biostudies-arrayexpressMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15235KRAS mutations frequently co-occur with alterations in STK11/LKB1 and/or KEAP1, defining an aggressive subset of tumors associated with resistance to immuno- and chemotherapy. While LKB1 loss is associated with vulnerability to DNA-damage response (DDR)-based therapies, the impact of KEAP1 alterations remains unknown. Here we demonstrate that KEAP1/NRF2 pathway drives a compensatory modulation of ATR-CHK1 signaling, enhancing vulnerability to ATR inhibitors (ATRi) particularly in the setting of increased replication stress associated with LKB1 loss. ATRis show enhanced anti-tumor activity in LKB1 and/or KEAP1-deficient NSCLC models and synergy combined with gemcitabine. ATRi also enhances antitumor immunity and helps mitigate the immunosuppressed phenotype of LKB1 and/or KEAP1-deficient tumors. Finally, in the HUDSON trial, LKB1/KEAP1-deficient NSCLC patients demonstrate enhanced benefits to the ATRi ceralasertib plus durvalumab. These findings suggest that alterations in the KEAP1/NRF2 pathway and/or LKB1 are associated with enhanced sensitivity to ATRi and could serve as useful biomarkers for predicting response to ATRi combination regimens.biostudies-arrayexpressSample Collection - CT26 and A20, BALB/c mice were subcutaneously implanted into the right flank with 5x105 and 1x107 cells respectively. Tumors were measured using digital calipers measured 3x a week until the end of study or when tumors reached the ethical limit of 1500- 2000mm3 using calipers.Nucleic Acid Extraction - CT26 tumor tissues were isolated and total RNA was extracted using the RNeasy 96 QIAcube HT Kit (Qiagen; C/N 74171) according to manufactuers guidelines with a DNase digest included. RNA concentration was determined by Qubit Flex Fluorometer (Invitrogen), RNA purity was determined using a NanoDrop Eight (Thermo Scientific) and RNA integrity was measured using a 4200 Tapestation (Agilent). All samples had a RINe of >7.0. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs; #E7760L) as per manufactuer’s guidelines with 1000ng of RNA input. Ribosomal RNA was removed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs; E7490L)Sequencing - Each library was loaded onto one lane of an S4 v1.5 flow cell (300 cycles) (Illumina, #20028312) on an Illumina NovaSeq 6000 at 2 x 150 paired-end configuration at Astrazeneca R&DLibrary Construction - Libraries were subjected to 9 cycles of PCR with unique dual indexes (New England BioLabs; E6440L). Libraries were subsequently quantified by Qubit Flex Fluorometer (Invitrogen), and library sizes were determined by 4200 Tapestation (Agilent) and pooled equimolar.MINSEQE ScoreAssays and DataProcessed DataorganisationMAGE-TAB FilesData Transformation - We analyzed the human transcriptome in the PDX models with edgeR, using the TMM normalization for accounting for sequencing depth and library composition. Low expressed genes have been filtered out with the filterByExpr function. 20286 genes were kept, while 38449 were filtered out. Subsequently we estimated the genewise dispersion estimates over all genes, allowing for a possible abundance trend. We then fitted a quasi-likelihood negative binomial generalized log-linear, using combination of treatment day of dosage and dosage and confounders. We then performed gene set enrichment analysis (GSEA) to see which are the most enriched signature without depending on external cutoffs. Gene Set Enrichment analysis has the advantage that does not require a pre-defined threshold to call a gene DE or not. Instead, it uses all genes, ranking them for fold change (in this case from upregulated to downregulated in tumor vs healthy). Then it computes and an “Enrichment Score” by walking down the ranked list of genes, increasing a running-sum statistic when a gene is in the gene set and decreasing it when it is not. We therefore computed the median log2(CPM) for the IFN-I and STING signatures, respectively.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAMus musculusERP173461Marta MilofalseKEAP1 and STK11/LKB1 alterations enhance vulnerability to ATR inhibition in KRAS mutant NSCLCKRAS mutations frequently co-occur with alterations in STK11/LKB1 and/or KEAP1, defining an aggressive subset of tumors associated with resistance to immuno- and chemotherapy. While LKB1 loss is associated with vulnerability to DNA-damage response (DDR)-based therapies, the impact of KEAP1 alterations remains unknown. Here we demonstrate that KEAP1/NRF2 pathway drives a compensatory modulation of ATR-CHK1 signaling, enhancing vulnerability to ATR inhibitors (ATRi) particularly in the setting of increased replication stress associated with LKB1 loss. ATRis show enhanced anti-tumor activity in LKB1 and/or KEAP1-deficient NSCLC models and synergy combined with gemcitabine. ATRi also enhances antitumor immunity and helps mitigate the immunosuppressed phenotype of LKB1 and/or KEAP1-deficient tumors. Finally, in the HUDSON trial, LKB1/KEAP1-deficient NSCLC patients demonstrate enhanced benefits to the ATRi ceralasertib plus durvalumab. These findings suggest that alterations in the KEAP1/NRF2 pathway and/or LKB1 are associated with enhanced sensitivity to ATRi and could serve as useful biomarkers for predicting response to ATRi combination regimens.2025-06-15T00:00:00Z2025-06-13T16:18:29.023Z2025-06-13T16:02:18.766ZE-MTAB-15235ERP173461EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184