The intermediate filament synemin promotes non-homologous end joining in an ATM-dependent manner

Background: Therapy resistance is a great challenge in cancer treatment. Among numerous factors, cell adhesion to extracellular matrix is a well-known determinant of radiochemo-resistance. It has been shown that targeting focal adhesion proteins (FAPs), e.g. β1 integrin, enhances tumor cell radio(chemo)sensitivity in various entities such as head and neck squamous cell carcinoma (HNSCC), lung carcinoma, glioblastoma, breast carcinoma and leukemia. Previous studies demonstrated a functional crosstalk between specific FAPs and DNA repair processes; however, the molecular circuitry underlying this crosstalk remains largely unsolved. Hence, this study in HNSCC aimed to identify alternative FAPs associated with DNA damage repair mechanisms and radioresistance. Materials and Methods: A novel 3D High Throughput RNAi Screen (3DHT-RNAi-S) using laminin-rich extracellular matrix (lrECM) was established to determine radiation-induced re-sidual DNA double strand breaks (DSBs; foci assay) and clonogenic radiation survival. In the screen, we used UTSCC15 HNSSC cells stably expressing the DSB marker protein 53BP1 tagged to pEGFP. Validations were performed in 10 additional HNSCC cell lines (Cal33, FaDu, SAS, UTSCC5, UTSCC8, UTSCC14, UTSCC15, UTSCC45 and XF354fl2) grown in 3D lrECM. Immunofluorescence staining, immunoblotting, chromatin fractionation were utilized to evaluate protein expression, dynamics and kinetics post irradiation. Investigations of molecular mechanisms of DNA repair and radio(chemo)resistance employed DSB repair reporter assays for non-homologous end joining (NHEJ) and homologous recombination (HR), cell cycle analysis, chromatin fractionation levels evaluation and kinase activity profiling (PamGene) upon protein knockdown in combination with/-out X-ray exposure. Foci assay and clonogenic survival assay were performed after single or multiple knockdowns of synemin and associated proteins such as DNA-PKcs and c-Abl. Protein-protein interactions between synemin and associated proteins were determined using immunoprecipitation and proximity ligation assay. Mutant/depletion constructs of synemin (ΔLink-Tail, ΔHead-Link, Synemin_301-961, Synemin_962-1565, S1114A and S1159A) were generated in order to identify essential synemin’s sites controlling DNA repair functions. Results: Among the targets found in the 3DHT-RNAi-S, synemin was one of the most promising FAP candidates to determine HNSCC cell survival and DNA damage repair. Synemin silencing radiosensitized HNSCC cells, while its exogenous overexpression induced radio-protection. Radiation induced an increased synemin/chromatin interaction and a marked ac-cumulation of synemin in the perinuclear area. Intriguingly, synemin depletion elicited a 40% reduction in NHEJ activity without affecting HR or Alt-EJ. In line, ATM, DNA-PKcs and c-Abl phosphorylation as well as Ku70 expression strongly declined in synemin depleted and irra-diated cells relative to controls, whereas an opposite effect was observed under synemin overexpression. Single, double and triple depletion of synemin, DNA-PKcs and c-Abl resulted in a similar radiosensitizing effect and DSB levels as detected upon single knockdown of synemin, describing its upstream role. In kinome analysis, tyrosine kinases showed signifi-cantly reduced activity after synemin silencing relative to controls. Furthermore, immunoprecipitation assays revealed a protein complex formed between synemin, DNA-PKcs and c-Abl under pre- and post-irradiation conditions. This protein complex dispersed when ATM was pharmacologically inhibited, implying synemin function to be dependent on ATM kinase activity. By means of the different mutation/deletion constructs of synemin, the phosphorylation site at serine 1114 located on the distal portion of synemin’s tail was identified as essential protein-protein interaction site for synemin’s function in DNA repair. Conclusions: The established 3DHT-RNAi-S provides a robust screening platform for identifying novel targets involved in therapy resistance. Based on this screen and detailed mechanistic analyses, the intermediate filament synemin was discovered as a novel important determinant of DNA repair, tyrosine kinase activity and radiochemoresistance of HNSCC cells. These results further support the notion that DNA repair is controlled by cooperative interactions between nuclear and cytoplasmic proteins

The intermediate filament synemin promotes non-homologous end joining in an ATM-dependent manner

Background: Therapy resistance is a great challenge in cancer treatment. Among numerous factors, cell adhesion to extracellular matrix is a well-known determinant of radiochemo-resistance. It has been shown that targeting focal adhesion proteins (FAPs), e.g. β1 integrin, enhances tumor cell radio(chemo)sensitivity in various entities such as head and neck squamous cell carcinoma (HNSCC), lung carcinoma, glioblastoma, breast carcinoma and leukemia. Previous studies demonstrated a functional crosstalk between specific FAPs and DNA repair processes; however, the molecular circuitry underlying this crosstalk remains largely unsolved. Hence, this study in HNSCC aimed to identify alternative FAPs associated with DNA damage repair mechanisms and radioresistance. Materials and Methods: A novel 3D High Throughput RNAi Screen (3DHT-RNAi-S) using laminin-rich extracellular matrix (lrECM) was established to determine radiation-induced re-sidual DNA double strand breaks (DSBs; foci assay) and clonogenic radiation survival. In the screen, we used UTSCC15 HNSSC cells stably expressing the DSB marker protein 53BP1 tagged to pEGFP. Validations were performed in 10 additional HNSCC cell lines (Cal33, FaDu, SAS, UTSCC5, UTSCC8, UTSCC14, UTSCC15, UTSCC45 and XF354fl2) grown in 3D lrECM. Immunofluorescence staining, immunoblotting, chromatin fractionation were utilized to evaluate protein expression, dynamics and kinetics post irradiation. Investigations of molecular mechanisms of DNA repair and radio(chemo)resistance employed DSB repair reporter assays for non-homologous end joining (NHEJ) and homologous recombination (HR), cell cycle analysis, chromatin fractionation levels evaluation and kinase activity profiling (PamGene) upon protein knockdown in combination with/-out X-ray exposure. Foci assay and clonogenic survival assay were performed after single or multiple knockdowns of synemin and associated proteins such as DNA-PKcs and c-Abl. Protein-protein interactions between synemin and associated proteins were determined using immunoprecipitation and proximity ligation assay. Mutant/depletion constructs of synemin (ΔLink-Tail, ΔHead-Link, Synemin_301-961, Synemin_962-1565, S1114A and S1159A) were generated in order to identify essential synemin’s sites controlling DNA repair functions. Results: Among the targets found in the 3DHT-RNAi-S, synemin was one of the most promising FAP candidates to determine HNSCC cell survival and DNA damage repair. Synemin silencing radiosensitized HNSCC cells, while its exogenous overexpression induced radio-protection. Radiation induced an increased synemin/chromatin interaction and a marked ac-cumulation of synemin in the perinuclear area. Intriguingly, synemin depletion elicited a 40% reduction in NHEJ activity without affecting HR or Alt-EJ. In line, ATM, DNA-PKcs and c-Abl phosphorylation as well as Ku70 expression strongly declined in synemin depleted and irra-diated cells relative to controls, whereas an opposite effect was observed under synemin overexpression. Single, double and triple depletion of synemin, DNA-PKcs and c-Abl resulted in a similar radiosensitizing effect and DSB levels as detected upon single knockdown of synemin, describing its upstream role. In kinome analysis, tyrosine kinases showed signifi-cantly reduced activity after synemin silencing relative to controls. Furthermore, immunoprecipitation assays revealed a protein complex formed between synemin, DNA-PKcs and c-Abl under pre- and post-irradiation conditions. This protein complex dispersed when ATM was pharmacologically inhibited, implying synemin function to be dependent on ATM kinase activity. By means of the different mutation/deletion constructs of synemin, the phosphorylation site at serine 1114 located on the distal portion of synemin’s tail was identified as essential protein-protein interaction site for synemin’s function in DNA repair. Conclusions: The established 3DHT-RNAi-S provides a robust screening platform for identifying novel targets involved in therapy resistance. Based on this screen and detailed mechanistic analyses, the intermediate filament synemin was discovered as a novel important determinant of DNA repair, tyrosine kinase activity and radiochemoresistance of HNSCC cells. These results further support the notion that DNA repair is controlled by cooperative interactions between nuclear and cytoplasmic proteins

The intermediate filament synemin promotes non-homologous end joining in an ATM-dependent manner

Background: Therapy resistance is a great challenge in cancer treatment. Among numerous factors, cell adhesion to extracellular matrix is a well-known determinant of radiochemo-resistance. It has been shown that targeting focal adhesion proteins (FAPs), e.g. β1 integrin, enhances tumor cell radio(chemo)sensitivity in various entities such as head and neck squamous cell carcinoma (HNSCC), lung carcinoma, glioblastoma, breast carcinoma and leukemia. Previous studies demonstrated a functional crosstalk between specific FAPs and DNA repair processes; however, the molecular circuitry underlying this crosstalk remains largely unsolved. Hence, this study in HNSCC aimed to identify alternative FAPs associated with DNA damage repair mechanisms and radioresistance. Materials and Methods: A novel 3D High Throughput RNAi Screen (3DHT-RNAi-S) using laminin-rich extracellular matrix (lrECM) was established to determine radiation-induced re-sidual DNA double strand breaks (DSBs; foci assay) and clonogenic radiation survival. In the screen, we used UTSCC15 HNSSC cells stably expressing the DSB marker protein 53BP1 tagged to pEGFP. Validations were performed in 10 additional HNSCC cell lines (Cal33, FaDu, SAS, UTSCC5, UTSCC8, UTSCC14, UTSCC15, UTSCC45 and XF354fl2) grown in 3D lrECM. Immunofluorescence staining, immunoblotting, chromatin fractionation were utilized to evaluate protein expression, dynamics and kinetics post irradiation. Investigations of molecular mechanisms of DNA repair and radio(chemo)resistance employed DSB repair reporter assays for non-homologous end joining (NHEJ) and homologous recombination (HR), cell cycle analysis, chromatin fractionation levels evaluation and kinase activity profiling (PamGene) upon protein knockdown in combination with/-out X-ray exposure. Foci assay and clonogenic survival assay were performed after single or multiple knockdowns of synemin and associated proteins such as DNA-PKcs and c-Abl. Protein-protein interactions between synemin and associated proteins were determined using immunoprecipitation and proximity ligation assay. Mutant/depletion constructs of synemin (ΔLink-Tail, ΔHead-Link, Synemin_301-961, Synemin_962-1565, S1114A and S1159A) were generated in order to identify essential synemin’s sites controlling DNA repair functions. Results: Among the targets found in the 3DHT-RNAi-S, synemin was one of the most promising FAP candidates to determine HNSCC cell survival and DNA damage repair. Synemin silencing radiosensitized HNSCC cells, while its exogenous overexpression induced radio-protection. Radiation induced an increased synemin/chromatin interaction and a marked ac-cumulation of synemin in the perinuclear area. Intriguingly, synemin depletion elicited a 40% reduction in NHEJ activity without affecting HR or Alt-EJ. In line, ATM, DNA-PKcs and c-Abl phosphorylation as well as Ku70 expression strongly declined in synemin depleted and irra-diated cells relative to controls, whereas an opposite effect was observed under synemin overexpression. Single, double and triple depletion of synemin, DNA-PKcs and c-Abl resulted in a similar radiosensitizing effect and DSB levels as detected upon single knockdown of synemin, describing its upstream role. In kinome analysis, tyrosine kinases showed signifi-cantly reduced activity after synemin silencing relative to controls. Furthermore, immunoprecipitation assays revealed a protein complex formed between synemin, DNA-PKcs and c-Abl under pre- and post-irradiation conditions. This protein complex dispersed when ATM was pharmacologically inhibited, implying synemin function to be dependent on ATM kinase activity. By means of the different mutation/deletion constructs of synemin, the phosphorylation site at serine 1114 located on the distal portion of synemin’s tail was identified as essential protein-protein interaction site for synemin’s function in DNA repair. Conclusions: The established 3DHT-RNAi-S provides a robust screening platform for identifying novel targets involved in therapy resistance. Based on this screen and detailed mechanistic analyses, the intermediate filament synemin was discovered as a novel important determinant of DNA repair, tyrosine kinase activity and radiochemoresistance of HNSCC cells. These results further support the notion that DNA repair is controlled by cooperative interactions between nuclear and cytoplasmic proteins

Comparative Therapeutic Exploitability of Acute Adaptation Mechanisms to Photon and Proton Irradiation in 3D Head and Neck Squamous Cell Carcinoma Cell Cultures

For better tumor control, high-precision proton beam radiation therapy is currently being intensively discussed relative to conventional photon therapy. Here, we assumed that radiation type-specific molecular response profiles in more physiological 3D, matrix-based head and neck squamous cell carcinoma (HNSCC) cell cultures can be identified and therapeutically exploited. While proton irradiation revealed superimposable clonogenic survival and residual DNA double strand breaks (DSB) relative to photon irradiation, kinome profiles showed quantitative differences between both irradiation types. Pharmacological inhibition of a subset of radiation-induced kinases, predominantly belonging to the mitogen-activated protein kinase (MAPK) family, failed to sensitize HNSCC cells to either proton or photon irradiation. Likewise, inhibitors for ATM, DNA-PK and PARP did not discriminate between proton and photon irradiation but generally elicited a radiosensitization. Conclusively, our results suggest marginal cell line-specific differences in the radiosensitivity and DSB repair without a superiority of one radiation type over the other in 3D grown HNSCC cell cultures. Importantly, radiation-induced activity changes of cytoplasmic kinases induced during the first, acute phase of the cellular radiation response could neither be exploited for sensitization of HNSCC cells to photon nor proton irradiation

Correction: Meerz et al. Comparative Therapeutic Exploitability of Acute Adaptation Mechanisms to Photon and Proton Irradiation in 3D Head and Neck Squamous Cell Carcinoma Cell Cultures. Cancers 2021, 13, 1190

The authors wish to make the following corrections to this paper [...

c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair

The intermediate filament synemin has been previously identified as novel regulator of cancer cell therapy resistance and DNA double strand break (DSB) repair. c-Abl tyrosine kinase is involved in both of these processes. Using PamGene technology, we performed a broad-spectrum kinase activity profiling in three-dimensionally, extracellular matrix grown head and neck cancer cell cultures. Upon synemin silencing, we identified 86 deactivated tyrosine kinases, including c-Abl, in irradiated HNSCC cells. Upon irradiation and synemin inhibition, c-Abl hyperphosphorylation on tyrosine (Y) 412 and threonine (T) 735 was significantly reduced, prompting us to hypothesize that c-Abl tyrosine kinase is an important signaling component of the synemin-mediated radioresistance pathway. Simultaneous targeting of synemin and c-Abl resulted in similar radiosensitization and DSB repair compared with single synemin depletion, suggesting synemin as an upstream regulator of c-Abl. Immunoprecipitation assays revealed a protein complex formation between synemin and c-Abl pre- and post-irradiation. Upon pharmacological inhibition of ATM, synemin/c-Abl protein-protein interactions were disrupted implying synemin function to depend on ATM kinase activity. Moreover, deletion of the SH2 domain of c-Abl demonstrated a decrease in interaction, indicating the dependency of the protein-protein interaction on this domain. Mechanistically, radiosensitization upon synemin knockdown seems to be associated with an impairment of DNA repair via regulation of non-homologous end joining independent of c-Abl function. Our data generated in more physiological 3D cancer cell culture models suggest c-Abl as further key determinant of radioresistance downstream of synemin