The animal procol was registered at the Catalan government (FUE-2018-00858322 & ID 6PFJ7JZN5)

The animal procol was registered at the Catalan government (FUE-2018-00858322 & ID 6PFJ7JZN5). Consent to publish Not applicable. Data availability All the data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding authors upon reasonable request. to DDA and/or DNA damage-response inhibitor (DDRi) therapies. Methods We used a multidisciplinary effort combining specific immunohistochemistry, pharmacology tests, anticancer combination therapies and mechanistic studies to assess SLFN11 as a potential biomarker AT9283 for stratification of patients treated with several DDA and/or DDRi in the preclinical and clinical setting. Results SLFN11 protein associated with both preclinical and patient treatment response to DDA, but not to non-DDA or DDRi therapies, such as WEE1 inhibitor or olaparib in breast cancer. SLFN11-low/absent cancers were identified across different tumour types tested. Combinations of DDA with DDRi targeting the replication-stress response (ATR, CHK1 and WEE1) could re-sensitise SLFN11-absent/low cancer models to the DDA treatment and were effective in upper gastrointestinal and genitourinary malignancies. Conclusion SLFN11 informs on the standard of care chemotherapy based on DDA and the effect of selected combinations with ATR, WEE1 or CHK1 inhibitor in a wide range of cancer types and models. and virus, as previously described.14 Ceralasertib (AZD6738),24 adavosertib (AZD1775),25 AZD7648,26 AZD0156,27 AZ-31, AZD6244, prexasertib,28 SRA737 AT9283 and olaparib were synthesised at AstraZeneca. Gemcitabine, cisplatin, hydroxyurea (HU) and etoposide were obtained from Tocris, camptothecin from Sigma, and GDC-0623 from Cayman Chemicals. Stock solutions of gemcitabine (50?mM), cisplatin (1.67?mM) and HU (1?M) were prepared in an aqueous solution; all other drugs were dissolved at 10?mM concentration in DMSO. In total, 10?mM SN-38 dissolved in DMSO was obtained from Abcam (ab141108). The experiment to verify SLFN11 protein levels following continuous chemotherapy treatment was performed similarly as previously described.29 Briefly, DU145 cells were plated in T75 flasks (0?h time point). Twenty-four hours later, treatment with DMSO or SN-38 at final concentrations of 1 1 and 4?nM was initiated based on reported median SN-38 plasma concentrations circulating in patients for up to 3 weeks after infusion of the standard dose of irinotecan.29 Every 3 till 4 days cells were split, and an aliquot was taken for immunoblotting. Every 24C48?h, the medium was refreshed to minimise potential confounding effects deriving from SN-38 chemical instability. Due to cytotoxicity effects, for the 4?nM dose aliquots were only taken at day 3 and 20. Knockout of SLFN11 was performed by CRISPR/Cas9 in-house. Transient knockdown of SLFN11 was performed by siRNA transfections using RNAi-Max kit (Thermofisher Scientific), as previously described.1 Combination synergy and correlation analysis Combination activity (synergism) was performed and calculated using the HSA dose-additivity model in Genedata Screener software. For correlation analysis of SLFN11 RMA normalised gene expression with drug response (log(IC50)), mutational burden, copy number variations or ploidy data sets were downloaded from GDSC database.30 To test response to various DDA or DDRi monotherapies or to determine SLFN11 mRNA expression in cell lines, Sanger pharmacology monotherapy data (IC50 values)30 and SLFN11 expression data were retrieved from publicly available and unpublished data from GDSC and AstraZeneca. SN-38 drug combination data were taken from an in-house database of unpublished collaboration data between Sanger and AstraZeneca. Cell lines with HSA values (excess effect over Highest Single Agent) greater than 0.1 and maximum activity of the combination over 0.5 were considered to benefit from the combination treatment. CCLE SLFN11 RNA-Seq expression data from the DepMap Consortium 20Q1 release were used to divide the cell lines into SLFN11 high and low groups. We fitted a mixture of Gaussians model to the RNA-Seq expression data to try to determine the best threshold to define the groups, but since a significantly better fit was obtained by using more than two components, a range of threshold values were considered to be equally valid. Cells with log2(TMP?+?1) values greater than 2 were classified as SLFN11 high and the rest as SLFN11 low, but similar results were obtained with a threshold of 0.5. A summary of all the combined data can be found in Rabbit Polyclonal to TAZ Supplementary Table?S1. CellTiter-Glo viability assays, clonogenic assay and IncuCyte time-lapse imaging For CellTiter-Glo luminescent assays (Promega), cells in 96-well plates were AT9283 compound dosed using a HP dispenser or manually dosed, and cell viability was determined 72?h later. Spheroids were formed with fibroblasts, as previously described.31 After AT9283 3 or 4 4 days, formed spheroids were compound dosed using HP dispenser and 72?h later, cell viability determined by 3D-cell titre Glo assay.In total, 10?mM SN-38 dissolved in DMSO was obtained from Abcam (ab141108). cancers were AT9283 identified across different tumour types tested. Combinations of DDA with DDRi targeting the replication-stress response (ATR, CHK1 and WEE1) could re-sensitise SLFN11-absent/low cancer models to the DDA treatment and were effective in upper gastrointestinal and genitourinary malignancies. Conclusion SLFN11 informs on the standard of care chemotherapy based on DDA and the effect of selected combinations with ATR, WEE1 or CHK1 inhibitor in a wide range of cancer types and models. and virus, as previously described.14 Ceralasertib (AZD6738),24 adavosertib (AZD1775),25 AZD7648,26 AZD0156,27 AZ-31, AZD6244, prexasertib,28 SRA737 and olaparib were synthesised at AstraZeneca. Gemcitabine, cisplatin, hydroxyurea (HU) and etoposide were obtained from Tocris, camptothecin from Sigma, and GDC-0623 from Cayman Chemicals. Stock solutions of gemcitabine (50?mM), cisplatin (1.67?mM) and HU (1?M) were prepared in an aqueous solution; all other drugs were dissolved at 10?mM concentration in DMSO. In total, 10?mM SN-38 dissolved in DMSO was obtained from Abcam (ab141108). The experiment to verify SLFN11 protein levels following continuous chemotherapy treatment was performed similarly as previously described.29 Briefly, DU145 cells were plated in T75 flasks (0?h time point). Twenty-four hours later, treatment with DMSO or SN-38 at final concentrations of 1 1 and 4?nM was initiated based on reported median SN-38 plasma concentrations circulating in patients for up to 3 weeks after infusion of the standard dose of irinotecan.29 Every 3 till 4 days cells were split, and an aliquot was taken for immunoblotting. Every 24C48?h, the medium was refreshed to minimise potential confounding effects deriving from SN-38 chemical instability. Due to cytotoxicity effects, for the 4?nM dose aliquots were only taken at day time 3 and 20. Knockout of SLFN11 was performed by CRISPR/Cas9 in-house. Transient knockdown of SLFN11 was performed by siRNA transfections using RNAi-Max kit (Thermofisher Scientific), as previously explained.1 Combination synergy and correlation analysis Combination activity (synergism) was performed and determined using the HSA dose-additivity magic size in Genedata Screener software. For correlation analysis of SLFN11 RMA normalised gene manifestation with drug response (log(IC50)), mutational burden, copy number variations or ploidy data units were downloaded from GDSC database.30 To test response to various DDA or DDRi monotherapies or to determine SLFN11 mRNA expression in cell lines, Sanger pharmacology monotherapy data (IC50 values)30 and SLFN11 expression data were retrieved from publicly available and unpublished data from GDSC and AstraZeneca. SN-38 drug combination data were taken from an in-house database of unpublished collaboration data between Sanger and AstraZeneca. Cell lines with HSA ideals (excess effect over Highest Solitary Agent) greater than 0.1 and maximum activity of the combination over 0.5 were considered to benefit from the combination treatment. CCLE SLFN11 RNA-Seq manifestation data from your DepMap Consortium 20Q1 launch were used to divide the cell lines into SLFN11 high and low organizations. We fitted a mixture of Gaussians model to the RNA-Seq manifestation data to try to determine the best threshold to define the organizations, but since a significantly better match was obtained by using more than two parts, a range of threshold ideals were considered to be equally valid. Cells with log2(TMP?+?1) ideals greater than 2 were classified as SLFN11 high and the rest as SLFN11 low, but related results were acquired having a threshold of 0.5. A summary of all the combined data can be found in Supplementary Table?S1. CellTiter-Glo viability assays, clonogenic assay and IncuCyte time-lapse imaging For CellTiter-Glo luminescent assays (Promega), cells in 96-well plates were compound dosed using a HP dispenser or by hand dosed, and cell viability was identified 72?h later on. Spheroids were created with fibroblasts, as previously explained.31 After 3 or 4 4 days, formed spheroids were compound dosed using HP dispenser and 72?h later on, cell viability determined by 3D-cell titre Glo assay (Promega). Percentage growth for both 2D and 3D ethnicities was identified using the equation (test or Wilcoxon test was used to determine statistical variations between two groups of the data, whereas a one-way ANOVA with Dunnetts T3 multiple comparisons test was used to calculate statistical variations between more.