Synthetic Lethal Screen Research Articles

Dual Fluorescence Isogenic Synthetic Lethal Kinase Screen and High-Content Secondary Screening for MUC16/CA125-Selective Agents.

Significant strides have been made in the development of precision therapeutics for cancer. Aberrantly expressed glycoproteins represent a potential avenue for therapeutic development. The MUC16/CA125 glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer. Previously, we demonstrated a proof-of-principle approach to selectively targeting MUC16+ cells. In this report, we performed a synthetic lethal kinase screen using a human kinome RNAi library and identified key pathways preferentially targetable in MUC16+ cells using isogenic dual-fluorescence ovarian cancer cell lines. Using a separate approach, we performed high-content small-molecule screening of six different libraries of 356,982 compounds for MUC16/CA125-selective agents and identified lead candidates that showed preferential cytotoxicity in MUC16+ cells. Compounds with differential activity were selected and tested in various other ovarian cell lines or isogenic pairs to identify lead compounds for structure-activity relationship (SAR) selection. Lead siRNA and small-molecule inhibitor candidates preferentially inhibited invasion of MUC16+ cells in vitro and in vivo, and we show that this is due to decreased activation of MAPK, and non-receptor tyrosine kinases. Taken together, we present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development.

Aurora kinase A inhibition induces synthetic lethality in SMAD4-deficient colorectal cancer cells via spindle assembly checkpoint activation.

SMAD4 loss-of-function mutations have been frequently observed in colorectal cancer (CRC) and are recognized as a drug target for therapeutic exploitation. In this study, we performed a synthetic lethal drug screening with SMAD4-isogenic CRC cells and found that aurora kinase A (AURKA) inhibition is synthetic lethal with SMAD4 loss. Inhibition of AURKA selectively inhibited the growth of SMAD4-/- CRC in vitro and in vivo. Mechanistically, SMAD4 negatively regulated AURKA level, resulting in the significant elevation of AURKA in SMAD4-/- CRC cells. Inhibition of AURKA induced G2/M cell cycle delay in SMAD4+/+ CRC cells, but induced apoptosis in SMAD4-/- CRC cells. We further observed that a high level of AURKA in SMAD4-/- CRC cells led to abnormal mitotic spindles, leading to cellular aneuploidy. Moreover, SMAD4-/- CRC cells expressed high levels of spindle assembly checkpoint (SAC) proteins, suggesting the hyperactivation of SAC. The silencing of key SAC proteins significantly rescued the AURKA inhibition-induced cell death in SMAD4-/- cells, suggesting that SMAD4-/- CRC cells are hyper-dependent on AURKA activity for mitotic exit and survival during SAC hyperactivation. This study presents a unique synthetic lethal interaction between SMAD4 and AURKA and suggests that AURKA could be a potential drug target in SMAD4-deficient CRC.

Abstract P5-08-10: TBK1 inhibition potentiates the efficacy of AXL-targeted therapy in aggressive breast cancer preclinical models

Abstract Background: Novel mechanism-oriented targeted therapies are needed for aggressive breast cancers such as triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC). We previously showed the oncogenic receptor tyrosine kinase AXL crucially contributes to the aggressiveness of TNBC and TN-IBC, which suggests that AXL is a potential therapeutic target. However, AXL-targeted therapy is insufficient to control the progress of aggressive breast cancer in preclinical models. Therefore, to further enhance the efficacy of AXL-targeted therapy in aggressive breast cancer, we conducted a synthetic lethality screen of pooled kinome siRNA and identified TANK-binding kinase 1 (TBK1) as a potential target. TBK1 is a necessary serine/threonine protein kinase that regulates innate and adaptive immune responses, T cell migration, and anti-tumor immune responses. In the present work, we validated that AXL and TBK1 inhibition have a synergistic anti-tumor effect by modulating the tumor microenvironment (TME). Methods: To determine the synergistic effect of AXL and TBK1 inhibition on cell proliferation and colony formation in vitro, we first used siRNA-mediated knockdown (KD) or CRISPR/Cas9 knockout (KO) to independently silence AXL and TBK1 expression. We also used pharmacological inhibitors to inactivate AXL and TBK1 signaling and conducted similar studies. To determine the effect of targeting TBK1 and AXL pathways in vivo, we injected control or TBK1 KO murine 4T1.2 TNBC cells into BALB/c mice and assessed the efficacy of the AXL inhibitor TP-0903 in reducing the tumor growth. We used multicolor flow cytometry and multiplexed immunostaining to examine the population of TME components in control and TBK1 KO 4T1.2 tumors treated with vehicle or TP-0903. We also used an in vitro migration assay to test the effect of targeting AXL and TBK1 on the recruitment of CD8+ T cells. We performed a mouse chemokine array to identify intratumoral chemokines involved in regulating infiltrating immune cells and used RT-PCR to validate these findings. Results: Compared with AXL or TBK1 suppression alone, the genomic or pharmacological suppression of both AXL and TBK1 resulted in a significantly greater reduction of TNBC and TN-IBC cell growth and colony-forming ability (P < 0.01). In our syngeneic 4T1.2 TNBC mouse tumor model, TP-0903 was more effective at controlling tumor growth from TBK1-KO cells than from control cells (P < 0.0005). TBK1-KO 4T1.2 tumors treated with TP-0903 had a significantly higher population of functional cytotoxic T cells (GranzymeB+CD3+CD8+) than control KO tumors treated with vehicle or TP-0903. Furthermore, our migration assay showed that TBK1-KO/AXL-KD SUM149 cell-conditioned media attracted more CD8+ T cells than control cell-conditioned media, suggesting the inhibition of AXL/TBK1 signaling enhances the recruitment of CD8+ T cells through a paracrine effect. Our chemokine array revealed increased CXCL16 levels in TP-0903-treated TBK1-KO tumors. Conclusion: Targeting TBK1 could enhance the sensitivity of aggressive breast cancer cells to AXL-targeted therapy in vitro and in vivo. Our preliminary TME data suggest that combined AXL and TBK1 inhibition enhances the anti-tumor immune environment by upregulating secretion of CXCL16 by tumor cells, resulting in the increased recruitment of cytotoxic CD8+ T cells. In future studies, we will further elucidate the role that CXCL16 upregulation plays in this novel synergy. Significance of this findings: This study will enable us to develop hypothesis-driven novel combination therapies for patients with aggressive breast cancer that can be rapidly tested as a clinical trial, leading to an improvement of treatment and patients’ outcome. Citation Format: Lan Phi, Takashi Semba, Jason Foulks, Steven Warner, David Bearss, Savitri Krishnamurthy, James Long, James Reuben, Debu Tripathy, Naoto Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy in aggressive breast cancer preclinical models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-10.

ScreenGarden: a shinyR application for fast and easy analysis of plate-based high-throughput screens

BackgroundColony growth on solid media is a simple and effective measure for high-throughput genomic experiments such as yeast two-hybrid, synthetic dosage lethality and Synthetic Physical Interaction screens. The development of robotic pinning tools has facilitated the experimental design of these assays, and different imaging software can be used to automatically measure colony sizes on plates. However, comparison to control plates and statistical data analysis is often laborious and pinning issues or plate specific growth effects can lead to the detection of false-positive growth defects.ResultsWe have developed ScreenGarden, a shinyR application, to enable easy, quick and robust data analysis of plate-based high throughput assays. The code allows comparisons of different formats of data and different sized arrays of colonies. A comparison of ScreenGarden with previous analysis tools shows that it performs, at least, equivalently. The software can be run either via a website or offline via the RStudio program; the code is available and can be modified by expert uses to customise the analysis.ConclusionsScreenGarden provides a simple, fast and effective tool to analyse colony growth data from genomic experiments.

Selective vulnerabilities in the proteostasis network of castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is associated with an increased reliance on heat shock protein 70 (HSP70), but it is not clear what other protein homeostasis (proteostasis) factors might be involved. To address this question, we performed functional and synthetic lethal screens in four prostate cancer cell lines. These screens confirmed key roles for HSP70, HSP90, and their co-chaperones, but also suggested that the mitochondrial chaperone, HSP60/HSPD1, is selectively required in CRPC cell lines. Knockdown of HSP60 does not impact the stability of androgen receptor (AR) or its variants; rather, it is associated with loss of mitochondrial spare respiratory capacity, partly owing to increased proton leakage. Finally, transcriptional data revealed a correlation between HSP60 levels and poor survival of prostate cancer patients. These findings suggest that re-wiring of the proteostasis network is associated with CRPC, creating selective vulnerabilities that might be targeted to treat the disease.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

Literature-based translation from synthetic lethality screening into therapeutics targets: CD82 is a novel target for KRAS mutation in colon cancer

Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and RAS-mutant high-throughput screening (HTS) data. We matched essential genes from text-mining and mutant genes from the COSMIC and CCLE HTS datasets to build a prediction model of SL gene pairs. CCLE gene expression data were used to enrich the essential-mutant SL gene pairs using Spearman’s correlation coefficient and literature mining. In total, 223 essential trigger terms were extracted and ranked. The threshold of the essential gene score (Sg) was set to 10. We identified 586 genes essential for the SL prediction model of colon cancer. Seven essential RAS-mutant SL gene pairs were identified in our model, including CD82-KRAS/NRAS, PEBP1-NRAS, MT-CO2-HRAS, IFI27-NRAS/KRAS, and SUMO1-HRAS gene pairs. Using RAS-mutant HTS data validation, we identified two potential SL gene pairs, including the CD82 (essential gene)–KRAS (mutant gene) pair and CD82–NRAS pair in the DLD-1 colon cancer cell line (Spearman’s correlation p-values = 0.004786 and 0.00249, respectively). Based on further annotations by PubChem, we observed that digitonin targeted the complex comprising CD82, especially in KRAS-mutated HCT116 cancer cells. Moreover, we experimentally demonstrated that CD82 exhibited selective vulnerability in KRAS-mutant colorectal cancer. We used literature mining and HTS data to identify candidates for SL targets for RAS-mutant colon cancer.

RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells.

Rationale: PI3K/mTOR signaling is frequently upregulated in breast cancer making inhibitors of this pathway highly promising anticancer drugs. However, PI3K-inhibitors have a low therapeutic index. Therefore, finding novel combinatory treatment options represents an important step towards clinical implementation of PI3K pathway inhibition in breast cancer therapy. Here, we propose proteases as potential synergistic partners with simultaneous PI3K inhibition in breast cancer cells.Methods: We performed mRNA expression studies and unbiased functional genetic synthetic lethality screens by a miR-E based knockdown system targeting all genome-encoded proteases, i.e. the degradome of breast cancer cells. Importantly theses RNA interference screens were done in combination with two PI3K pathway inhibitors. Protease hits were validated in human and murine breast cancer cell lines as well as in non-cancerous cells by viability and growth assays.Results: The degradome-wide genetic screens identified 181 proteases that influenced susceptibility of murine breast cancer cells to low dose PI3K inhibition. Employing independently generated inducible knockdown cell lines we validated 12 protease hits in breast cancer cells. In line with the known tumor promoting function of these proteases we demonstrated Usp7 and Metap2 to be important for murine and human breast cancer cell growth and discovered a role for Metap1 in this context. Most importantly, we demonstrated that Usp7, Metap1 or Metap2 knockdown combined with simultaneous PI3K inhibition resulted in synergistic impairment of murine and human breast cancer cell growthConclusion: We successfully established proteases as combinatory targets with PI3K inhibition in human and murine breast cancer cells. Usp7, Metap1 and Metap2 are synthetic lethal partners of simultaneous protease/PI3K inhibition, which may refine future breast cancer therapy.

The CIP2A-TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer.

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.

The Pan-Clk/Dyrk Inhibitor Cirtuvivint (SM08502) Exposes Mechanistic Underpinnings of Alternative Splicing As a Therapeutic Vulnerability in Heme Malignancies

Mutations in spliceosomal genes are one of the most common classes of somatic alterations in patients with Myelodysplastic Syndrome (MDS) and occur across the entire spectrum of myeloid malignancies, including 10‒25% of patients with acute myeloid leukemia (AML). These mutations occur in higher proportions in AML subjects greater than 60 years of age, or when AML has transformed from an antecedent MDS. Spliceosomal gene mutations are implicated in the production of pathological RNA splicing patterns that block cell differentiation and maintain a myeloid precursor phenotype. This suggests deranged pre-mRNA splicing is a mechanistic determinant of many heme malignancies and, as such, has provoked interest in therapeutic modulation of pre-mRNA splicing as a treatment paradigm. The CLK/DYRK family of protein kinases has been recognized as an integration hub for signal transduction-dependent modulation of alternative pre-mRNA splice junction selection through direct phosphorylation of the serine/arginine-rich splicing factor (SRSF) splice junction enhancer-binding proteins. Thus, these kinases potentially represent a druggable intervention point in alternative splicing-dependent cancers. The isoquinoline SM08502 (cirtuvivint) is a potent ATP-competitive inhibitor of the Cdc2-like kinases (CLK1-4) and the dual specificity tyrosine phosphorylation-regulated kinases (DYRK1-4) with activity against only a minimal number of the remaining members of the CMGC-family kinases and the kinome as a whole. Here the consequence of pan-CLK/DYRK kinase inhibition on cell viability and tumorigenicity was evaluated across a panel of human tumor-derived AML, DLBCL, MCL, myeloma, T-ALL, and CML/CLL models. EC 50s in response to cirtuvivint ranged from 0.014 μM‒0.495 μM in 4-day in vitro cell viability assays, with low-dose responders enriched in subsets of AML, myeloma, DLBCL, MCL and T-ALL. Cell viability EC 50s were associated with induction of programed cell death at drug exposures that inhibited accumulation of phosphorylated SRSF proteins and the anti-apoptotic protein MCL-1. To directly evaluate the contribution of CLK/DYRK kinases to alternative splicing profiles, high-depth RNAseq analysis was performed across 4 cell lines (3 acute myeloid leukemia cell lines and 1 mantle cell lymphoma cell line) +/- a 6-hour exposure to 1µM cirtuvivint. Both baseline and drug-induced changes in alternative splicing events (ASEs) were measured using a multivariate analysis of splicing transcripts (rMATS). The frequency of cirtuvivint-induced ASEs was approximately 20% of total detected ASEs. Concordant drug-induced ASEs among the tested cell lines were in genes enriched in pathways known to drive oncogenesis in hematopoietic lineages, including the MAP kinase and mTOR signaling pathways. Tumor growth inhibition assays in immunocompromised mice showed a range of model-specific responses, including tumor stasis and partial to complete tumor regression at clinically relevant exposures. A cell-based synthetic-lethal screen of cirtuvivint across 36 small molecule inhibitors identified multiple BCL-2 inhibitors among the most prominent synergistic combinations. Consistent with this, combination of the BCL-2 inhibitor venetoclax with cirtuvivint was sufficient to induce tumor regressions in AML xenograft models (KG-1 and HL-60) that were resistant to either single-agent drug at the same concentrations. These observations support further evaluation of CLK/DYRK inhibitors as a therapeutic strategy for heme malignancies dependent upon alternative pre-mRNA splicing. DisclosuresBossard: Biosplice Therapeutics: Current Employment. McMillan: Prizer: Ended employment in the past 24 months. Beaupre: Pfizer: Ended employment in the past 24 months. White: Pfizer: Ended employment in the past 24 months.

Identification of De Novo Pyrimidine Synthesis as a Targetable Vulnerability in a Novel IDH1 Mutant Engineered Astrocytoma Model

<h3>Purpose/Objective(s)</h3> Despite high prevalence of IDH1-R132H mutations in grade II-III gliomas, effective therapies remain limited. We sought to identify tumor-specific vulnerabilities induced by the IDH1-R132H oncogene and test the translational relevance of targeting them using a new genetically engineered mouse model (GEMM) of IDH1 mutant anaplastic astrocytoma. <h3>Materials/Methods</h3> We conducted a synthetic lethality screen using isogenic IDH1 mutant and IDH1 wild-type (WT) glioma cells and a novel drug screening platform developed by our group, called MAPS. To create a GEMM of anaplastic astrocytoma, we developed a strategy to engineer Tp53, Atrx, Pik3ca, and Idh1 mutations in the brains of adult mice. We intracranially injected adeno-associated virus (AAV) expressing Cre recombinase and CRISPR sgRNAs targeting murine Atrx and Tp53 genes into 4 mouse strains: 1) LSL-Cas9; 2) LSL-Cas9; LSL-Pik3ca^H1047R, 3) LSL-Cas9; LSL-Idh1^R132H, and 4) LSL-Cas9; LSL-Idh1^R132H; LSL-Pik3ca^H1047R. <h3>Results</h3> Our screen revealed that IDH1 mutant cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We demonstrated that these cytotoxic effects are on-target and are associated with increased DNA damage in IDH1 mutant cells. Next, we showed that IDH1 mutant patient-derived glioma stem-like cell lines (GSCs) are also hyperdependent on de novo pyrimidine synthesis compared to IDH1 WT lines. In addition, we found that the novel brain penetrant DHODH inhibitor, BAY2402234 (currently undergoing testing in leukemia patients), decreased tumor growth in an orthotopic xenograft model of IDH1 mutant, but not IDH1 WT, glioblastoma. We then sought to create and use a GEMM of IDH1 mutant anaplastic astrocytoma to test whether dependence on de novo pyrimidine synthesis manifests across tumor grade. Following intracranial AAV injection (see methods), astrocytomas preferentially formed after 9-14 months in mice carrying both Idh1 and Pik3ca conditional alleles. These tumors histologically resembled grade III astrocytomas, expressed astrocytoma lineage markers, and displayed elevated (R)-2-hydroxyglutarate. To create an additional model with shorter tumor latency, we performed secondary transplants of GSCs derived from our GEMM into recipient mice. We found that BAY2402234 blocked tumor growth in these orthotopic astrocytoma allografts. <h3>Conclusion</h3> Our findings establish IDH1 mutations as predictive biomarkers of DHODH inhibitor efficacy in gliomas across tumor grade, highlight BAY2402234 as a candidate glioma therapeutic, and unveil new genetically faithful mouse models of IDH1 mutant glioma. In addition, we show that BAY2402234 induces preferential DNA damage in IDH1 mutant cells, thereby supporting evaluation of BAY2402234 as a potential tumor-selective radiosensitizer.

YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In this study, a combination of shRNA‐mediated synthetic lethality screening and transcriptomic analysis revealed the transcription factors YAP/TAZ as key drivers of Sorafenib resistance in hepatocellular carcinoma (HCC) by repressing Sorafenib‐induced ferroptosis. Mechanistically, in a TEAD‐dependent manner, YAP/TAZ induce the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, thus enabling HCC cells to overcome Sorafenib‐induced ferroptosis. At the same time, YAP/TAZ sustain the protein stability, nuclear localization, and transcriptional activity of ATF4 which in turn cooperates to induce SLC7A11 expression. Our study uncovers a critical role of YAP/TAZ in the repression of ferroptosis and thus in the establishment of Sorafenib resistance in HCC, highlighting YAP/TAZ‐based rewiring strategies as potential approaches to overcome HCC therapy resistance.

A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers.

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the Skp, Cullin, F-box containing (SCF) ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (Histone Deacetylase 4, HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work identified a novel potential drug target for tumors that overexpress MELK.

Overview of Ferroptosis and Synthetic Lethality Strategies.

Ferroptosis, a term first proposed in 2012, is iron-dependent, non-apoptotic regulatory cell death induced by erastin. Ferroptosis was originally discovered during synthetic lethal screening for drugs sensitive to RAS mutant cells, and is closely related to synthetic lethality. Ferroptosis sensitizes cancer stem cells and tumors that undergo epithelial−mesenchymal transition and are resistant to anticancer drugs or targeted therapy. Therefore, ferroptosis-inducing molecules are attractive new research targets. In contrast, synthetic lethal strategies approach mechanisms and genetic abnormalities that cannot be directly targeted by conventional therapeutic strategies, such as RAS mutations, hypoxia, and abnormalities in the metabolic environment. They also target the environment and conditions specific to malignant cells, have a low toxicity to normal cells, and can be used in combination with known drugs to produce new ones. However, the concept of synthetic lethality has not been widely adopted with ferroptosis. In this review, we surveyed the literature on ferroptosis-related factors and synthetic lethality to examine the potential therapeutic targets in ferroptosis-related molecules, focusing on factors related to synthetic lethality, discovery methods, clinical application stages, and issues in drug discovery.

Genome-wide synthetic lethal screen unveils novel CAIX-NFS1/xCT axis as a targetable vulnerability in hypoxic solid tumors.

The metabolic mechanisms involved in the survival of tumor cells within the hypoxic niche remain unclear. We carried out a synthetic lethal CRISPR screen to identify survival mechanisms governed by the tumor hypoxia-induced pH regulator carbonic anhydrase IX (CAIX). We identified a redox homeostasis network containing the iron-sulfur cluster enzyme, NFS1. Depletion of NFS1 or blocking cyst(e)ine availability by inhibiting xCT, while targeting CAIX, enhanced ferroptosis and significantly inhibited tumor growth. Suppression of CAIX activity acidified intracellular pH, increased cellular reactive oxygen species accumulation, and induced susceptibility to alterations in iron homeostasis. Mechanistically, inhibiting bicarbonate production by CAIX or sodium-driven bicarbonate transport, while targeting xCT, decreased adenosine 5'-monophosphate-activated protein kinase activation and increased acetyl-coenzyme A carboxylase 1 activation. Thus, an alkaline intracellular pH plays a critical role in suppressing ferroptosis, a finding that may lead to the development of innovative therapeutic strategies for solid tumors to overcome hypoxia- and acidosis-mediated tumor progression and therapeutic resistance.

Correction: Synthetic Lethal Screens Reveal Cotargeting FAK and MEK as a Multimodal Precision Therapy for GNAQ-Driven Uveal Melanoma

In the original version of [this article][1] ([1][2]), the author order is incorrect for Simone Lubrano, Frederico Gomes, Nadia Arang, Manoela Tiago, and Silvia Coma. This error has been corrected in the latest online HTML and PDF versions of the article. The authors and the publisher regret this

Synthetic Lethality Screening Highlights Colorectal Cancer Vulnerability to Concomitant Blockade of NEDD8 and EGFR Pathways.

Simple SummaryIdentification of effective therapies for clinically aggressive, treatment-resistant colorectal cancer (CRC) remains an unmet clinical need. Targeted therapies against the epidermal growth factor receptor (EGFR) signaling axis lead to clinical benefits only in a small fraction of patients due to primary and acquired resistance. We previously showed that the NEDD8 pathway inhibitor pevonedistat induced tumor stabilization in preclinical models of aggressive CRC. Here, through synthetic lethality screenings, we found that pevonedistat could be successfully combined with EGFR pathway-targeted treatments in BRAF-mutant and RAS-RAF wild-type CRCs originally resistant to BRAF and EGFR blockade. We found that combined blockade of NEDD8 and EGFR pathways reverted compensatory feedback loops that reduced the efficacy of single treatments. Our results provide preclinical validation of a promising therapeutic strategy for clinically aggressive CRC resistant to EGFR and BRAF-targeted treatments.Colorectal cancer (CRC) is a heterogeneous disease showing significant variability in clinical aggressiveness. Primary and acquired resistance limits the efficacy of available treatments, and identification of effective drug combinations is needed to further improve patients’ outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a “drop-out” loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. These results unveil possible therapeutic opportunities in specific CRC clinical settings.

Abstract 1143: Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers

Abstract Background: E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed acetyl transferases (HAT) that also act as co-activators for number of transcription factors including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Both CBP and p300 possess bromodomain (BRD) and a lysine acetyltransferase (KAT) domain. These two closely related epigenetic modulators are known to play oncogenic roles in a variety of cancers. Functional synthetic lethal screens have identified preferential killing in CBP-deficient and MYC-dependent hematological cancer cells by suppression of the paralogue p300. CBP/p300 BRD inhibitor could also prevent its coactivator function at AR, thereby potentially inhibit growth of AR-dependent prostate cancer cells. Thus, targeting CBP/p300 represents an attractive approach for developing personalized therapies. Experimental procedures and Results: Multiple potent and selective CBP/p300 BRD inhibitors that are structurally unrelated to known inhibitors have been identified by iterative medicinal chemistry and SAR based approaches. The lead compound, AU-18069 was optimized towards attaining good potency, physicochemical properties and DMPK profile. AU-18069 potently inhibited viability and proliferation of a wide range of cell lines derived from prostate cancer, CBP mutant and MYC-dependent hematological cancers and demonstrated improved PK profile in rodent models in comparison with a compound, currently in clinical trials. Excellent efficacy with significant tumor growth inhibition (TGI) was observed in MV4-11 xenograft model at well-tolerated doses along with downregulation of cMYC in a single dose PK-PD study. AU-18069 also showed modulation of different immune phenotypes including CD4+ T cell subsets. In summary, our lead candidate AU-18069 demonstrated that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in vitro and in vivo. Further evaluation of immune activation potential, efficacy studies in various xenograft models, long term toxicological evaluation in different species and other IND enabling studies are in progress. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Ramesh S Senaiar, Subhendu Mukherjee, Mahaboobi Jaleel, Sivapriya Marappan, Raghavendra N R, Girish Aggunda Renukappa, Aravind A B, Naveen Kumar R, Venkata Siva Reddy, Asha Babu, Akhila Srinivas, Prasad Yadlapalli, Suraj T Gore, Pathange Hemasankar, Mamon Dey, Samiulla D S, Chandranath D Naik, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, Mikko Myllymäki, Gerd Wohlfahrt, Murali Ramachandra, Susanta Samajdar. Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1143.

Abstract 2019: Drug repositioning to induce the synthetic lethality in cancer cells with GSH-depleting therapy

Abstract The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis. Inhibitor of the GSH synthesis including buthionine sulfoximine (BSO) and cystine-glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, thus potential anticancer agents. However, the efficacy of xCT or GSH synthesis-targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. In this study, we performed a synthetic lethal screen of a drug library to identify agents that sensitize the GSH deficiency-resistant cancer cells to the xCT inhibitor sulfasalazine (SSZ). This screen identified the oral anesthetic dyclonine (DYC) which act as a covalent inhibitor for aldehyde dehydrogenases (ALDHs). Treatment with DYC induced intracellular accumulation of the toxic aldehyde 4-hydroxynonenal (4-HNE) in a cooperative manner with SSZ. The combination of DYC and SSZ cooperatively suppressed the growth of SSZ-resistant tumors. Furthermore, we recently found that a vasodilator oxyfedrine (OXY) also possess the similar structure with dyclonine and induce cell death in BSO-resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase-2 expression as a potential biomarker for the efficacy of such combination therapy. Moreover, OXY-mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion. Citation Format: Yuji Otsuki, Osamu Nagano, Hideyuki Saya. Drug repositioning to induce the synthetic lethality in cancer cells with GSH-depleting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2019.