Guiding ATR and PARP inhibitor combinationswith chemogenomic screens.

Combinations of ataxia telangiectasia- and Rad3-related kinase inhibitors (ATRis) and poly(ADP-ribose) polymerase inhibitors (PARPis) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this, we performed a genome-wide CRISPR-Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations, or the "alternative lengthening of telomeres" telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable in vivo dosing schedule. We present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.

CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

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.

Frameshift indels introduced by genome editing can lead to in-frame exon skipping.

The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of "multiple of three nucleotides" exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a "multiple of three nucleotides" exon of LGALS8 that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.

Discovery of Potent, Orally Bioavailable Inhibitors of Human Cytomegalovirus.

A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 µM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.

The acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus OC43 strongly enhances the production of infectious virus.

Most betacoronaviruses possess an hemagglutinin-esterase (HE) protein, which appears to play a role in binding to or release from the target cell. Since this HE protein possesses an acetyl-esterase activity that removes acetyl groups from O-acetylated sialic acid, a role as a receptor-destroying enzyme has been postulated. However, the precise function of HE and of its enzymatic activity remains poorly understood. Making use of neutralizing antibody and of molecular clones of recombinant human coronavirus OC43 (HCoV-OC43), our results suggest that the HE protein of this HCoV could be associated with infection of target cells and, most notably, is important in the production of infectious viral particles. Indeed, after transfecting BHK-21 cells with various cDNA infectious clones of HCoV-OC43, either lacking the HE protein or bearing an HE protein with a nonfunctional acetyl-esterase enzymatic activity, we were reproducibly unable to detect recombinant infectious viruses compared to the reference infectious HCoV-OC43 clone pBAC-OC43(FL). Complementation experiments, using BHK-21 cells expressing wild-type HE, either transiently or in a stable ectopic expression, demonstrate that this protein plays a very significant role in the production of infectious recombinant coronaviral particles that can subsequently more efficiently infect susceptible epithelial and neuronal cells. Even though the S protein is the main viral factor influencing coronavirus infection of susceptible cells, our results taken together indicate that a functionally active HE protein enhances the infectious properties of HCoV-OC43 and contributes to efficient virus dissemination in cell culture.