
79].
To date, the only described instance of collateral lethality in lung cancer involves SMARCA4 (a chromatin remodelling helicase) and its paralogue, SMARCA2. Unlike the ENO1/ENO2 example, SMARCA4 is a true tumour suppressor gene and is directly inactivated through mutations or deletions in approximately 10–15 % of LACs. However, unlike other tumour suppressors, SMARCA4 and SMARCA2 perform cellular housekeeping functions rather than preventing abnormal cell growth. Thus, inhibition of SMARCA2 in the background of SMARCA4 genetic inactivation leads to synthetic lethality as the cell loses the ability to complete an essential function necessary for survival, as is the case with ENO1/ENO2 in GBM. Inhibitors targeting SMARCA2 may therefore prove effective in the large subset of LAC patients with SMARCA4 mutations [
80‐
83].