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Supplementary Figures S1 - S9, Table S1. Supplementary Figure S1 (related to Figure 1) shows TNFR expression in melanoma cells. Supplementary Figure S2 (related to Figure 2) shows an NF-κBα binding site in the MITF promoter and MITF target gene expression. Supplementary Figure S3 (related to Figure 3) shows macrophage differentiation and their effect on melanoma cells, keratinocytes and fibroblasts. Supplementary Figure S4 (related to Figure 3) shows M1 and M2 macrophage marker expression in melanoma. Supplementary Figure S5 (related to Figure 4) shows that MEK inhibition does not affect the protective function of macrophages. Supplementary Figure S6 (related to Figure 4) BRAFi and MEKi induced changes in macrophage marker expression in vivo. Supplementary Figure S7 (related to Figure 6) shows that IKK inhibition and MEK inhibition synergise in vitro. Supplementary FigureS8 (related to Figure 6) shows that TNFα protects from BRAF inhibition and IKK and BRAF inhibition synergise in vitro. Supplementary Figure S9 (related to Figure 7) shows the expression of macrophage markers in IKKi treated allografts. Supplementary Table S1 lists patient details.

Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 (Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression.

Background & Aims Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. Methods We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient–derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. Results Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. Conclusions Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy., Graphical abstract

Summary Disrupted chromatin regulatory processes contribute to the development of cancer, in particular pancreatic ductal adenocarcinoma. The assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) is typically used to study chromatin organization. Here, we present a revised ATAC-seq protocol to study chromatin accessibility in adherent patient-derived cell lines. We provide details on how to calculate the library molarity using Agilent’s Bioanalyzer and an analysis pipeline for peak calling and transcription factor mapping. For complete details on the use and execution of this protocol, please refer to Brunton et al. (2020)., Graphical Abstract, Highlights • ATAC-seq protocol to measure chromatin accessibility in patient-derived cell lines • Includes detailed size selection library clean-up step • Instructions on how to calculate the library molarity using Agilent Bioanalyzer • A comprehensive data analysis pipeline is provided, Disrupted chromatin regulatory processes contribute to the development of cancer, in particular pancreatic ductal adenocarcinoma. The assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) is typically used to study chromatin organization. Here, we present a revised ATAC-seq protocol to study chromatin accessibility in adherent patient-derived cell lines. We provide details on how to calculate the library molarity using Agilent’s Bioanalyzer and an analysis pipeline for peak calling and transcription factor mapping.

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.

Large-scale sequencing analyses have transformed our understanding of PDAC and have defined several molecular taxonomies that now guide pre-clinical and clinical therapeutic development. The identification of molecularly defined subgroups of patients with distinct biological underpinnings and potential therapeutic vulnerabilities promises a step change in clinical practice. However, the ability of these molecular taxonomies to guide therapy and ultimately improve patient outcomes remains to be established. This review examines the current status of molecular subtyping in PDAC and explores subtype-specific biology, potential subtype-specific vulnerabilities and their potential relevance to clinical practice.

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.

Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting the DNA damage response (DDR) and replication stress. We show that patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum and PARP inhibitor therapy in vitro and in vivo. We generated a novel signature of replication stress with potential clinical utility in predicting response to ATR and WEE1 inhibitor treatment. Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy.Abstract FigureSTATEMENT OF SIGNIFICANCEWe define therapeutic strategies that target subgroups of PC using novel signatures of DNA damage response deficiency and replication stress. This potentially offers patients with DNA repair defects therapeutic options outside standard of care platinum chemotherapy and is being tested in clinical trials on the Precision-Panc platform.

The identification of molecularly defined subgroups of Pancreatic ductal Adenocarcinoma (PDAC) has the potential to transform clinical practice. There is now a growing consensus that PDAC can be divided into transcriptomic subtypes with 2 broad linages referred to as Classical (Pancreatic) and Squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal linage specification, HNF4A and GATA6, switch metabolic profiles from Classical (Pancreatic) to predominantly Squamous, with GSK3B a key regulator of glycolysis. Pharmacological inhibition of GSK3B results in selective sensitivity in the Squamous subtype, however a subset of these Squamous PDCLs acquired rapid drug tolerance. Using chromatin accessibility maps, we identify subtype specific chromatin landscapes and unique promoter usage between subpopulations of Squamous PDCLs can affect drug tolerance. Our findings demonstrate that a chromatin-based framework can be used to identify subtypes of PDAC that are indistinguishable by gene expression profiles which could refine patient selection for precision medicine.

Heterochromatin (HC) poses a barrier to γH2AX focus expansion and DNA double-strand break (DSB) repair, the latter being relieved by ATM-dependent KAP-1 phosphorylation. Using high-resolution imaging, we show here that the HC superstructure markedly restricts ATM signaling to cell cycle checkpoint proteins. The impact of HC is greater than anticipated from the percentage of HC-DNA and, in distinction to DSB repair, ATM only partly overcomes the constraints posed by HC. Importantly, we examine ATM signaling in human syndromes with disordered HC. After depletion of MeCP2 and DNMT3B, proteins defective in the Rett and immunodeficiency with centromere instability and facial anomalies (ICF) syndromes, respectively, we demonstrate enhanced γH2AX signal expansion at HC-chromocenters in mouse NIH 3T3 cells, which have visible HC-chromocenters. Previous studies have shown that the G(2)/M checkpoint is inefficient requiring multiple DSBs to initiate arrest. MeCP2 and DNMT3B depletion leads to hypersensitive radiation-induced G(2)/M checkpoint arrest despite normal DSB repair. Cell lines from Rett, ICF, and Hutchinson-Guildford progeria syndrome patients similarly showed hyperactivated ATM signaling and hypersensitive and prolonged G(2)/M checkpoint arrest. Collectively, these findings reveal that heterochromatin contributes to the previously described inefficient G(2)/M checkpoint arrest and demonstrate how the signaling response can be uncoupled from DSB repair.

Ataxia telangiectasia (ATM) mutated and Artemis, the proteins defective in ataxia telangiectasia and a class of Radiosensitive-Severe Combined Immunodeficiency (RS-SCID), respectively, function in the repair of DNA double strand breaks (DSBs), which arise in heterochromatic DNA (HC-DSBs) following exposure to ionizing radiation (IR). Here, we examine whether they have protective roles against oxidative damage induced and/or endogenously induced DSBs. We show that DSBs generated following acute exposure of G0/G1 cells to the oxidative damaging agent, tert-butyl hydroperoxide (TBH), are repaired with fast and slow components of similar magnitude to IR-induced DSBs and have a similar requirement for ATM and Artemis. Strikingly, DSBs accumulate in ATM(-/-) mouse embryo fibroblasts (MEFs) and in ATM or Artemis-defective human primary fibroblasts maintained for prolonged periods under confluence arrest. The accumulated DSBs localize to HC-DNA regions. Collectively, the results provide strong evidence that oxidatively induced DSBs arise in HC as well as euchromatic DNA and that Artemis and ATM function in their repair. Additionally, we show that Artemis functions downstream of ATM and is dispensable for HC-relaxation and for pKAP-1 foci formation. These findings are important for evaluating the impact of endogenously arising DNA DSBs in ATM and Artemis-deficient patients.

Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.