A synthetic lethal screen identifies ATR-inhibition as a novel therapeutic approach for POLD1-deficient cancers

ATR (Ataxia Telangiectasia-mutated and Rad3-related) kinase acts as a central regulator and mediator of the replication checkpoint in response to DNA damage and replication stress. To initiate DNA repair, ATR induces a G2/M cell cycle arrest and stabilizes the replication fork during DNA synthesis. Pharmacological inhibition of ATR has recently been demonstrated to eliminate tumor cells in colorectal cancers (CRCs) but the underlying genetic determinants remain unexplained. Identification of these determinants is essential to develop novel tumor therapy strategies. Due to ATRs` essential role in DNA repair, synthetic lethal interactions of DNA repair mechanisms with ATR are suggested to mediate ATR inhibitor specific tumor cell killing. Using the concept of synthetic lethality, a synthetic lethal screen was conducted in a genetically well-defined ATR knock-in model of DLD1 CRC cells to identify potential genetic determinants eliciting ATR inhibitor-specific tumor cell killing. Applying a siRNA library directed against 288 DNA-repair genes, a set of DNA-repair genes was identified whose knockdown caused either the selective killing of DLD1 ATR-deficient cells (n=6) or an ATR genotype-independent cell killing of DLD1 ATR-proficient and DLD1 ATR-deficient cells (n=20). The strongest synthetic lethal effect was observed between ATR and POLD1 confirmed by kinetic and titration analysis upon POLD1 knockdown in ATR-deficient cells. ATR genotype-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 as well as a panel of other CRC cell lines by using chemical inhibitors of ATR or of its major effector kinase CHK1. Mechanistically, POLD1 depletion in DLD1 ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA damage along with impaired DNA repair, as demonstrated by elevated and sustained levels of γ H2AX focus formation and pan-nuclear γ H2AX staining. Irradiation-induced spatial co-localization of POLD1 with ATR as well as of POLD1 with γ H2AX at sites of DNA damage was further detected. Notably, inactivating POLD1 mutations have recently been described in four families with multiple colorectal adenomas and CRC. In three of these families endometrial tumors were diagnosed. Considering that whole genome-sequencing might determine the POLD1 mutation rates in different tumor entities, our data could have clinical implications in tumor genotype-based cancer therapy with regard to patients harboring those POLD1-deficient tumors, which might respond to chemical inhibition of the ATR/CHK1-axis. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1 inhibitors, which are currently tested in clinical trials. Long-term, the development of selective POLD1-targeted drugs might further broaden the applicability of the identified synthetic lethality with ATR-inhibitors

A synthetic lethal screen identifies ATR-inhibition as a novel therapeutic approach for POLD1-deficient cancers

ATR (Ataxia Telangiectasia-mutated and Rad3-related) kinase acts as a central regulator and mediator of the replication checkpoint in response to DNA damage and replication stress. To initiate DNA repair, ATR induces a G2/M cell cycle arrest and stabilizes the replication fork during DNA synthesis. Pharmacological inhibition of ATR has recently been demonstrated to eliminate tumor cells in colorectal cancers (CRCs) but the underlying genetic determinants remain unexplained. Identification of these determinants is essential to develop novel tumor therapy strategies. Due to ATRs` essential role in DNA repair, synthetic lethal interactions of DNA repair mechanisms with ATR are suggested to mediate ATR inhibitor specific tumor cell killing. Using the concept of synthetic lethality, a synthetic lethal screen was conducted in a genetically well-defined ATR knock-in model of DLD1 CRC cells to identify potential genetic determinants eliciting ATR inhibitor-specific tumor cell killing. Applying a siRNA library directed against 288 DNA-repair genes, a set of DNA-repair genes was identified whose knockdown caused either the selective killing of DLD1 ATR-deficient cells (n=6) or an ATR genotype-independent cell killing of DLD1 ATR-proficient and DLD1 ATR-deficient cells (n=20). The strongest synthetic lethal effect was observed between ATR and POLD1 confirmed by kinetic and titration analysis upon POLD1 knockdown in ATR-deficient cells. ATR genotype-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 as well as a panel of other CRC cell lines by using chemical inhibitors of ATR or of its major effector kinase CHK1. Mechanistically, POLD1 depletion in DLD1 ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA damage along with impaired DNA repair, as demonstrated by elevated and sustained levels of γ H2AX focus formation and pan-nuclear γ H2AX staining. Irradiation-induced spatial co-localization of POLD1 with ATR as well as of POLD1 with γ H2AX at sites of DNA damage was further detected. Notably, inactivating POLD1 mutations have recently been described in four families with multiple colorectal adenomas and CRC. In three of these families endometrial tumors were diagnosed. Considering that whole genome-sequencing might determine the POLD1 mutation rates in different tumor entities, our data could have clinical implications in tumor genotype-based cancer therapy with regard to patients harboring those POLD1-deficient tumors, which might respond to chemical inhibition of the ATR/CHK1-axis. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1 inhibitors, which are currently tested in clinical trials. Long-term, the development of selective POLD1-targeted drugs might further broaden the applicability of the identified synthetic lethality with ATR-inhibitors

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8 degrees C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27mediated through both the intrinsic and the extrinsic apoptotic pathwaysat least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients

A novel European H5N8 influenza A virus has increased virulence in ducks but low zoonotic potential

We investigated in a unique setup of animal models and a human lung explant culture biological properties, including zoonotic potential, of a representative 2016 highly pathogenic avian influenza virus (HPAIV) H5N8, clade 2.3.4.4 group B (H5N8B), that spread rapidly in a huge and ongoing outbreak series in Europe and caused high mortality in waterfowl and domestic birds. HPAIV H5N8B showed increased virulence with rapid onset of severe disease and mortality in Pekin ducks due to pronounced neuro- and hepatotropism. Cross-species infection was evaluated in mice, ferrets, and in a human lung explant culture model. While the H5N8B isolate was highly virulent for Balb/c mice, virulence and transmissibility were grossly reduced in ferrets, which was mirrored by marginal replication in human lung cultures infected ex vivo. Our data indicate that the 2016 HPAIV H5N8B is avian-adapted with augmented virulence for waterfowl, but has low zoonotic potential. The here tested combination of animal studies with the inoculation of human explants provides a promising future workflow to evaluate zoonotic potential, mammalian replication competence and avian virulence of HPAIV.Peer Reviewe

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1,2,3,4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle

Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8 degrees C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27mediated through both the intrinsic and the extrinsic apoptotic pathwaysat least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients

Preclinical Assessment of Bacteriophage Therapy against Experimental Lung Infection.

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages