Searching for synthetic lethality in cancer

The incentive to develop personalised therapy for cancer treatment is driven by the premise that it will increase therapeutic efficacy and reduce toxicity. Understanding the underlying cellular and molecular basis of the disease has been extremely important in the design of these novel therapies; however, identifying new drug targets for personalised therapies remains problematic. This review describes how the biological concept of synthetic lethality has been successfully implemented to identify new therapeutic approaches and targets in models from yeast through to human cells. We also discuss how recent technical advances combined with an increased understanding of the complexity of cellular networks may facilitate therapeutic advances in the future.     

Targeting cancer cells by synthetic lethality

Standard cytotoxic agents for treating cancer were developed based on their effectiveness to kill rapidly dividing cells, not on their ability to selectively kill cancer cells and spare normal tissue. Much of contemporary cancer research is aimed at identifying specific molecular features of cancers to directly target tumor cells with the hope of reducing or eliminating unwanted side effects. Targeted therapy for the treatment of cancer can be divided into two main categories: monoclonal antibodies and small molecules. In this Perspective, we review the approach of synthetic lethality to target cancer, specifically renal cell carcinoma. The concept of synthetic lethality is used to describe a genetic interaction of two non-allelic and non-lethal genes that when mutated simultaneously results in cell death. Recently, we identified a compound, STF-62247, that functions in a synthetic lethal manner to the loss of VHL, a mutation found in the majority of renal cell carcinomas.     

Targeting cancer-specific synthetic lethality in double-strand DNA break repair

There is interest in the use of DNA repair inhibitors as sensitizers of classic cytotoxic therapy against cancer. However, there is also risk – theoretical, at least – that such a strategy may increase the side effects of traditional therapies, including but not limited to treatment-related secondary malignancies. Before being brought to clinical application, therefore, important questions remain to be answered regarding how these therapies will be tailored to achieve benefit without concomitantly increasing harm. A potential solution may involve targeting so-called “synthetic lethalities” in tumor DNA repair pathways; taking advantage of defects acquired in DNA repair pathways during tumorigenesis by targeting alternative repair pathways on which the tumor critically depends. Conceivably, as repair pathways are functional in normal tissue, such targeted therapy should be relatively tumor-specific and non-toxic. We review here the rationale for this strategy, describe examples of its application, and outline potential strengths and weaknesses of this approach. For simplicity, a focus will be placed on the repair of double-strand breaks as a model system, but the conceptual framework is generally applicable to many other pathways of DNA repair.     

Modeling synthetic gene oscillators

Genetic oscillators have long held the fascination of experimental and theoretical synthetic biologists alike. From an experimental standpoint, the creation of synthetic gene oscillators represents a yardstick by which our ability to engineer synthetic gene circuits can be measured. For theorists, synthetic gene oscillators are a playground in which to test mathematical models for the dynamics of gene regulation. Historically, mathematical models of synthetic gene circuits have varied greatly. Often, the differences are determined by the level of biological detail included within each model, or which approximation scheme is used. In this review, we examine, in detail, how mathematical models of synthetic gene oscillators are derived and the biological processes that affect the dynamics of gene regulation.     

Identification of synthetic lethality of PLK1 inhibition and microtubule-destabilizing drugs

Polo-like kinase 1 (PLK1) is frequently overexpressed in cancer, which correlates with poor prognosis. Therefore, we investigated PLK1 as therapeutic target using rhabdomyosarcoma (RMS) as a model. Here, we identify a novel synthetic lethal interaction of PLK1 inhibitors and microtubule-destabilizing drugs in preclinical RMS models and elucidate the underlying molecular mechanisms of this synergism. PLK1 inhibitors (i.e., BI 2536 and BI 6727) synergistically induce apoptosis together with microtubule-destabilizing drugs (i.e., vincristine (VCR), vinblastine (VBL) and vinorelbine (VNR)) in several RMS cell lines (combination index <0.9) including a patient-derived primary RMS culture. Importantly, PLK1 inhibitors and VCR cooperate to significantly suppress RMS growth in two in vivo models, including a mouse xenograft model, without causing additive toxicity. In addition, no toxicity was observed in non-malignant fibroblast or myoblast cultures. Mechanistically, BI 2536/VCR co-treatment triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of antiapoptotic BCL-2 family proteins, followed by BAX/BAK activation, production of reactive oxygen species (ROS) and activation of caspase-dependent or caspase-independent effector pathways. This conclusion is supported by data showing that BI 2536/VCR-induced apoptosis is significantly inhibited by preventing cells to enter mitosis, by overexpression of BCL-2 or a non-degradable MCL-1 mutant, by BAK knockdown, ROS scavengers, caspase inhibition or endonuclease G silencing. This identification of a novel synthetic lethality of PLK1 inhibitors and microtubule-destabilizing drugs has important implications for developing PLK1 inhibitor-based combination treatments.Treatment response critically depends on intact cell death programs in cancer cells. One of the best-characterized forms of programmed cell death is apoptosis.¹ Engagement of the extrinsic (death receptor) or the intrinsic (mitochondrial) pathway of apoptosis eventually leads to activation of caspases, a family of enzymes that function as cell death effector molecules.¹ Signaling via the mitochondrial pathway of apoptosis is tightly controlled by both antiapoptotic (BCL-2, BCL-X_L, MCL-1) and proapoptotic (BAX, BAK) proteins of the BCL-2 family.² Apoptosis normally eliminates cells with intolerable DNA damage or perturbations in cell cycle progression.^{3, 4} In cancer cells, however, antiapoptotic proteins are frequently expressed at high levels, contributing to evasion of apoptosis and treatment resistance.²Polo-like kinase 1 (PLK1) is a serine/threonine-specific kinase that is pivotal for progression through mitosis.⁵ Consistently, high expression of PLK1 correlates with increased proliferative potential and poor prognosis in many tumor entities.⁵ Thus, PLK1 has emerged as an attractive therapeutic target in oncology. In recent years, several PLK1 inhibitors have been developed, with some agents showing encouraging results in early-phase clinical trials.⁵ However, little is yet known on whether the antitumor activity of PLK1 inhibitors can be potentiated in rational combination regimens. Recently, overexpression of PLK1 has been documented in human tissue samples of rhabdomyosarcoma (RMS), the most frequent pediatric soft-tissue sarcoma, and was shown to correlate with reduced survival.^{6, 7, 8} Searching for new synthetic lethal drug interactions, we used RMS as a model to investigate PLK1 inhibitor-based combination therapies in this study.     

Lethality and synthetic lethality in the genome-wide metabolic network of Escherichia coli

Recent genomic analyses on the cellular metabolic network show that reaction flux across enzymes are diverse and exhibit power-law behavior in its distribution. While intuition might suggest that the reactions with larger fluxes are more likely to be lethal under the blockade of its catalysing gene products or gene knockouts, we find, by in silico flux analysis, that the lethality rarely has correlations with the flux level owing to the widespread backup pathways innate in the genome-wide metabolism of Escherichia coli. Lethal reactions, of which the deletion generates cascading failure of following reactions up to the biomass reaction, are identified in terms of the Boolean network scheme as well as the flux balance analysis. The avalanche size of a reaction, defined as the number of subsequently blocked reactions after its removal, turns out to be a useful measure of lethality. As a means to elucidate phenotypic robustness to a single deletion, we investigate synthetic lethality in reaction level, where simultaneous deletion of a pair of nonlethal reactions leads to the failure of the biomass reaction. Synthetic lethals identified via flux balance and Boolean scheme are consistently shown to act in parallel pathways, working in such a way that the backup machinery is compromised.     

Genome‐scale gene/reaction essentiality and synthetic lethality analysis

Synthetic lethals are to pairs of non‐essential genes whose simultaneous deletion prohibits growth. One can extend the concept of synthetic lethality by considering gene groups of increasing size where only the simultaneous elimination of all genes is lethal, whereas individual gene deletions are not. We developed optimization‐based procedures for the exhaustive and targeted enumeration of multi‐gene (and by extension multi‐reaction) lethals for genome‐scale metabolic models. Specifically, these approaches are applied to iAF1260, the latest model of Escherichia coli, leading to the complete identification of all double and triple gene and reaction synthetic lethals as well as the targeted identification of quadruples and some higher‐order ones. Graph representations of these synthetic lethals reveal a variety of motifs ranging from hub‐like to highly connected subgraphs providing a birds‐eye view of the avenues available for redirecting metabolism and uncovering complex patterns of gene utilization and interdependence. The procedure also enables the use of falsely predicted synthetic lethals for metabolic model curation. By analyzing the functional classifications of the genes involved in synthetic lethals, we reveal surprising connections within and across clusters of orthologous group functional classifications.     

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD

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Genetic synthetic lethality screen at the single gene level in cultured human cells

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.     

Epitope tag-induced synthetic lethality between cohesin subunits and Ctf7/Eco1 acetyltransferase

Ctf7/Eco1-dependent acetylation of Smc3 is essential for sister chromatid cohesion. Here, we use epitope tag-induced lethality in cells diminished for Ctf7/Eco1 activity to map cohesin architecture in vivo. Tagging either Smc1 or Mcd1/Scc1, but not Scc3/Irr1, appears to abolish access to Smc3 in ctf7/eco1 mutant cells, suggesting that Smc1 and Smc3 head domains are in direct contact with each other and also with Mcd1/Scc1. Thus, cohesin complexes may be much more compact than commonly portrayed. We further demonstrate that mutation in ELG1 or RFC5 anti-establishment genes suppress tag-induced lethality, consistent with the notion that the replication fork regulates Ctf7/Eco1.     

Poly(ADP-ribose) polymerase-1 inhibition: preclinical and clinical development of synthetic lethality

The hereditary forms of breast cancer identified by BRCA1 and BRCA2 genes have a defect in homologous DNA repair and demonstrate a dependence on alternate DNA repair processes by base excision repair, which requires poly(ADP-ribose) polymerase 1 (PARP-1). siRNA and deletion mutations demonstrate that interference with PARP-1 function results in enhanced cell death when the malignancy has a defect in homologous recombination. These findings resulted in a plethora of agents in clinical trials that interfere with DNA repair, and these agents offer the potential of being more selective in their effects than classic chemotherapeutic drugs. An electronic search of the National Library of Medicine for published articles written in English used the terms "PARP inhibitors" and "breast cancer" to find prospective, retrospective and review articles. Additional searches were done for articles dealing with mechanism of action. A total of 152 articles dealing with breast cancer and PARP inhibition were identified. PARP inhibition not only affects nonhomologous repair, but also has several other nongenomic functions. Mutational resistance to these agents was seen in preclinical studies. To date, PARP-1 inhibitors were shown to enhance cytotoxic effects of some chemotherapy agents. This new class of agents may offer more therapeutic specificity by exploiting a DNA repair defect seen in some human tumors with initial clinical trials demonstrating antitumor activity. Although PARP inhibitors may offer a therapeutic option for selected malignancies, the long-term effects of these agents have not yet been defined.     

shRNA-mediated RNA interference as a tool for genetic synthetic lethality screening in mouse embryo fibroblasts

Previously, we demonstrated the establishment of synthetic lethality screening in cultured somatic human cells, or mouse embryo fibroblasts (MEFs), for chemicals or mutant genes synergistically lethal with a mutated gene of interest. Here, we show in MEFs that the usage of RNA interference-based genetic suppressor elements encoding short hairpin RNAs (shRNAs) enables for genetic synthetic lethality screening at a frequency much higher than that achieved before with short truncated sense and antisense RNAs. These findings open up the possibility of using in mammalian cells genome-wide shRNA libraries for genetic synthetic lethality screening at the multi-gene level.     

Mutation load,functional overlap,and synthetic lethality in the evolution and treatment of cancer

The efficacy of conventional anti-cancer drugs is puzzling in view of the ubiquitous tissue distribution and vital nature of their targets. Differences in cell cycling rates are not thought sufficient to explain chemotherapeutic selectivity. I suggest an alternative possibility based on the combinatorial effects of mutations in cancer cells. This model incorporates the concepts of synthetic-lethal interactions and mutation loads to explain the drug sensitivity of cancer cells. From this perspective, drugs that target complex processes that utilize genetically redundant or overlapping components, such as DNA replication and chromosome segregation, offer attractive target opportunities.     

Mechanisms for stalled replication fork stabilization: new targets for synthetic lethality strategies in cancer treatments

Timely and faithful duplication of the entire genome depends on completion of replication. Replication forks frequently encounter obstacles that may cause genotoxic fork stalling. Nevertheless, failure to complete replication rarely occurs under normal conditions, which is attributed to an intricate network of proteins that serves to stabilize, repair and restart stalled forks. Indeed, many of the components in this network are encoded by tumour suppressor genes, and their loss of function by mutation or deletion generates genomic instability, a hallmark of cancer. Paradoxically, the same fork‐protective network also confers resistance of cancer cells to chemotherapeutic drugs that induce high‐level replication stress. Here, we review the mechanisms and major pathways rescuing stalled replication forks, with a focus on fork stabilization preventing fork collapse. A coherent understanding of how cells protect their replication forks will not only provide insight into how cells maintain genome stability, but also unravel potential therapeutic targets for cancers refractory to conventional chemotherapies.     

Racing to block tumorigenesis after pRb loss: An innocuous point mutation wins with synthetic lethality

A major goal of tumor suppressor research is to neutralize the tumorigenic effects of their loss. Since loss of pRb does not induce tumorigenesis in many types of cells, natural mechanisms may neutralize the tumorigenic effects of pRb loss in these cells. For susceptible cells, neutralizing the tumorigenic effects of pRb loss could logically be achieved by correcting the deregulated activities of pRb targets to render pRb-deficient cells less abnormal. This line of research has unexpectedly revealed that knocking out the pRb target Skp2 did not render Rb1 deficient cells less abnormal but, rather, induced apoptosis in them, thereby completely blocking tumorigenesis in Rb1+/- mice and after targeted deletion of Rb1 in pituitary intermediate lobe (IL). Skp2 is a substrate-recruiting component of the SCFSkp2 E3 biquitin ligase; one of its substrates is Thr187-phosphorylated p27Kip1. A p27T187A knockin (KI) mutation phenocopied Skp2 knockout (KO) in inducing apoptosis following Rb1 loss. Thus, Skp2 KO or p27T187A KI are synthetic lethal with pRb inactivation. Since homozygous p27T187A KI mutations show no adverse effects in mice, inhibiting p27T187 phosphorylation or p27T187p ubiquitination could be a highly therapeutic and minimally toxic intervention strategy for pRb deficiency-induced tumorigenesis.     

The allele-specific synthetic lethality of prlA-prlG double mutants predicts interactive domains of SecY and SecE.

The secretion of proteins from the cytoplasm of Escherichia coli requires the interaction of two integral inner membrane components, SecY and SecE. We have devised a genetic approach to probe the molecular nature of the SecY-SecE interaction. Suppressor alleles of secY and secE, termed prlA and prlG, respectively, were analyzed in pair-wise combinations for synthetic phenotypes. From a total of 115 combinations, we found only seven pairs of alleles that exhibit a synthetic defect when present in combination with one another. The phenotypes observed are not the result of additive defects caused by the prl alleles, nor are they the consequence of multiple suppressors functioning within the same strain. In all cases, the synthetic defect is recessive to wild-type secY or secE provided in trans. The recessive nature argues for a defective interaction between the Prl suppressors. The extreme allele specificity and topological coincidence of the mutations represented by these seven pairs of alleles identify domains of interaction between SecY/PrlA and SecE/PrlG.     

Design and synthesis of Pictet-Spengler condensation products that exhibit oncogenic-RAS synthetic lethality and induce non-apoptotic cell death

A series of Pictet-Spengler condensation derivatives (tetrahydro-β-carbolines) was designed, synthesized and evaluated for lethality against a panel of seven cancer cell lines. Seven compounds (2a, 13, 20, 21, 27, 29 and 34) showed lethality in at least five cell lines. Among these, compound 27 showed a unique selectivity towards oncogenic-RAS expressing BJ-TERT/LT/ST/RAS(V12) tumor cells, compared to non-transformed BJ-TERT cells. Further investigation revealed that 27 induces cell death without activation of caspases. This represents a useful new probe of non-apoptotic cell death and oncogenic-RAS synthetic lethality.     

Graph based recurrent network for context specific synthetic lethality prediction

The concept of synthetic lethality(SL) has been successfully used for targeted therapies. To further explore SL for cancer therapy, identifying more SL interactions with therapeutic potential are essential. Recently, graph neural network-based deep learning methods have been proposed for SL prediction, which reduce the SL search space of wet-lab based methods. However, these methods ignore that most SL interactions depend strongly on genetic context, which limits the application of the predicted results. In this study, we proposed a graph recurrent network-based model for specific context-dependent SL prediction(SLGRN). In particular, we introduced a Graph Recurrent Network-based encoder to acquire a context-specific, low-dimensional feature representation for each node, facilitating the prediction of novel SL. SLGRN leveraged gate recurrent unit(GRU) and it incorporated a context-dependent-level state to effectively integrate information from all nodes. As a result, SLGRN outperforms the state-of-the-arts models for SL prediction. We subsequently validate novel SL interactions under different contexts based on combination therapy or patient survival analysis. Through in vitro experiments and retrospective clinical analysis, we emphasize the potential clinical significance of this context-specific SL prediction model.