Antifibrotic effects of hypocalcemic vitamin D analogs in murine and human hepatic stellate cells and in the CCl4 mouse model.

Liver cirrhosis is a life-threatening consequence of liver fibrosis. The aim of this study was to investigate the antifibrotic potential of clinically available vitamin D analogs compared to that of calcitriol in vitro and in vivo. Murine hepatic stellate cells, Kupffer cells, and human LX-2 cells were treated with vitamin D analogs, and the profibrotic behavior of these cells was studied. In vivo liver fibrosis was induced using CCl4 until measurable fibrosis was established. Animals were then treated with calcitriol and paricalcitol. Vitamin D and its analogs showed antifibrotic effects in vitro. Treatment with active vitamin D (calcitriol, CAL) and its analogs reduced the protein expression of α-smooth muscle actin (α-SMA) in mHSC. In human LX-2 cells alfacalcidol reduced transforming growth factor-ß (TGF-ß) induced platelet-derived growth factor receptor-ß protein expression and contractility while paricalcitol (PCT), in its equipotent dose to CAL, reduced TGF-ß induced α-SMA protein expression, and ACTA2 and TGF-ß mRNA expression. No effects of a treatment with vitamin D and its analogs were observed in Kupffer cells. In vivo, PCT-treated mice had significantly lower calcium levels than CAL-treated mice. CAL and PCT reduced the hepatic infiltration of CD11b-positive cells and alanine transaminase levels, while PCT but not CAL significantly inhibited fibrosis progression, with a favorable side effect profile in the CCl4 model. We conclude that hypocalcemic vitamin D analogs should be considered in future studies investigating vitamin D for the treatment of liver fibrosis.

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°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 HSP27-mediated through both the intrinsic and the extrinsic apoptotic pathways-at 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.

Genetic targeting of B-RafV600E affects survival and proliferation and identifies selective agents against BRAF-mutant colorectal cancer cells.

BACKGROUND: Colorectal cancers carrying the B-Raf V600E-mutation are associated with a poor prognosis. The purpose of this study was to identify B-RafV600E-mediated traits of cancer cells in a genetic in vitro model and to assess the selective sensitization of B-RafV600E-mutant cancer cells towards therapeutic agents. METHODS: Somatic cell gene targeting was used to generate subclones of the colorectal cancer cell line RKO containing either wild-type or V600E-mutant B-Raf kinase. Cell-biologic analyses were performed in order to link cancer cell traits to the BRAF-mutant genotype. Subsequently, the corresponding tumor cell clones were characterized pharmacogenetically to identify therapeutic agents exhibiting selective sensitivity in B-RafV600E-mutant cells. RESULTS: Genetic targeting of mutant BRAF resulted in restoration of sensitivity to serum starvation-induced apoptosis and efficiently inhibited cell proliferation in the absence of growth factors. Among tested agents, the B-Raf inhibitor dabrafenib was found to induce a strong V600E-dependent shift in cell viability. In contrast, no differential sensitizing effect was observed for conventional chemotherapeutic agents (mitomycin C, oxaliplatin, paclitaxel, etoposide, 5-fluorouracil), nor for the targeted agents cetuximab, sorafenib, vemurafenib, RAF265, or for inhibition of PI3 kinase. Treatment with dabrafenib efficiently inhibited phosphorylation of the B-Raf downstream targets Mek 1/2 and Erk 1/2. CONCLUSION: Mutant BRAF alleles mediate self-sufficiency of growth signals and serum starvation-induced resistance to apoptosis. Targeting of the BRAF mutation leads to a loss of these hallmarks of cancer. Dabrafenib selectively inhibits cell viability in B-RafV600E mutant cancer cells.

The inhibition of YAP Signaling Prevents Chronic Biliary Fibrosis in the Abcb4-/- Model by Modulation of Hepatic Stellate Cell and Bile Duct Epithelium Cell Pathophysiology.

Primary sclerosing cholangitis (PSC) represents a chronic liver disease characterized by poor prognosis and lacking causal treatment options. Yes-associated protein (YAP) functions as a critical mediator of fibrogenesis; however, its therapeutic potential in chronic biliary diseases such as PSC remains unestablished. The objective of this study is to elucidate the possible significance of YAP inhibition in biliary fibrosis by examining the pathophysiology of hepatic stellate cells (HSC) and biliary epithelial cells (BEC). Human liver tissue samples from PSC patients were analyzed to assess the expression of YAP/connective tissue growth factor (CTGF) relative to non-fibrotic control samples. The pathophysiological relevance of YAP/CTGF in HSC and BEC was investigated in primary human HSC (phHSC), LX-2, H69, and TFK-1 cell lines through siRNA or pharmacological inhibition utilizing verteporfin (VP) and metformin (MF). The Abcb4-/- mouse model was employed to evaluate the protective effects of pharmacological YAP inhibition. Hanging droplet and 3D matrigel culture techniques were utilized to investigate YAP expression and activation status of phHSC under various physical conditions. YAP/CTGF upregulation was observed in PSC patients. Silencing YAP/CTGF led to inhibition of phHSC activation and reduced contractility of LX-2 cells, as well as suppression of epithelial-mesenchymal transition (EMT) in H69 cells and proliferation of TFK-1 cells. Pharmacological inhibition of YAP mitigated chronic liver fibrosis in vivo and diminished ductular reaction and EMT. YAP expression in phHSC was effectively modulated by altering extracellular stiffness, highlighting YAP's role as a mechanotransducer. In conclusion, YAP regulates the activation of HSC and EMT in BEC, thereby functioning as a checkpoint of fibrogenesis in chronic cholestasis. Both VP and MF demonstrate effectiveness as YAP inhibitors, capable of inhibiting biliary fibrosis. These findings suggest that VP and MF warrant further investigation as potential therapeutic options for the treatment of PSC.

Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma.

A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer.

Inhibition of ataxia telangiectasia- and Rad3-related function abrogates the in vitro and in vivo tumorigenicity of human colon cancer cells through depletion of the CD133(+) tumor-initiating cell fraction.

The identification of novel approaches to specifically target the DNA-damage checkpoint response in chemotherapy-resistant cancer stem cells (CSC) of solid tumors has recently attracted great interest. We show here in colon cancer cell lines and primary colon cancer cells that inhibition of checkpoint-modulating phosphoinositide 3-kinase-related (PIK) kinases preferentially depletes the chemoresistant and exclusively tumorigenic CD133(+) cell fraction. We observed a time- and dose-dependent disproportionally pronounced loss of CD133(+) cells and the consecutive lack of in vitro and in vivo tumorigenicity of the remaining cells. Depletion of CD133(+) cells was initiated through apoptosis of cycling CD133(+) cells and further substantiated through subsequent recruitment of quiescent CD133(+) cells into the cell cycle followed by their elimination. Models using specific PIK kinase inhibitors, somatic cell gene targeting, and RNA interference demonstrated that the observed detrimental effects of caffeine on CSC were attributable specifically to the inhibition of the PIK kinase ataxia telangiectasia- and Rad3-related (ATR). Mechanistically, phosphorylation of CHK1 checkpoint homolog (S. pombe; CHK1) was significantly enhanced in CD133(+) as compared with CD133(-) cells on treatment with DNA interstrand-crosslinking (ICL) agents, indicating a preferential activation of the ATR/CHK1-dependent DNA-damage response in tumorigenic CD133(+) cells. Consistently, the chemoresistance of CD133(+) cells toward DNA ICL agents was overcome through inhibition of ATR/CHK1-signaling. In conclusion, our study illustrates a novel target to eliminate the tumorigenic CD133(+) cell population in colon cancer and provides another rationale for the development of specific ATR-inhibitors.

Comparative Response of HCC Cells to TKIs: Modified in vitro Testing and Descriptive Expression Analysis.

Introduction: Although the treatment paradigm for hepatocellular carcinoma (HCC) has recently shifted in favour of checkpoint inhibitor (CPI)-based treatment options, the tyrosine kinase inhibitors (TKI) currently approved for the treatment of HCC are expected to remain the cornerstone of HCC treatment alone or in combination with CPIs. Despite considerable research efforts, no biomarker capable of predicting the response to specific TKIs has been validated. Thus, personalized approaches to HCC may aid in determining optimal treatment lines for 2nd and 3rd lines. To identify new biomarkers, we examined differential sensitivity and investigated potential transcriptomic predictors. Methods: To this aim, the sensitivity of nine HCC cell lines to sorafenib, lenvatinib, regorafenib, and cabozantinib was evaluated by a prolonged treatment scheme to determine their respective growth rate inhibition concentrations (GR50). Subgroups discriminated by GR50 values underwent differential expression and gene set enrichment analysis (GSEA). Results: The nine cell lines showed broadly different sensitivities to different TKIs. GR50 values of sorafenib and regorafenib clustered closer in all cell lines, whereas treatments with lenvatinib and cabozantinib showed diversified GR50 values. GSEA showed the activation of specific pathways in sensitive vs non-sensitive cell lines. A signature consisting of 14 biomarkers (GAGE12H, GJB6, PTCHD3, PRH1-PRR4, C6orf222, HBB, C17orf99, GOLGA6A, CRYAA, CCL23, RP11-347C12.3, RP11-514O12.4, FAM180B, and TMPRSS4) discriminates the cell lines' response into three distinct treatment profiles: 1) equally sensible to sorafenib, regorafenib and cabozantinib, 2) sensible to lenvatinib, and 3) more sensible to regorafenib than sorafenib. Conclusion: We observed diverse responses to either of the four TKIs. Subgroup analysis of TKI effectiveness showed distinct transcriptomic profiles and signaling pathways associated with responsiveness. This prompts more extensive studies to explore and validate pharmacogenomic and transcriptomic strategies for a personalized treatment approach, particularly after the failure of CPI treatment.

p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice.

BACKGROUND & AIMS: Progression of chronic liver disease (CLD) to liver cirrhosis and liver cancer is a major global cause of morbidity and mortality. Treatment options capable of inhibiting progression of liver fibrosis when etiological treatment of CLD is not available or fails have yet to be established. We investigated the role of serine/threonine kinase p70 ribosomal protein S6 kinase (p70S6K) as checkpoint of fibrogenesis in hepatic stellate cells (HSCs) and as target for the treatment of liver fibrosis. APPROACH & RESULTS: Immunohistochemistry was used to assess p70S6K expression in liver resection specimen. Primary human or murine HSCs from wild-type or p70S6K-/- mice as well as LX-2 cells were used for in vitro experiments. Specific small interfering RNA or CEP-1347 were used to silence or inhibit p70S6K and assess its functional relevance in viability, contraction and migration assays, fluorescence-activated cell sorting, and Western blot. These results were validated in vivo by a chemical model of fibrogenesis using wild-type and p70S6K-/- mice. Expression of p70S6K was significantly increased in human cirrhotic vs noncirrhotic liver-tissue and progressively increased in vitro through activation of primary human HSCs. Conversely, p70S6K induced fibrogenic activation of HSCs in different models, including the small interfering RNA-based silencing of p70S6K in HSC lines, experiments with p70S6K-/- cells, and the pharmacological inhibition of p70S6K by CEP-1347. These findings were validated in vivo as p70S6K-/- mice developed significantly less fibrosis upon exposure to CCl4. CONCLUSIONS: We establish p70S6K as a checkpoint of fibrogenesis in vitro and in vivo and CEP-1347 as potential treatment option that can safely be used for long-term treatment.

Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents.

BACKGROUND: Inactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored. RESULTS: A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type. CONCLUSIONS: As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents.

Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma.

OBJECTIVES: Hepatocellular carcinoma (HCC) is a leading cancer-related cause of death. Unfortunately, recurrence is common even after curative treatment of early-stage patients, and no adjuvant treatment has yet been established. Aberrant expression of OLFM4 in human cancers has been reported; yet, its specific function during tumor development remains poorly understood, and its role in HCC is unknown. The purpose of this study is to examine the prognostic significance of OLFM4 and its functional relevance in determining recurrence in patients with early-stage HCC. METHODS: Immunohistochemical staining to assess expression, cellular distribution, and prognostic significance of OLFM4 was performed in a tissue microarray comprising 157 HCC tissues and matched nontumor tissues. In addition, expression of OLFM4-coding mRNA was assessed in a separate patients' cohort. The findings were validated by in vitro functional studies using siRNA directed against OLFM4 to assess its effect on cell motility and proliferation. RESULTS: The fraction of HCC samples exhibiting positive OLFM4 staining was higher in comparison with that observed in hepatocytes from matched nontumor tissue (61% vs 39%). However, cytoplasmic-only staining for OLFM4 was associated with vascular invasion (P = 0.048), MMP-7 expression (P = 0.002), and poorer survival (P = 0.008). A multivariate analysis confirmed the independent significance of OLFM4 in determining patients' outcome (5-year survival [58.3% vs 17.3%; HR: 2.135 {95% confidence interval: 1.135-4.015}; P = 0.019]). Correspondingly, inhibition of OLFM4 by siRNA modulated the expression of MMP-7 and E-cadherin, causing inhibition of cell proliferation, motility, and migration. DISCUSSION: To the best of our knowledge, we provide the first report on the prognostic significance of OLFM4 in HCC and identify its mechanistic role as crucial mediator of MMP family protein and E-Cadherin in determining cell invasion and metastasis formation.

The PI3K inhibitor copanlisib synergizes with sorafenib to induce cell death in hepatocellular carcinoma.

Sorafenib, a multikinase inhibitor targeting the Ras/Raf/MAPK (mitogen-activated protein kinase) and vascular endothelial growth factor signaling pathways is an established treatment option for patients with advanced-stage hepatocellular carcinoma (HCC); however, despite its clinical benefit, chemoresistance and disease progression eventually occur almost invariably during treatment. Activation of the PI3K/AKT (phosphatidylinositol-3-kinase/serine/threonine kinase) pathway plays a role in the pathogenesis of HCC and may contribute to determine resistance to sorafenib. We thus evaluated in vitro the effects of the combination of sorafenib and copanlisib, a PI3K inhibitor recently approved for clinical use. The effects of copanlisib alone and in combination with sorafenib were assessed in several HCC cell lines by proliferation and colony formation assays, fluorescence-activated cell sorting analyses, and western blot. In addition, sorafenib-resistant cell clones were used. Copanlisib strongly reduced cell viability and colony formation in different native and sorafenib-resistant HCC cell lines by affecting cyclin D1/CDK4/6 signaling and causing cell cycle arrest. Elevation of phosphorylated (p)-AKT was observed upon incubation with sorafenib and was consistently found in six different unstimulated sorafenib-resistant cell clones. Copanlisib counteracted sorafenib-induced phosphorylation of p-AKT and synergistically potentiated its antineoplastic effect. In summary, copanlisib shows potent anticancer activity as a single agent and acts synergistically in combination with sorafenib in human HCC. Combination of sorafenib with copanlisib represents a rational potential therapeutic option for advanced HCC.

Predictors of ribociclib-mediated antitumour effects in native and sorafenib-resistant human hepatocellular carcinoma cells.

PURPOSE: The cyclin-dependent kinases (CDKs) CDK4 and CDK6 are important regulators of the cell cycle and represent promising targets in cancer treatment. We aimed to investigate the relevance of CDK4/6 in the development of hepatocellular carcinoma (HCC) and the potential of ribociclib, a novel orally available CDK4/6 inhibitor, as a treatment for HCC. METHODS: The effect of ribociclib was assessed in native and sorafenib-resistant HCC cell lines using viability assays, colony formation assays and FACS-based analyses. The expression of potential biomarkers of ribociclib response was assessed in cell lines and primary human hepatocytes using Western blotting. In addition, the prognostic relevance of the cyclin D-CDK4/6-retinoblastoma protein (Rb) pathway was assessed by analysing mRNA expression data from The Cancer Genome Atlas (TCGA). RESULTS: We found that ribociclib downregulated Rb and caused a profound loss of cell viability by inducing G1 cell cycle arrest in HCC cell lines exhibiting Rb-high/p16-low protein expression profiles, but not in Rb-low/p16-high cells, regardless their sensitivity to sorafenib. siRNA-based Rb silencing decreased cell proliferation, but did not diminish the sensitivity of HCC cells to ribociclib. Furthermore, we found that ribociclib synergized with sorafenib to cause cell death. mRNA analysis of primary human HCC specimens showed that CDK4 expression was correlated with patient survival and that the expression of Rb and the p16-encoding CDKN2A gene were inversely correlated. CONCLUSIONS: From our data we conclude that impairment of the cyclin D-CDK4/6-Rb pathway is a frequent feature of HCC and that it is associated with a unfavourable prognosis. We also found that ribociclib exhibits a preferential antineoplastic activity in Rb-high HCC cells. Our results warrant further investigation of Rb and p16 expression as markers of HCC sensitivity to ribociclib.

Inactivation of BRCA2 in human cancer cells identifies a subset of tumors with enhanced sensitivity towards death receptor-mediated apoptosis.

PURPOSE: DNA repair defects due to detrimental BRCA2-mutations confer increased susceptibility towards DNA interstrand-crosslinking (ICL) agents and define patient subpopulations for individualized genotype-based cancer therapy. However, due to the side effects of these drugs, there is a need to identify additional agents, which could be used alone or in combination with ICL-agents. Therefore, we investigated whether BRCA2-mutations might also increase the sensitivity towards TRAIL-receptors (TRAIL-R)-targeting compounds. EXPERIMENTAL DESIGN: Two independent model systems were applied: a BRCA2 gene knockout and a BRCA2 gene complementation model. The effects of TRAIL-R-targeting compounds and ICL-agents on cell viability, apoptosis and cell cycle distribution were compared in BRCA2-proficient versus-deficient cancer cells in vitro. In addition, the effects of the TRAIL-R2-targeting antibody LBY135 were assessed in vivo using a murine tumor xenograft model. RESULTS: BRCA2-deficient cancer cells displayed an increased sensitivity towards TRAIL-R-targeting agents. These effects exceeded and were mechanistically distinguishable from the well-established effects of ICL-agents. In vitro, ICL-agents expectedly induced an early cell cycle arrest followed by delayed apoptosis, whereas TRAIL-R-targeting compounds caused early apoptosis without prior cell cycle arrest. In vivo, treatment with LBY135 significantly reduced the tumor growth of BRCA2-deficient cancer cells in a xenograft model. CONCLUSIONS: BRCA2 mutations strongly increase the in vitro- and in vivo-sensitivity of cancer cells towards TRAIL-R-mediated apoptosis. This effect is mechanistically distinguishable from the well-established ICL-hypersensitivity of BRCA2-deficient cells. Our study thus defines a new genetic subpopulation of cancers susceptible towards TRAIL-R-targeting compounds, which could facilitate novel therapeutic approaches for patients with BRCA2-deficient tumors.

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

The phosphoinositide 3-kinase-related kinase ATR represents a central checkpoint regulator and mediator of DNA-repair. Its inhibition selectively eliminates certain subsets of cancer cells in various tumor types, but the underlying genetic determinants remain enigmatic. Here, we applied a synthetic lethal screen directed against 288 DNA-repair genes using the well-defined ATR knock-in model of DLD1 colorectal cancer cells to identify potential DNA-repair defects mediating these effects. We identified a set of DNA-repair proteins, whose knockdown selectively killed ATR-deficient cancer cells. From this set, we further investigated the profound synthetic lethal interaction between ATR and POLD1. ATR-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 cells and a panel of other colorectal cancer cell lines by using chemical inhibitors of ATR or its major effector kinase CHK1. Mechanistically, POLD1 depletion in ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA-damage along with impaired DNA-repair. Our data could have clinical implications regarding tumor genotype-based cancer therapy, as inactivating POLD1 mutations have recently been identified in small subsets of colorectal and endometrial cancers. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1-inhibitors that are currently tested in clinical trials.