C5-Iminosugar modification of casein kinase 1δ lead 3-(4-fluorophenyl)-5-isopropyl-4-(pyridin-4-yl)isoxazole promotes enhanced inhibitor affinity and selectivity.

The quest for isoform-selective and specific ATP-competitive protein kinase inhibitors is of great interest, as inhibitors with these qualities will come with reduced toxicity and improved efficacy. However, creating such inhibitors is very challenging due to the high molecular similarity of kinases ATP active sites. To achieve selectivity for our casein kinase (CK) 1 inhibitor series, we elected to endow our previous CK1δ-hit, 3-(4-fluorophenyl)-5-isopropyl-4-(pyridin-4-yl)isoxazole (1), with chiral iminosugar scaffolds. These scaffolds were attached to C5 of the isoxazole ring, a position deemed favorable to facilitate binding interactions with the ribose pocket/solvent-open area of the ATP binding pocket of CK1δ. Here, we describe the synthesis of analogs of 1 ((-)-/(+)-34, (-)-/(+)-48), which were prepared in 13 steps from enantiomerically pure ethyl (3R,4S)- and ethyl (3S,4R)-1-benzyl-4-[(tert-butyldimethylsilyl)oxy]-5-oxopyrrolidine-3-carboxylate ((-)-11 and (+)-11), respectively. The synthesis involved the coupling of Weinreb amide-activated chiral pyrrolidine scaffolds with 4- and 2-fluoro-4-picoline and reaction of the resulting 4-picolyl ketone intermediates ((-)-/(+)-40 and (-)-/(+)-44) with 4-fluoro-N-hydroxybenzenecarboximidoyl chloride to form the desired isoxazole ring. The activity of the compounds against human CK1δ, -ε, and -α was assessed in recently optimized in vitro assays. Compound (-)-34 was the most active compound with IC50 values (CK1δ/ε) of 1/8 µM and displayed enhanced selectivity toward CK1δ.

A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples.

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.

A Polyclonal Selex Aptamer Library Directly Allows Specific Labelling of the Human Gut Bacterium Blautia producta without Isolating Individual Aptamers.

Recent studies have demonstrated that changes in the abundance of the intestinal bacterium Blautia producta, a potential probiotic, are closely associated with the development of various diseases such as obesity, diabetes, some neurodegenerative diseases, and certain cancers. However, there is still a lack of an effective method to detect the abundance of B. producta in the gut rapidly. Especially, DNA aptamers are now widely used as biometric components for medical testing due to their unique characteristics, including high chemical stability, low production cost, ease of chemical modification, low immunogenicity, and fast reproducibility. We successfully obtained a high-affinity nucleic acid aptamer library (B.p-R14) after 14 SELEX rounds, which efficiently discriminates B. producta in different analysis techniques including fluorometric suspension assays or fluorescence microscopy from other major gut bacteria in complex mixtures and even in human stool samples. These preliminary findings will be the basis towards aptamer-based biosensing applications for the fast and reliable monitoring of B. producta in the human gut microbiome.

CK1δ-Derived Peptides as Novel Tools Inhibiting the Interactions between CK1δ and APP695 to Modulate the Pathogenic Metabolism of APP.

Alzheimer's disease (AD) is the major cause of dementia, and affected individuals suffer from severe cognitive, mental, and functional impairment. Histologically, AD brains are basically characterized by the presence of amyloid plaques and neurofibrillary tangles. Previous reports demonstrated that protein kinase CK1δ influences the metabolism of amyloid precursor protein (APP) by inducing the generation of amyloid-ß (Aß), finally contributing to the formation of amyloid plaques and neuronal cell death. We therefore considered CK1δ as a promising therapeutic target and suggested an innovative strategy for the treatment of AD based on peptide therapeutics specifically modulating the interaction between CK1δ and APP. Initially, CK1δ-derived peptides manipulating the interactions between CK1δ and APP695 were identified by interaction and phosphorylation analysis in vitro. Selected peptides subsequently proved their potential to penetrate cells without inducing cytotoxic effects. Finally, for at least two of the tested CK1δ-derived peptides, a reduction in Aß levels and amyloid plaque formation could be successfully demonstrated in a complex cell culture model for AD. Consequently, the presented results provide new insights into the interactions of CK1δ and APP695 while also serving as a promising starting point for further development of novel and highly innovative pharmacological tools for the treatment of AD.

FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila.

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example.

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.

A CK1 FRET biosensor reveals that DDX3X is an essential activator of CK1ε.

Casein kinase 1 (CK1) plays central roles in various signal transduction pathways and performs many cellular activities. For many years CK1 was thought to act independently of modulatory subunits and in a constitutive manner. Recently, DEAD box RNA helicases, in particular DEAD box RNA helicase 3 X-linked (DDX3X), were found to stimulate CK1 activity in vitro In order to observe CK1 activity in living cells and to study its interaction with DDX3X, we developed a CK1-specific FRET biosensor. This tool revealed that DDX3X is indeed required for full CK1 activity in living cells. Two counteracting mechanisms control the activity of these enzymes. Phosphorylation by CK1 impairs the ATPase activity of DDX3X and RNA destabilizes the DDX3X-CK1 complex. We identified possible sites of interaction between DDX3X and CK1. While mutations identified in the DDX3X genes of human medulloblastoma patients can enhance CK1 activity in living cells, the mechanism of CK1 activation by DDX3X points to a possible therapeutic approach in CK1-related diseases such as those caused by tumors driven by aberrant Wnt/ß-catenin and Sonic hedgehog (SHH) activation. Indeed, CK1 peptides can reduce CK1 activity.

Influence of obesity on remodeling of lung tissue and organization of extracellular matrix after blunt thorax trauma.

BACKGROUND: Previously, it has been shown that obesity is a risk factor for recovery, regeneration, and tissue repair after blunt trauma and can affect the rate of muscle recovery and collagen deposition after trauma. To date, lung tissue regeneration and extracellular matrix regulation in obese mice after injury has not been investigated in detail yet. METHODS: This study uses an established blunt thorax trauma model to analyze morphological changes and alterations on gene and protein level in lean or obese (diet-induced obesity for 16 ± 1 week) male C57BL/6 J mice at various time-points after trauma induction (1 h, 6 h, 24 h, 72 h and 192 h). RESULTS: Morphological analysis after injury showed lung parenchyma damage at early time-points in both lean and obese mice. At later time-points a better regenerative capacity of lean mice was observed, since obese animals still exhibited alveoli collapse, wall thickness as well as remaining filled alveoli structures. Although lean mice showed significantly increased collagen and fibronectin gene levels, analysis of collagen deposition showed no difference based on colorimetric quantification of collagen and visual assessment of Sirius red staining. When investigating the organization of the ECM on gene level, a decreased response of obese mice after trauma regarding extracellular matrix composition and organization was detectable. Differences in the lung tissue between the diets regarding early responding MMPs (MMP8/9) and late responding MMPs (MMP2) could be observed on gene and protein level. Obese mice show differences in regulation of extracellular matrix components compared to normal weight mice, which results in a decreased total MMP activity in obese animals during the whole regeneration phase. Starting at 6 h post traumatic injury, lean mice show a 50% increase in total MMP activity compared to control animals, while MMP activity in obese mice drops to 50%. CONCLUSIONS: In conclusion, abnormal regulation of the levels of extracellular matrix genes in the lung may contribute to an aberrant regeneration after trauma induction with a delay of repair and pathological changes of the lung tissue in obese mice.

Downregulation of ORP3 Correlates with Reduced Survival of Colon Cancer Patients with Advanced Nodal Metastasis and of Female Patients with Grade 3 Colon Cancer.

Genome instability is an essential hallmark in tumor development, including colorectal cancer. We have recently identified the oxysterol binding protein-related protein 3 (ORP3), also known as oxysterol binding protein-like 3 (OSBPL3), as a novel ploidy-control gene, whose knock-out leads to aneuploidy induction and promotes tumor formation, indicating that ORP3 is a bona fide tumor suppressor protein. Here we analyzed expression of ORP3 in a cohort (n = 206) of colon cancer patients in relation to patient survival. We show that low ORP3 mRNA levels correlate with reduced survival of patients with advanced nodal metastasis (N2). While patient survival does not associate with grading when the whole cohort is evaluated, importantly, low ORP3 mRNA levels associate with worse survival of female patients with grade 3 colon cancer. Similarly, low ORP3 mRNA levels associate with worse survival of grade 3 colon cancer patients 70 years of age and younger while low ORP3 mRNA levels seem to be beneficial for colon cancer patients with a T2 tumor size. Together, the data show that ORP3 expression is downregulated during colon cancer progression, which correlates with reduced patient survival. Thus, ORP3 mRNA levels may be a prognostic marker for better stratification of colon cancer patients.

The sperm-associated antigen 6 interactome and its role in spermatogenesis.

Mammalian SPAG6, the orthologue of Chlamydomonas reinhardtii PF16, is a component of the central apparatus of the '9 + 2' axoneme that controls ciliary/flagellar motility, including sperm motility. Recent studies revealed that SPAG6 has functions beyond its role in the central apparatus. Hence, we reexamined the role of SPAG6 in male fertility. In wild-type mice, SPAG6 was present in cytoplasmic vesicles in spermatocytes, the acrosome of round and elongating spermatids and the manchette of elongating spermatids. Spag6-deficient testes showed abnormal spermatogenesis, with abnormalities in male germ cell morphology consistent with the multi-compartment pattern of SPAG6 localization. The armadillo repeat domain of mouse SPAG6 was used as a bait in a yeast two-hybrid screen, and several proteins with diverse functions appeared multiple times, including Snapin, SPINK2 and COPS5. Snapin has a similar localization to SPAG6 in male germ cells, and SPINK2, a key protein in acrosome biogenesis, was dramatically reduced in Spag6-deficient mice which have defective acrosomes. SPAG16L, another SPAG6-binding partner, lost its localization to the manchette in Spag6-deficient mice. Our findings demonstrate that SPAG6 is a multi-functional protein that not only regulates sperm motility, but also plays roles in spermatogenesis in multiple cellular compartments involving multiple protein partners.

2-Azo-, 2-diazocine-thiazols and 2-azo-imidazoles as photoswitchable kinase inhibitors: limitations and pitfalls of the photoswitchable inhibitor approach.

In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors.

Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b.

Insulin resistance represents a hallmark during the development of type 2 diabetes mellitus and in the pathogenesis of obesity-associated disturbances of glucose and lipid metabolism. MicroRNA (miRNA)-dependent post-transcriptional gene silencing has been recognized recently to control gene expression in disease development and progression, including that of insulin-resistant type 2 diabetes. The deregulation of miRNAs miR-143 (ref. 4), miR-181 (ref. 5), and miR-103 and miR-107 (ref. 6) alters hepatic insulin sensitivity. Here we report that the expression of miR-802 is increased in the liver of two obese mouse models and obese human subjects. Inducible transgenic overexpression of miR-802 in mice causes impaired glucose tolerance and attenuates insulin sensitivity, whereas reduction of miR-802 expression improves glucose tolerance and insulin action. We identify Hnf1b (also known as Tcf2) as a target of miR-802-dependent silencing, and show that short hairpin RNA (shRNA)-mediated reduction of Hnf1b in liver causes glucose intolerance, impairs insulin signalling and promotes hepatic gluconeogenesis. In turn, hepatic overexpression of Hnf1b improves insulin sensitivity in Lepr(db/db) mice. Thus, this study defines a critical role for deregulated expression of miR-802 in the development of obesity-associated impairment of glucose metabolism through targeting of Hnf1b, and assigns Hnf1b an unexpected role in the control of hepatic insulin sensitivity.

The kinase domain of CK1δ can be phosphorylated by Chk1.

Members of the casein kinase 1 (CK1) family are key regulators in numerous cellular signal transduction pathways and in order to prevent the development of certain diseases, CK1 kinase activity needs to be tightly regulated. Modulation of kinase activity by site-specific phosphorylation within the C-terminal regulatory domain of CK1δ has already been shown for several cellular kinases. By using biochemical methods, we now identified residues T161, T174, T176, and S181 within the kinase domain of CK1δ as target sites for checkpoint kinase 1 (Chk1). At least residues T176 and S181 show full conservation among CK1δ orthologues from different eukaryotic species. Enzyme kinetic analysis furthermore led to the hypothesis that site-specific phosphorylation within the kinase domain finally contributes to fine-tuning of CK1δ kinase activity. These data provide a basis for the extension of our knowledge about the role of site-specific phosphorylation for regulation of CK1δ and associated signal transduction pathways.

Newly Developed CK1-Specific Inhibitors Show Specifically Stronger Effects on CK1 Mutants and Colon Cancer Cell Lines.

Protein kinases of the CK1 family can be involved in numerous physiological and pathophysiological processes. Dysregulated expression and/or activity as well as mutation of CK1 isoforms have previously been linked to tumorigenesis. Among all neoplastic diseases, colon and rectal cancer (CRC) represent the fourth leading cause of cancer related deaths. Since mutations in CK1δ previously found in CRC patients exhibited increased oncogenic features, inhibition of CK1δ is supposed to have promising therapeutic potential for tumors, which present overexpression or mutations of this CK1 isoform. Therefore, it is important to develop new small molecule inhibitors exhibiting higher affinity toward CK1δ mutants. In the present study, we first characterized the kinetic properties of CK1δ mutants, which were detected in different tumor entities. Subsequently, we characterized the ability of several newly developed IWP-based inhibitors to inhibit wild type and CK1δ mutants and we furthermore analyzed their effects on growth inhibition of various cultured colon cancer cell lines. Our results indicate, that these compounds represent a promising base for the development of novel CRC therapy concepts.

Design, Synthesis and Biological Evaluation of Isoxazole-Based CK1 Inhibitors Modified with Chiral Pyrrolidine Scaffolds.

In this study, we report on the modification of a 3,4-diaryl-isoxazole-based CK1 inhibitor with chiral pyrrolidine scaffolds to develop potent and selective CK1 inhibitors. The pharmacophore of the lead structure was extended towards the ribose pocket of the adenosine triphosphate (ATP) binding site driven by structure-based drug design. For an upscale compatible multigram synthesis of the functionalized pyrrolidine scaffolds, we used a chiral pool synthetic route starting from methionine. Biological evaluation of key compounds in kinase and cellular assays revealed significant effects of the scaffolds towards activity and selectivity, however, the absolute configuration of the chiral moieties only exhibited a limited effect on inhibitory activity. X-ray crystallographic analysis of ligand-CK1δ complexes confirmed the expected binding mode of the 3,4-diaryl-isoxazole inhibitors. Surprisingly, the original compounds underwent spontaneous Pictet-Spengler cyclization with traces of formaldehyde during the co-crystallization process to form highly potent new ligands. Our data suggests chiral "ribose-like" pyrrolidine scaffolds have interesting potential for modifications of pharmacologically active compounds.

CK1α overexpression correlates with poor survival in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer related deaths worldwide and prognosis in advanced tumor stage still remains poor. Since CK1 isoforms have been reported to be deregulated in several tumor entities CK1 has emerged as a novel drug target in cancer therapy. In this study we set out to investigate whether CK1α might have the potential to serve as prognostic marker. METHODS: CK1α RNA and protein expression levels in healthy and tumor tissue of CRC patients were analyzed using quantitative real-time PCR and Western Blot analysis, respectively. Prognostic relevance was investigated by correlating obtained CK1α expression levels with patients' survival rate generating Kaplan-Meier survival plots. RESULTS: It could be shown that CK1α is overexpressed in colorectal tumor tissue compared to normal tissue and CK1α overexpression in tumor tissue correlates with poor survival in CRC patients. Results become more significant when only considering patients with high-grade tumors, as well as patients assigned to UICC II and UICC III stage. Furthermore, Cox regression analysis revealed that CK1α is an independent prognostic factor. In addition, tumors expressing decreased levels of the kinase reveal positive effects on overall survival when localized in the right colon compared to those in the left side. CONCLUSION: In summary, this study provides evidence for the first time that CK1α RNA levels might serve as prognostic marker for CRC.

The Emerging Role of Cyclin-Dependent Kinases (CDKs) in Pancreatic Ductal Adenocarcinoma.

The family of cyclin-dependent kinases (CDKs) has critical functions in cell cycle regulation and controlling of transcriptional elongation. Moreover, dysregulated CDKs have been linked to cancer initiation and progression. Pharmacological CDK inhibition has recently emerged as a novel and promising approach in cancer therapy. This idea is of particular interest to combat pancreatic ductal adenocarcinoma (PDAC), a cancer entity with a dismal prognosis which is owed mainly to PDAC's resistance to conventional therapies. Here, we review the current knowledge of CDK biology, its role in cancer and the therapeutic potential to target CDKs as a novel treatment strategy for PDAC.

State-dependent block of voltage-gated sodium channels by the casein-kinase 1 inhibitor IC261.

Background and Purpose IC261 (3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one) has previously been introduced as an isoform specific inhibitor of casein kinase 1 (CK1) causing cell cycle arrest or cell death of established tumor cell lines. However, it is reasonable to assume that not all antitumor activities of IC261 are mediated by the inhibition of CK1. Meanwhile there is growing evidence that functional voltage-gated sodium channels are also implicated in the progression of tumors as their blockage suppresses tumor migration and invasion of different tumor cell lines. Thus, we asked whether IC261 functionally inhibits voltage-gated sodium channels. Experimental Approach Electrophysiological experiments were performed using the patch-clamp technique at human heart muscle sodium channels heterologously expressed in human TsA cells. Key Results IC261 inhibits sodium channels in a state-dependent manner. IC261 does not interact with the open channel and has only a low affinity for the resting state of the hNav1.5 (human voltage-gated sodium channel; Kr: 120 µM). The efficacy of IC261 strongly increases with membrane depolarisation, indicating that the inactivated state is an important target. The results of different experimental approaches finally revealed an affinity of IC261 to the inactivated state between 1 and 2 µM. Conclusion and Implications IC261 inhibits sodium channels at a similar concentration necessary to reduce CK1δ/ε activity by 50% (IC50 value 1 µM). Thus, inhibition of sodium channels might contribute to the antitumor activity of IC261.

Cancer stem cells: The potential role of autophagy, proteolysis, and cathepsins in glioblastoma stem cells.

One major obstacle in cancer therapy is chemoresistance leading to tumor recurrence and metastasis. Cancer stem cells, in particular glioblastoma stem cells, are highly resistant to chemotherapy, radiation, and immune recognition. In case of immune recognition, several survival mechanisms including, regulation of autophagy, proteases, and cell surface major histocompatibility complex class I molecules, are found in glioblastoma stem cells. In different pathways, cathepsins play a crucial role in processing functional proteins that are necessary for several processes and proper cell function. Consequently, strategies targeting these pathways in glioblastoma stem cells are promising approaches to interfere with tumor cell survival and will be discussed in this review.

CDK9 is a prognostic marker and therapeutic target in pancreatic cancer.

Despite recent advances in diagnosis and therapy, prognosis of pancreatic cancer still remains very poor. Besides valid prognostic markers, novel therapeutic approaches are urgently needed. The family of cyclin-dependent kinases comprises 20 kinases which contribute to malignancy by promoting proliferation, migration, invasion, and apoptotic resistance of cancer cells. In this work, we investigated the role of CDK9 in pancreatic cancer. Immunohistochemical analysis of CDK9 expression in tumor and normal tissue of pancreatic cancer patients revealed an overexpression of CDK9 in pancreatic cancer tissue. In addition, high CDK9 expression in tumor tissue is associated with significantly shortened survival, especially in well-differentiated tumors. Moreover, the therapeutic potential of selective CDK9 inhibition on pancreatic cancer cells was evaluated by analysis of cell viability, long-term survival, and induction of apoptosis and characterized by western blotting and flow cytometry. Pharmacological CDK9 inhibition by SNS-032 drastically reduced cell viability in pancreatic cancer cells and potently suppressed long-term survival. Analyzing the mechanism of action revealed that CDK9 inhibition induced apoptosis and cell cycle arrest in a time-dependent manner by suppression of anti-apoptotic proteins. Furthermore, CDK9 inhibition potently enhances the therapeutic effect of chemotherapeutics in pancreatic cancer cells. In conclusion, we identified CDK9 as a negative prognostic marker in pancreatic cancer. Furthermore, pharmacological CDK9 inhibition is a novel and promising therapeutic approach for pancreatic cancer.