Synthetic Lethal Gene Research Articles

PO-142 RNAi-mediated therapeutic knockdown of the neuropilin axis and GIPC1 in pancreatic carcinoma

IntroductionB-cell acute lymphoblastic leukaemia (B-ALL) is caused by malignant transformation of B-cell progenitors and has multiple genetic subtypes, each characterised by specific genetic abnormalities, including MLL-rearranged and TCF3/PBX1 t(1;19). Survival for B-ALL is favourable compared to many other cancers, however, there is a need for novel therapeutic approaches that are less toxic and lead to better long term outcome for patients compared to current treatment options. These could potentially be found by identifying subtype-specific synthetic lethal (SL) genes. We have developed a novel bioinformatic approach which uses genome-wide methylation and expression data to identify candidate SL genes in specific genetic subtypes of disease. We aimed toidentify candidate SL genes in MLL-rearranged B-ALL, utilising our novel bioinformatics approach.Material and methodsGenome-wide methylation and expression data from patient samples were used to identify candidate genes. Candidate SL genes were knocked down with siRNA in cell lines with/without the corresponding subtype defining genetic abnormality. Apoptosis was measured by Annexin-V staining, and proliferation by cell counts using a specialised flow cytometer. Gene re-expression was performed using a lentiviral construct, co-expressing eGFP.Results and discussionsSurprisingly, in addition to a single candidate SL gene specific for MLL-fusion related B-ALL, our novel bioinformatic analysis also identified two classes of gene not generally seen in our analysis of other subtypes; a candidate subtype-specific tumour suppressor (KLF11) and a candidate shared SL gene (LAMP5, shared with t(1;19). LAMP5 knockdown significantly increased apoptosis (12.7%, p=0.0012) and decreased proliferation (23.3%, p=0.0006) in a t(1;19) cell line but not in MLL cell lines. As predicted, KLF11 re-expression in the MLL-rearranged SEM cell line caused significantly reduced cell growth/survival compared with the KLF11-negative parental cell line. However, high level re-expression in three cell lines expressing endogenous KLF11 resulted in similar effects on cell growth, implying that high level KLF11 expression was universally toxic in leukaemia cell lines.ConclusionLAMP5 may be a SL gene, however only specifically in t(1;19) B-ALL. This is the first time this gene has been linked to leukaemia and if confirmed to have a role in this subtype of B-ALL it would represent a novel therapeutic target.

Synthetic Lethality-based Identification of Targets for Anticancer Drugs in the Human Signaling Network

Chemotherapy agents can cause serious adverse effects by attacking both cancer tissues and normal tissues. Therefore, we proposed a synthetic lethality (SL) concept-based computational method to identify specific anticancer drug targets. First, a 3-step screening strategy (network-based, frequency-based and function-based screening) was proposed to identify the SL gene pairs by mining 697 cancer genes and the human signaling network, which had 6306 proteins and 62937 protein-protein interactions. The network-based screening was composed of a stability score constructed using a network information centrality measure (the average shortest path length) and the distance-based screening between the cancer gene and the non-cancer gene. Then, the non-cancer genes were extracted and annotated using drug-target interaction and drug description information to obtain potential anticancer drug targets. Finally, the human SL data in SynLethDB, the existing drug sensitivity data and text-mining were utilized for target validation. We successfully identified 2555 SL gene pairs and 57 potential anticancer drug targets. Among them, CDK1, CDK2, PLK1 and WEE1 were verified by all three aspects and could be preferentially used in specific targeted therapy in the future.

Synthetic Lethality: From Research to Precision Cancer Nanomedicine.

Cancer is an evolutionary disease with multiple genetic alterations, accumulated due to chromosomal instability and/or aneuploidy and it sometimes acquires drug-resistant phenotype also. Whole genome sequencing and mutational analysis helped in understanding the differences among persons for predisposition of a disease and its treatment non-responsiveness. Thus, molecular targeted therapies came into existence. Among them, the concept of synthetic lethality have enthralled great attention as it is a pragmatic approach towards exploiting cancer cell specific mutations to specifically kill cancer cells without affecting normal cells and thus enhancing anti-cancer drug therapeutic index. Thus, this approach helped in discovering new therapeutic molecules for development of precision medicine. Nanotechnology helped in delivering these molecules to the target site in an effective concentration thus reducing off target effects of drugs, dose and dosage frequency drugs. Researchers have tried to deliver siRNA targeting synthetic lethal partner for target cancer cell killing by incorporating it in nanoparticles and it has shown efficacy by preventing tumor progression. This review summarizes the brief introduction of synthetic lethality, and synthetic lethal gene interactions, with a major focus on its therapeutic anticancer potential with the application of nanotechnology for development of personalized medicine.

Facilitating Anti-Cancer Combinatorial Drug Discovery by Targeting Epistatic Disease Genes.

Due to synergistic effects, combinatorial drugs are widely used for treating complex diseases. However, combining drugs and making them synergetic remains a challenge. Genetic disease genes are considered a promising source of drug targets with important implications for navigating the drug space. Most diseases are not caused by a single pathogenic factor, but by multiple disease genes, in particular, interacting disease genes. Thus, it is reasonable to consider that targeting epistatic disease genes may enhance the therapeutic effects of combinatorial drugs. In this study, synthetic lethality gene pairs of tumors, similar to epistatic disease genes, were first targeted by combinatorial drugs, resulting in the enrichment of the combinatorial drugs with cancer treatment, which verified our hypothesis. Then, conventional epistasis detection software was used to identify epistatic disease genes from the genome wide association studies (GWAS) dataset. Furthermore, combinatorial drugs were predicted by targeting these epistatic disease genes, and five combinations were proven to have synergistic anti-cancer effects on MCF-7 cells through cell cytotoxicity assay. Combined with the three-dimensional (3D) genome-based method, the epistatic disease genes were filtered and were more closely related to disease. By targeting the filtered gene pairs, the efficiency of combinatorial drug discovery has been further improved.

Abstract P3-07-04: EphA2: An emerging target in triple-negative breast cancer

Abstract Purpose: Breast tumors classified as 'triple negative' (TNBC) lack defining markers ER/PR/HER2 and do not have clinically-approved targeted therapy. This heterogeneous classification of breast cancers, while immediately responsive to standard chemotherapy, commonly develop resistance and have a poor five-year survival rate. As such, the identification of new therapeutic targets are warranted. As part of our drug discovery platform, we have identified EphA2, as a synthetic-lethal gene that enhances the therapeutic action of FDA-approved, anti-inflammatory compounds. Thus we sought to ascertain the relevance of EphA2-targeted therapy in TNBC, through the evaluation of the marker in preclinical and clinical specimens. Methods: Sixty-one human and murine breast cancer cell lines or patient-derived xenografts were collated. Protein lysates were created from cells in vitro or from respective tumors established from cells implanted into NSG mice. Forty-nine tumors established (minimum 500mm3) and were surgically removed, fixed in formalin and paraffin embedded. A TMA was constructed with tumor specimens represented twice on the array and reflected all molecular subtypes including; ER-positive (n=5), PR-positive (n=3), HER2-positive (n=9) and TNBC (n=31). Immunostaining for EphA2 was performed with the rabbit monoclonal antibody EphA2 (D4A2) XP (Cell Signaling, #6997) using manufacturer's instructions. Immunostaining was evaluated using the H-score method (score between 0-300), with positive staining for EphA2 reflecting a score of 100 or greater. Analysis of breast cancer lysates by western blot was analyzed by absolute and relative quantitation methods; gene expression data was assessed through Oncomine or using the BreastMark algorithm (http://glados.ucd.ie/BreastMark/). This algorithm integrates gene expression and survival data from 26 datasets on 12 different microarray platforms corresponding to ˜17,000 genes in up to 4,738 samples. Results: In an integrated gene expression platform (BreastMark), we observed that elevated EphA2 expression was associated with poor prognosis in a cohort of TNBC patient tumor samples. Western blot analysis of EphA2 protein on breast cancer cell lines, identified a greater percentage of TNBC cells expressing EphA2 compared to non-TNBC cell lines. EphA2 immunostaining was observed in the majority of tumor tissues. When present on cancer cells, EphA2 localized to the cell surface; while displaying ubiquitous localization within stromal populations. Cell surface expression of EphA2 on cancer cells was largely restricted to TNBC tumors (11/31 tumors, 35.5%) compared to other molecular subtypes (1/13 non-TNBC tumors, 7.7%; p = 0.0294). Expression of EphA2 in stromal cell populations was similar between groups (TNBC = 22/31, non-TNBC = 11/13; p = 0.1711). Conclusions: Our analysis determined that EphA2 was specifically expressed on cancer cells derived from tumors with a 'triple-negative' molecular subtype. Collectively our data suggests that EphA2 is an emerging target in TNBC and that therapies directed against EphA2 may provide a significant benefit for a majority of patients that express this marker. Citation Format: Eckhardt BL, Torres AM, Woodward WA, Krishnamurthy S, Meric-Bernstam F, Ueno NT. EphA2: An emerging target in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-04.

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current approaches suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from S. aureus and S. pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The “Big Papi” (Paired aureus and pyogenes for intereactions) approach described here will be widely applicable for the study of combinatorial phenotypes.

Computational Prediction of Synthetic Lethals in Genome-Scale Metabolic Models Using Fast-SL.

In this chapter, we describe Fast-SL, an in silico approach to predict synthetic lethals in genome-scale metabolic models. Synthetic lethals are sets of genes or reactions where only the simultaneous removal of all genes or reactions in the set abolishes growth of an organism. In silico approaches to predict synthetic lethals are based on Flux Balance Analysis (FBA), a popular constraint-based analysis method based on linear programming. FBA has been shown to accurately predict the viability of various genome-scale metabolic models. Fast-SL builds on the framework of FBA and enables the prediction of synthetic lethal reactions or genes in different organisms, under various environmental conditions. Predicting synthetic lethals in metabolic network models allows us to generate hypotheses on possible novel genetic interactions and potential candidates for combinatorial therapy, in case of pathogenic organisms. We here summarize the Fast-SL approach for analyzing metabolic networks and detail the procedure to predict synthetic lethals in any given metabolic model. We illustrate the approach by predicting synthetic lethals in Escherichia coli. The Fast-SL implementation for MATLAB is available from https://github.com/RamanLab/FastSL/ .

The Essential UPP Phosphatase Pair BcrC and UppP Connects Cell Wall Homeostasis during Growth and Sporulation with Cell Envelope Stress Response in Bacillus subtilis.

The bacterial cell wall separates the cell from its surrounding and protects it from environmental stressors. Its integrity is maintained by a highly regulated process of cell wall biosynthesis. The membrane-located lipid II cycle provides cell wall building blocks that are assembled inside the cytoplasm to the outside for incorporation. Its carrier molecule, undecaprenyl phosphate (UP), is then recycled by dephosphorylation from undecaprenyl pyrophosphate (UPP). In Bacillus subtilis, this indispensable reaction is catalyzed by the UPP phosphatases BcrC and UppP. Here, we study the physiological function of both phosphatases with respect to morphology, cell wall homeostasis and the resulting cell envelope stress response (CESR). We demonstrate that uppP and bcrC represent a synthetic lethal gene pair, which encodes an essential physiological function. Accordingly, cell growth and morphology were severely impaired during exponential growth if the overall UPP phosphatase level was limiting. UppP, but not BcrC, was crucial for normal sporulation. Expression of bcrC, but not uppP, was upregulated in the presence of cell envelope stress conditions caused by bacitracin if UPP phosphatase levels were limited. This homeostatic feedback renders BcrC more important during growth than UppP, particularly in defense against cell envelope stress.

Abstract PR12: Genome-wide in-vivo tumor xenograft CRISPR knockout screening for identifying KRAS mutant synthetic lethal interactions

Abstract The KRAS oncogene is frequently mutated in many of the most lethal human cancers and has been resistant to targeted therapies, leading to efforts to identify synthetic lethal genetic interactions in large-scale screens. These screens have been carried out largely in in-vitro cell culture systems using RNA interference (RNAi). Functional genomic screening using the bacterial type II clustered regularly interspaced short palindrome repeats and their associated proteins (CRISPR-Cas9) system has proven to be more powerful than RNAi for systematic genomic perturbation due to its simplicity, decreased off-target effects, and the ability to create knockout rather than knockdown phenotypes. We have conducted a genome-wide CRISPR-Cas9 loss-of-function screen with the human GeCKO v2 library directly in-vivo utilizing a paired isogenic human colorectal cell line (HCT116) with and without a KRAS oncogenic mutation (G13D) to identify genes whose knockout results in synthetic lethal interactions in tumor xenografts with mutated KRAS. Compared to cell culture models, tumor xenografts better recapitulate obstacles tumor cells must overcome for continued proliferation including limited accessibility to nutrients and oxygen. Many recent studies have also highlighted the effect of KRAS oncogenic mutations on the rewiring of metabolic pathways to fuel increased tumor growth. Tumor xenografts offer a better screening platform in identifying factors that might exploit these metabolic pathways. Pathway analysis of our genome-wide CRISPR screen indeed revealed candidate synthetic lethal genes involved in metabolic and nucleic acid synthesis pathways in KRAS mutant tumor xenografts. We individually validated some of the candidate genes in these metabolic pathways (including the TCA cycle, pentose phosphate pathway, and fructose metabolism) with individual CRISPR-Cas9 knockouts and confirmed that knockout of these genes resulted in decreased tumor xenograft growth in KRAS mutant tumors. One of the KRAS synthetic lethal genes that we identified was the gene NADK, which encodes NAD+ kinase that converts NAD+ into NADP+. Low-frequency activating mutations in NADK were also recently discovered by another group to harbor oncogenic potential in pancreatic adenocarcinoma. Given the high frequency of KRAS mutations in pancreatic adenocarcinoma, NADK may represent a therapeutic target in KRAS mutant tumors in general. While more sensitive than RNAi screens, there was still significant noise in our CRISPR-Cas9 genome-wide screen as expected in an in-vivo dropout screen. To overcome this noise, we generated a smaller pooled lentiviral CRISPR library (targeting ~250 genes) based on candidate hits in the genome-wide screen and again directly screened this smaller but deeper pooled library in tumor xenografts with higher coverage and more replicates. This strategy allowed for better discrimination of novel KRAS dependent synthetic lethal genes. Citation Format: Edwin H. Yau, Tariq Rana. Genome-wide in-vivo tumor xenograft CRISPR knockout screening for identifying KRAS mutant synthetic lethal interactions [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR12.

Abstract IA21: Systematic, network-based elucidation of synthetic lethal proteins and synergistic compounds

Abstract Given the complex nature of cancer and the cell's ability to effectively compensate for individual perturbation through evolutionarily honed adaptive mechanisms, there is strong expectation that combination therapy approaches may be required to address the limitations that have emerged from single agent therapy. Synthetic lethality provides strong biological rationale supporting this observation, although mechanisms of compound synergy and sensitization are in general more complex than individual gene-pair interactions. Synthetic lethal genes have been elucidated mainly via large-scale screens in model organisms, or by pooled shRNA/CRISPR screens in cells harboring specific genetic events, or based on mechanistic understanding of specific pathways. These methodologies are laborious, expensive, and prone to significant false positive and negative rates due to the nature of pooled screens. More importantly, results are often idiosyncratic, depending on the specific genetic and epigenetic context of the cellular models used in the analysis. To address these challenges, we have developed network-based methods to identify master regulator proteins that are responsible for maintaining tumor state, despite being rarely mutated using only patient-derived data. Master regulators are highly enriched in synthetic lethal interactions that can be elucidated computationally, via protein-target analysis. Furthermore, the approach can be trivially extended to elucidate synergistic drug interaction, based on perturbational assays of individual compounds. In this presentation, we will address the discovery of synthetic lethal genes and synergistic compounds in several tumor contexts, including lymphoma, breast cancer, prostate cancer, and glioma, which has led to the design and implementation of novel clinical trials. We will also discuss integration of these methodologies into an N of 1 protocol, which is currently enrolling 260 patients across 14 distinct malignancies. Citation Format: Andrea Califano. Systematic, network-based elucidation of synthetic lethal proteins and synergistic compounds [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA21.

Abstract A16: Discovery and functional characterization of essential pathways in KRAS mutant cancers

Abstract KRAS oncogenes are present in 30% of all human cancers and are required for tumor development and maintenance and although clearly important efforts to target KRAS mutant tumors remains a major challenge. Several systematic approaches have been made to target these RAS-dependent cancers through small molecule screening and genome-scale RNAi screens to identify KRAS Synthetic Lethal (SL) genes. We applied and network based approached to integrate published KRAS SL screen data to uncover protein networks enriched with KRAS SL genes. We are using these proteins networks to generate a KRAS genetic interaction map (E-MAP) using combinatorial RNAi to identify candidate drug combinations, which may be useful to treat KRAS mutant cancers. Our network analysis revealed a dozen protein networks enriched with KRAS SL genes. Our pilot RNAi screen revealed that KRAS mutant tumors are sensitive to PAPR inhibition. Furthermore our isogenic models reveal that KRAS mutant cells harbor active DNA repair pathways in the absence of DNA damaging agents and are likely suffering from replication stress. A combinatorial RNAi screen revealed a KRAS specific synthetic lethal interaction with PARP inhibitors and knock down of MEK-ERK pathway genes. Our results suggest that PARP inhibitions have some efficacy in treating KRAS mutant tumors undergoing replication stress single agents and potentially combined with inhibitors of MEK-ERK signaling. Future studies are underway to determine if PAPR inhibitors are effective in cancer cell in vitro and in vivo models. Citation Format: Angel A. Ku, Sourav Bandyopadhyay. Discovery and functional characterization of essential pathways in KRAS mutant cancers [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A16.

Abstract IA14: Understanding the complex biology of KRAS mutant cancers using genetic screens

Abstract Apart from identifying synthetic lethal genes, genetic screens have the potential to reveal much about the basic biology of cell lines. We utilize two techniques, one analytical and one experimental, to demonstrate how genetic screens can also reveal which pathways are on in cell lines and what are the phenotypic consequences of pathway perturbation. First, we apply a clustering analysis of genome-scale CRISPR screening data to map out active pathways in cancer cell lines and to discover novel members of signaling transduction pathways and protein complexes. Secondly, we performed a combinatorial siRNA screen that knocks out multiple genes at once and measures five functional parameters to comprehensively describe the effects of pathway inhibition. Application of these two techniques to KRAS mutant cancer cell lines reveals a trove of biological information that highlights the vast heterogeneity of this disease and subtype-specific opportunities for therapeutic intervention. Citation Format: Tina L. Understanding the complex biology of KRAS mutant cancers using genetic screens [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA14.

Abstract A25: Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer

Abstract Mutations in tumour suppressors and un-druggable oncogenes dominate the landscape of cancer driver genes. Only a minority of colon cancers have mutations in druggable cancer drivers, such as PIK3CA. Conversely, mutations in tumour suppressors such as APC and TP53 are frequent, as are mutations in the notoriously difficult to drug KRAS target. There is an urgent need for new therapeutics to target tumours driven by these mutations: immune checkpoint approaches are likely to only prove effective in the fraction of patients whose tumours bear high mutation loads, which is colon cancer may be restricted to the minority of mismatch repair deficient cancers. The concepts of non-oncogene addiction and synthetic lethality provide a conceptual framework for finding therapeutic targets in these cancers. We have used arrayed siRNA and pooled CRISPR-Cas9 libraries to screen a panel of isogenic and non-isogenic colon cancer cell lines under conditions designed to increase the cells dependency on oncogenic pathways. This panel contains cell lines with mutations in TP53, APC, KRAS, PIK3CA and/or FBXW7 for which we are aiming to identify co-dependencies that consistently segregate with genotype. Our screens have identified a number of novel targets for which reduced expression imposes specific fitness defects on cells with mutant PIK3CA, mutant TP53 or mutant FBXW7. Few of these targets were identified by both siRNA and CRISPR-Cas9 approaches. Furthermore, isogenic pairs of cell lines have not proved helpful for identifying synthetic lethal targets in the KRAS or PIK3CA genotypes. However, we believe the increased penetrance of the CRIPSR-Cas9 approach has uncovered novel candidate synthetic lethal genes that were not found by RNA interference. Although our current screens are not saturating, we have confirmed several previously reported genetic interactions including the requirements for expression of MDM2 in TP53 wild-type cancers and HK2 in colon cancer lines with KRAS mutations. The screens in the FBXW7 genotype were particularly interesting: in addition to identifying a potential synthetic lethal interaction with a target for which a drug has recently entered the clinic, we also found that FBXW7 itself seemed to be required in many of the colon cancers with FBXW7 mutations. This suggests that FBXW7 may sometimes act as an oncogene, rather than as a tumour suppressor in cancer initiation or progression. Finally, we have attempted to validate some of the candidate synthetic lethal genes identified by RNA interference via ultra-deep CRISPR-CAS9 pooled screening, extending the approach of the Vakoc lab (Nature Biotech. 33, 661-667, 2015). We can confirm this method can highlight functional domains of particular significance to a protein's function, but note that many-fold drop outs of sgRNAs can also occur where they target multiple sites in the genome, presumably via a DNA damage response. Citation Format: Jonathan D. Moore, Chantelle Hudson, Paul Russell, Gaganpreet Tiwana, David Walter, Ceri M. Wiggins, Joanne Yarker. Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A25.

Abstract A29: Targeting p300 addiction in CBP-deficient cancers causes synthetic lethality by apoptotic cell death due to abrogation of MYC expression

Abstract Loss-of-function mutations in the CBP/CREBBP gene, which encodes a histone acetyltransferase (HAT), are present in a variety of human tumors, including lung, bladder, gastric, and hematopoietic cancers. Consequently, development of a molecular targeting method capable of specifically killing CBP-deficient cancer cells would greatly improve cancer therapy. Functional screening of synthetic-lethal genes in CBP-deficient cancers identified the CBP paralog p300/EP300. Ablation of p300 in CBP-knockout and -deficient cancer cells induced G1/S cell-cycle arrest, followed by apoptosis. Genome-wide gene expression analysis revealed that MYC is a major factor responsible for the synthetic lethality. Indeed, p300 ablation in CBP-deficient cells caused downregulation of MYC expression via reduction of histone acetylation in its promoter, and this lethality was rescued by exogenous MYC expression. The p300-HAT inhibitor C646 specifically suppressed the growth of CBP-deficient lung and hematopoietic cancer cells in vitro and in vivo; thus p300 is a promising therapeutic target for treatment of CBP-deficient cancers. Citation Format: Hideaki Ogiwara, Mariko Sasaki, Takahiro Oike, Saito Higuchi, Yuichi Tominaga, Takashi Kohno. Targeting p300 addiction in CBP-deficient cancers causes synthetic lethality by apoptotic cell death due to abrogation of MYC expression [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A29.

Candidate synthetic lethality partners to PARP inhibitors in the treatment of ovarian clear cell cancer.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are new types of personalized treatment of relapsed platinum-sensitive ovarian cancer harboring BRCA1/2 mutations. Ovarian clear cell cancer (CCC), a subset of ovarian cancer, often appears as low-stage disease with a higher incidence among Japanese. Advanced CCC is highly aggressive with poor patient outcome. The aim of the present study was to determine the potential synthetic lethality gene pairs for PARP inhibitions in patients with CCC through virtual and biological screenings as well as clinical studies. We conducted a literature review for putative PARP sensitivity genes that are associated with the CCC pathophysiology. Previous studies identified a variety of putative target genes from several pathways associated with DNA damage repair, chromatin remodeling complex, PI3K-AKT-mTOR signaling, Notch signaling, cell cycle checkpoint signaling, BRCA-associated complex and Fanconi's anemia susceptibility genes that could be used as biomarkers or therapeutic targets for PARP inhibition. BRCA1/2, ATM, ATR, BARD1, CCNE1, CHEK1, CKS1B, DNMT1, ERBB2, FGFR2, MRE11A, MYC, NOTCH1 and PTEN were considered as candidate genes for synthetic lethality gene partners for PARP interactions. When considering the biological background underlying PARP inhibition, we hypothesized that PARP inhibitors would be a novel synthetic lethal therapeutic approach for CCC tumors harboring homologous recombination deficiency and activating oncogene mutations. The results showed that the majority of CCC tumors appear to have indicators of DNA repair dysfunction similar to those in BRCA-mutation carriers, suggesting the possible utility of PARP inhibitors in a subset of CCC.

Abstract 3852: Validation of synthetic lethality of PARP and NAMPT dual inhibition in a small cell lung cancer PDX model

Abstract Small cell lung cancer (SCLC) is a highly malignant cancer type with a 5-year survival rate of less than 10%. Different from non-small cell lung cancer (NSCLC), no effective target therapies have been approved to treat SCLC. Poly (ADP) ribose polymerase (PARP) overexpression in SCLC has prompted great efforts to evaluate the role of PARP inhibitors in clinic. To maximize their therapeutic value it is urgent in need to explore the best combination strategies between PARP inhibitors and other pathway modulators. In this study we identified a SCLC patient-derived xenograft (PDX) model with high PARP level and low nicotinamide phosphoribosyltransferase (NAMPT) level. Furthermore, synergistic effect of PARP and NAMPT dual inhibition was demonstrated, supporting translational research of PARP inhibitors and NAMPT inhibitors in this model. The mRNA expression of PARP as well as its 30 most-studied synthetic lethality genes was compared between SCLC and NSCLC in both PDXs and cancer cell lines. We found that NAMPT levels decreased most significantly in SCLC compared with NSCLC across the 30 synthetic lethality genes of PARP. As NAMPT is the rate-limiting enzyme for the synthesis of the PARP substrate β-NAD+, we hypothesized that low β-NAD+ level due to low NAMPT level might render SCLC sensitive to PARP inhibition. LU-01-0547, a SCLC PDX model with high PARP and low NAMPT expression, was identified here. To test our hypothesis, we investigated the preclinical efficacy of ABT888, a PARP inhibitor, with or without FK866, a NAMPT inhibitor, in this subcutaneous xenograft model. The single-agent efficacy of ABT888 was demonstrated (TGI=96%@100 mg/kg, BID). At a sub-optimal dose (50 mg/kg, BID), treatment of ABT888 alone produced little activity in the same model, while its combination with a NAMPT inhibitor, FK866, significantly boosted the antitumor response indicating the synergistic effect (TGI=117%, ORR=6/6, CR=4/6). When nicotinic acid (NA) was administered to promote β-NAD+ biosynthesis via the de novo pathway other than the NAMPT-mediated transformation, the PARP inhibitor’s effectiveness was greatly antagonized (TGI=70% vs. 13%), suggesting β-NAD+ level might correlate with PARP inhibitor sensitivity. In conclusion, this study identified a SCLC PDX model with high PARP and low NAMPT expression. Synergistic inhibition of PARP and NAMPT in this model was demonstrated superior to either treatment alone in efficacy, which warranted its future application in drug discovery. To our knowledge, this was the first in vivo evidence of synthetic lethality in SCLC PDX, supporting future clinical test of combination of PARP and NAMPT inhibitors in SCLC patients. Citation Format: Zhixiang Zhang, Dongfang Li, Chen Chen, Bo Zhang, Xuzhen Tang, Hao Ye, Qingyang Gu, Qunsheng Ji. Validation of synthetic lethality of PARP and NAMPT dual inhibition in a small cell lung cancer PDX model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3852. doi:10.1158/1538-7445.AM2017-3852

Personalizing Chinese medicine by integrating molecular features of diseases and herb ingredient information: application to acute myeloid leukemia.

Traditional Chinese Medicine (TCM) has been widely used as a complementary medicine in Acute Myeloid Leukemia (AML) treatment. In this study, we proposed a new classification of Chinese Medicines (CMs) by integrating the latest discoveries in disease molecular mechanisms and traditional medicine theory. We screened out a set of chemical compounds on basis of AML differential expression genes and chemical-protein interactions and then mapped them to Traditional Chinese Medicine Integrated Database. 415 CMs contain those compounds and they were categorized into 8 groups according to the Traditional Chinese Pharmacology. Pathway analysis and synthetic lethality gene pairs were applied to analyze the dissimilarity, generality and intergroup relations of different groups. We defined hub CM pairs and alternative CM groups based on the analysis result and finally proposed a formula to form an effective anti-AML prescription which combined the hub CM pairs with alternative CMs according to patients’ molecular features. Our method of formulating CMs based on patients’ stratification provides novel insights into the new usage of conventional CMs and will promote TCM modernization.

Abstract A24: A genome-wide RNAi screen identifies synthetic lethality of CX-5461 with homologous recombination repair deficiency in ovarian cancer

Abstract Cancer is characterized by deregulated cell growth and proliferation, both of which are associated with hyperactivation of ribosome biogenesis. Inhibition of ribosome biogenesis using CX-5461, a specific inhibitor of RNA polymerase I-dependent transcription, has shown therapeutic efficacy in a MYC driven B-cell lymphoma mouse model, which is enhanced when used in combination with the mTORC1 inhibitor Everolimus. However, the therapeutic potential of CX-5461 in solid cancers is yet to be determined. Our preliminary data utilizing a panel of 36 ovarian cancer (OVCA) cell lines suggest that acute CX-5461 treatment results in cell cycle arrest and does not induce apoptosis. We hypothesize that the identification of genes that can be targeted to cooperate with CX-5461 will define novel drug combinations for the improved treatment of OVCA. Therefore, we performed a genome-wide RNAi screen to identify synthetic lethal genes with CX-5461 in the high-grade serous ovarian cancer (HGSOC) cell line OVCAR4. Pathway enrichment analysis of the candidate hits showed significant enrichment in the homologous recombination DNA repair (HR) pathway. Synergy with CX-5461 was validated in multiple HGSOC cell lines by both genetic and pharmacological inhibition of HR pathway components. We are currently investigating the mechanism of this synergy and will further assess efficacy in vivo. As HR deficiency is observed in 20% of OVCA patients, we suggest that future application of our studies will lead to new therapeutic options to improve the survival of this cohort of patients. Citation Format: Shunfei Yan, Keefe T. Chan, Kaylene J. Simpson, Elaine Sanij, Karen E. Sheppard, Katherine M. Hannan, Ross D. Hannan, Richard B. Pearson. A genome-wide RNAi screen identifies synthetic lethality of CX-5461 with homologous recombination repair deficiency in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A24.

IMYC: Proof-of-concept study of ibrutinib in c-MYC and HER2 amplified oesophagogastric carcinoma.

TPS221 Background: The MYC proto-oncogene is among the most frequently dysregulated genes in human cancer and is amplified in 10-30% of oesophagogastric (OG) cancers. Such genes which code for transcription factors are challenging to target directly with small molecule inhibitors or monoclonal antibodies. High-throughput siRNA screening of OG cell lines has found silencing of Bruton’s tyrosine kinase (BTK) to result in selective lethality in the presence of MYC amplification. Sensitivity to the orally available BTK inhibitor ibrutinib has been confirmed in-vitro in MYC, HER2 and co-amplified OG cancer cell lines (Chong, Ann Onc 2014), suggesting it may be a potentially effective therapeutic strategy in both MYC and HER2 amplified OG cancer. Methods: i MYC is an open label, phase II non-randomised study to assess the efficacy of ibrutinib in advanced pre-treated OG cancer. A novel FISH assay for tumour MYC amplification has been developed and pts are pre-screened for MYC and HER2 amplification. Eligibility includes metastatic or locally advanced inoperable OG cancer (SCC or adeno), MYC (ratio >2.5) and/or HER2 (ratio>2) amplification, progression after at least 1 prior line of chemotherapy +/- trastuzumab for advanced disease, PS 0-2 and no history of significant cardiovascular or bleeding disorders. Of the first 9 pts, at least 4 will demonstrate MYC amplification and remaining 5 will show either MYC or HER2 amplification, or co-amplification of both. Pts will be treated with ibrutinib monotherapy until progression or unacceptable toxicity. A Simon 2 stage design will be used for the primary endpoint of response rate, with interim analysis after 9 pts and maximum recruitment to 17 pts. Secondary endpoints include PFS, OS and exploratory translational and imaging biomarker analysis. Mandatory biopsy will be obtained at baseline and optional biopsies will be undertaken at day 10-14, week 8 and progression. The trial represents the first attempt at targeting MYC amplification via synthetic lethal gene interactions in OG cancer through the novel application of an existing anti-cancer drug. As of September 2016, 61 patients have consented for pre-screening, 19 completed MYC analysis and first patient has started treatment. Clinical trial information: 02884453.

Transposon Insertion Site Sequencing for Synthetic Lethal Screening.

Transposon insertion site sequencing (TIS) permits genome-wide, quantitative fitness assessment of individual genomic loci. In addition to the identification of essential genes in given growth conditions, TIS enables the elucidation of genetic networks such as synthetic lethal or suppressor gene combinations. Therefore, TIS becomes an exceptionally powerful tool for the high-throughput determination of genotype-phenotype relationships in bacteria. Here, we describe a protocol for the generation of high-density transposon insertion libraries and subsequent preparation of DNA samples for Illumina sequencing using the Gram-negative bacterium Vibrio cholerae as an example.