This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese , 3-mercaptopyruvate sulfurtransferase  and cystathionine γ-lyase  in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line , mouse mammary gland tumor , mouse fibroblast , mouse melanoma , human colorectal adenocarcinoma , human embryonic kidney , human osteosarcoma  and rat myocardium . Changes in STRs activity are directly related to the bioavailability of cysteine and the sulfane sulfur level, and thus the present authors also measured these parameters, as well as the level of glutathione (its reduced (GSH) and oxidized (GSSG) form) and the [GSH]/[GSSG] ratio that determines the antioxidant capacity of the cells. STRs demonstrate diverse functionality and clinical relevance; therefore, we also performed an analysis of genetic variation of STRs genes that revealed a large number of polymorphisms. Although STRs still provide challenges in several fields, responding to them could not only improve the understanding of various diseases, but may also provide a way to treat them. Rhodanase , 3-mercaptopyruvate sulfurtransferase  and cystathionine γ-lyase CTH, EC: 4.4.1.1) belong to the enzyme superfamily known as sulfurtransferases (STRs). The enzymes are widespread in the world of prokaryotic and eukaryotic organisms, but, in recent years, they have rapidly become a subject of our growing interest; here, the trigger was the discovery that they were involved in the multi-enzyme pathway, which can generate hydrogen sulfide from cysteine, homocysteine and their disulfides .4.1.1 be,6.- (as TST and MPST do), but produces sulfur-donor molecules for STRs /[GSSG] concentration ratio—the parameters that are very often determined along with the activity and expression of STRs.www.surveymonkey.com, accessed on 22 February 2018). The questionnaire listed 80 cell lines diverse in species, tissue origin, and disease occurrence. The cell lines included in this study represent 10% of the most frequently chosen records from scientists employed by the Jagiellonian University, willing to participate in the survey. The present investigators also present data and references for five additional cell lines , and Naglu−/− line , since there are data from the scientific literature addressing this topic of interest.The cell lines presented in this paper were selected based on responses to an anonymous questionnaire from the free online survey tool, Survey Monkey (v/v) fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. To make the complete growth medium of the NMuMG cells, the addition of insulin was necessary . For experimental procedures cell lines were maintained in exponential growth and were refed 48 h prior to harvest. They were maintained at 37 °C, 5% CO2, 95% humidity.All experiments were performed using cell lines obtained from American Type Culture Collection (ATCC). We used eight cell lines: mouse mammary gland cell line , mouse mammary gland tumor (4T1—ATCC: CRL-2539)—this tumor mimics an animal stage IV human breast cancer—mouse fibroblast , mouse melanoma , human colorectal adenocarcinoma , human embryonic kidney , human osteosarcoma  and rat myocardium . All cell lines were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (6 cells) were suspended in 0.1 M phosphate buffer, pH 7.5, in the proportion 1 mln cells/0.04 mL of the buffer and sonicated 15 s at 4 °C . After centrifugation at 1600× g, at 4 °C for 10 min, the supernatant was used for determinations of protein content, sulfane sulfur levels and sulfurtransferases activity. For determinations of low-molecular-weight, sulfur-containing compounds using reverse-phase, high-performance liquid chromatography (RP-HPLC), the pellets were suspended in 250 μL 0.9% NaCl/1 mM BPDS/70% perchloric acid (PCA) and sonicated for 15 s at 4 °C . The sediment was separated by centrifugation at 1600× g for 10 min, and supernatant was saved at −80 °C until used for RP-HPLC analyses.The pellets of cells . After equilibration at 37 °C, 2.5 μL of lactate dehydrogenase  was added, and the decrease in absorbance was measured at 340 nm. The difference between the initial value of absorbance (before LDH addition) and the lowest value (after LDH addition) corresponded to the amount of pyruvate formed in the reaction catalyzed by MPST. The MPST activity was expressed as nmoles of pyruvate produced during one-minute-incubation at 37 °C per 1 mg of protein.The MPST activity was assayed according to the method of Valentine and Frankenfeld  with somg for 10 min. After that, 25 μL of supernatant was transferred to 625 μL of 0.194 mM NADH solution and kept at 37 °C. Control samples (without 24 mM cystathionine) were prepared in the same way as the examined samples. The measurement (absorbance at 340 nm) was conducted for 180 s; after 10 s, 25 μL of LDH (9.06 IU) was added. The difference between the initial value of absorbance (before LDH addition) and the lowest value (after LDH addition) corresponded to the amount of α-ketobutyrate formed in the reaction catalyzed by CTH. The enzyme activity was expressed as nmoles of α-ketobutyrate formed during a one-minute-incubation at 37 °C per 1 mg of protein.The CTH activity was determined according to Matsuo and Greenberg’s method , which w- (thiocyanate) per 1 mg of protein.Sulfane sulfur level was determined by the method of Wood  with somTotal protein content was determined by the method of Lowry et al. . The cryThe levels of the reduced (GSH) and oxidized (GSSG) glutathione, cysteine and cystine in the incubation mixtures were determined using the RP-HPLC method of Dominic et al.  with modTotal RNA was extracted from the cells using TRI reagent, according to the protocol provided by the manufacturer . The extracted RNA was suspended in ribonuclease free-water and quantified by measuring the absorbance at 260 nm. After the procedure, the purity of the obtained RNA was determined by the spectrophotometric analysis (A260 nm/A280 nm). The integrity of the achieved RNA was confirmed by the separation of the 28S and 18S rRNA bands in 2.0% agarose-gel electrophoresis. The RNA solutions were stored at −80 °C until further use.TM Reverse Transcriptase Kit according to the manufacturer’s protocol . For a reverse transcription reaction, 3 µg of total RNA was mixed with 1 μL of Oligo (dT)15 primer (0.5 μg/reaction) and water pretreated with diethylpyrocarbonate (DEPC-H2O), and then incubated for 5 min at 70 °C. After preincubation, other components were added to this mixture: 4 μL of GoScriptTM 5× concentrated reaction buffer, 3 μL of MgCl2 , 1 μL of deoxyribonucleotide triphosphates , 1 μL of RNase inhibitor (20 U/μL), and 1 μL of GoScriptTM Reverse Transcriptase (160 U/μL) in a total volume of 20 μL. The mixture was first incubated for 5 min at 25 °C, then for 60 min at 42 °C, and, finally, for 15 min at 70 °C. If necessary, the solutions of complementary DNA (cDNA) were stored at −20 °C.Total RNA from the cell samples was reverse-transcribed using a GoScript2O. In each the case, a similar reaction was also performed in the mixture without DNA (the negative control) in order to confirm the specificity of the obtained reaction products. The temperature profile of PCR amplification for these genes, as well as gene-specific primer sequences, are collected in TM MP Imaging system with Image Lab Software, version 6.0, Bio-Rad).The expressions of the three genes  were analyzed by PCR. As a reference , gene-encoding glyceraldehyde 3-phosphate dehydrogenase  was used. The amplification of cDNA samples was performed in a 12.5 μL reaction volume containing 1 μL of synthesized cDNA, 10 μM of each of gene-specific primer pair, 2 U/μL Taq DNA polymerase in 10 mM buffer Tris-HCl at pH 8.8, and 10 mM of each dNTPs and DEPC-H2S). H2S plays a role in protection of neurons against oxidative stress and stimulates an increase in γ-glutamylcysteine synthetase, and thereby an increase in the level of GSH. On the other hand, diminished CTH expression entails a decrease in the level of cysteine, glutathione (GSH), taurine and H2S in the cells [2S-generating enzymes  in the cells may causes a decrease/increase in H2S bioavailability and indicates the possible genetic background of H2S-geneting enzyme regulation [We performed an analysis of genetic variations of the TST-, MPST- and CTH-encoding genes . Some inhe cells ,31. The gulation . Therefogulation  and/or igulation . MoreoveNaglu−/− cell lines (the cellular model of mucopolysaccharidosis IIIB). Therefore, to expand this knowledge, the expression levels of the genes encoding TST, MPST and CTH were investigated in selected cell lines. The obtained data are presented in In the scientific literature, the expression data of tested STRs are presented only in a few cell lines. Jurkowska et al.  showed tThe performed gene expression analysis confirmed that the expression of all three STRs took place in all investigated cell lines: the mouse mammary gland cells MNuMG, mouse mammary gland tumor 4T1, mouse fibroblast MEF, mouse melanoma B16-F1, human colorectal adenocarcinoma Caco-2, human embryonic kidney cells HEK, human osteosarcoma MG-63, and rat myocardium H9c2. Taking into consideration the species tested, the highest TST and CTH expression level was observed in the mouse cell lines, while the MPST expression was the highest in the rat H9c2 cell line . Based oNaglu−/− cell line—the line derived from mice with mutation B6.129S6-Naglutm1Efn/J in the gene encoding the N-alpha-glucosaminidase [2S from cysteine, homocysteine and their disulfides [The STRs activity analysis confirmed the presence of the activity of TST, MPST and CTH in all the tested and some additional cell lines; the latter were added to minidase , as wellNaglu−/− cells (2 nmole/mg protein), and this is likely a biochemical cellular response to the mutation existing in this line (the mutation likely causes an increase in the sulfate levels originating from undigested disaccharide—heparan sulfate) [Naglu−/− cell line (p < 0.05), it can be concluded that, in the MG-63, SH-SY5Y and H9c2 cells, the low level of GSH and sulfane sulfur-containing compounds (Naglu−/− and Caco-2 cells and high activity of TST (the Caco-2 cells)  ,40. L-cyell line . Furtherompounds  correspoompounds  and loweompounds . In contompounds  were corompounds  and the ompounds . The low2 cells)  were als2 cells) . The mos2 cells)  determinll lines  was corrll lines . MeanwhiThe scientific literature presents a hypothesis postulating a decrease in STRs activity and the level of sulfane sulfur-containing compounds in neoplastic cell lines ,37,42. TOur studies of the STRs activity and expression levels and directly related parameters  provide researchers with a choice of cellular research models without the need to conduct their own large-scale screening studies. The presented investigation results confirm the expression and activity of STRs in all the tested cell lines and demonstrate differences in the activity and the level of STRs mRNA depending on the species and origin the cells. Taking into consideration the species tested, the greatest TST and CTH expression was observed in the mouse cell lines, while the MPST expression was the highest in the rat H9c2 cell line . Among t


Three-dimensional (3D) cell culture methods are identified for simulating the biological microenvironment and demonstrating more similarity to in vivo circumstances. Anaplastic thyroid carcinoma (ATC) is a lethal endocrine malignancy. Despite different treatment approaches, no improvement in the survival rate of the patients has been shown. In this study, we used the 3D in vitro ATC model to investigate the cytotoxic effect of BI-847325 anticancer drug in two-dimensional (2D)- and 3D- cultured cells.′,6-diamidino-2-phenylindole) staining. Survival of alginate-encapsulated cells was evaluated by CFSE  staining. The population doubling times of C643 and SW1736 cell lines cultured in 2D monolayer as well as in 3D system were calculated. The cytotoxic effect of BI-847325 on 2D- and 3D- cultured cell lines was assessed for 24–72 h by MTT  assay. Finally, the 3D culture results were compared with the 2D culture method.Human ATC cell lines, C643 and SW1736, were cultured in one percentage (w/v) sodium alginate. Spheroids were incubated in medium for one week. The reproducibility of the fabrication of alginate beads was evaluated. Encapsulation of the cells in alginate was examined by DAPI (450) values of BI-847325 were higher in 3D culture compared to 2D culture. The cytotoxicity data indicated that 3D in vitro models were more resistant to chemotherapy agents.The half-maximal inhibitory concentration (ICThe findings of this study are beneficial for developing in vitro ATC 3D models to analyze the efficacy of different chemotherapy drugs and formulations. Thyroid cancer is the most frequent endocrine malignancy with the highest growing incidence rate between the other types of solid tumors in the United States –3. AnaplIn general, cancer investigators rely on 2D cell culture in vitro experiments and animal models to understand the complex biological processes of tumor formation, progression and, its treatment , 9. In r2, nutrients, metabolic wastes, growth factors, etc. Therefore, hydrogel scaffolds are used in different pharmaceutical and biomedical fields  assay. The C643 and SW1736 cell lines were seeded in 96-well plates at a concentration of 5000 cells/well. After 24 h cells were treated with 0.125–8 μM doses of BI-847325 for 24–72 h. The viability of the cells was evaluated by the metabolic conversion of the tetrazolium dye to insoluble formazan. MTT (5 mg/ml) was added to the 96-well plates at 10 μl/well, and the plates were then incubated for three hours in 5% CO2 at 37 °C incubator. The medium was removed cautiously with a pipette to avoid the disruption of beads. To dissolve the formazan salt, 500 μl of 0.1 M HCl  in 2-propanol  was added. Plates were placed in the incubator for 45 min to allow the formazan to diffuse out of the alginate hydrogel and entirely be dissolved in the solvent [50 values were calculated via the Reed–Muench method [The effect of BI-847325 treatment on encapsulated ATC cell lines was also examined by MTT assay. Cells were treated with BI-847325 (1–64 μM) for 24 h as described earlier , and cur solvent . The absh method , 32.p < .05 (*); p < .01 (**); p < .001 (***); p < .0001 (****). IC50 values were calculated by nonlinear regression using GraphPad Prism v5 software. Statistical comparisons of population doubling times were carried out using the unpaired t-test method in each case (n = 3).The data show the mean of four independent experiments±SD. Statistical analysis of MTT results were performed using One-way ANOVA followed by Tukey’s post hoc tests. Statistical significances are expressed as n = 20), their size was measured using ImageJ software  which indicates appropriate reproducibility in spheroids formation. Encapsulation and survival of cell lines are shown in Fig. First, to evaluate the reproducibility of the fabrication of alginate spheroids  indicating the same growth rate of ATC cell lines in 2D and 3D cultures.As shown in Fig. RAS mutation and SW1736 with BRAFV600E mutation [50 remained constant after 48 h and IC50 values of BI-847325 on C643 and SW1736 were at 2 μM and 4 μM, respectively  for 24–72 h prevented proliferation in the two cell lines Fig. . In both50 values in the 3D method demonstrated that C643 cell line is more sensitive towards BI-847325 compared to the SW1736 cell line.Interestingly, similar to 2D cell culture, the IC50 values in 3D cell culture system may be associated with multiple mechanisms, including a reduced penetration of chemotherapy agents because of the simulation of key factors of natural tumor microenvironment such as physiological gradients and presence of ECM, enhanced pro-survival signaling pathways such as MAPK pathway due to upregulation of genes involving in anticancer drug resistance [The results of this study are the first report of 3D model for a more accurate assessment of the effect of chemotherapy drugs in ATC. These findings indicate that 3D in vitro models demonstrate more resistance to anticancer drugs. Increased ICsistance , 37–41 (Furthermore, CSCs cultured in the 2D system may reduce their stemness characteristics, while CSCs cultured in 3D may be more resistant to anticancer drugs , 42, 43.50 values were significantly higher in the 3D cell culture system compared to the 2D one for both cell lines (Table 50 values of BI-847325 for C643 and SW1736 were at 15 μM and 34 μM concentrations, respectively. The higher IC50 values in 3D cell cultures demonstrate the resistance potential gained by cells while growing in the 3D system which simulates the natural physiological conditions. This evidence supports the higher resistance rate of cultured cells in the 3D system to chemotherapy drugs as reported previously [BI-847325 cytotoxicity on ATC C643 and SW1736 cell lines was measured in 2D and 3D cell culture systems. The study has revealed that the concentration ranges used in the 2D cell culture method were not appropriate to exhibit significant cytotoxicity in the 3D cell culture system . Based oeviously , 23.In a similar study, higher viability rates following treatment with Paclitaxel were observed in ovarian cancer cells cultured in 3D compared to 2D cell culture . In anot50 values are higher in 3D cell culture system compared to the 2D cell culture method. Like what observed in 2D culture, the obtained IC50 in the 3D culture for SW1736 was higher than that for C643 similar to conventional 2D culture. This observation indicates the resistant nature of this cell line which is probably due to more stemness properties of ATC SW1736 cell line. The elevated IC50 values confirm the Lei and colleagues report about 3D matrix-driven CSCs theory in which 3D culture of breast cancer cell line, MCF-7, resulted in more tumorigenesis and elevated anticancer drug resistance [On the other hand, the subpopulation of malignant cells in the tumor, known as CSCs, is responsible for cancer recurrence and different drug resistance mechanisms –48. We hsistance , 23, 50.In order to understand the difference between cytotoxic responses of chemotherapeutic drugs in 2D- and 3D- based culture systems, Godudu et al. examined the uptake efficiency of Doxorubicin inside the alginate-encapsulated lung cancer cell lines . DoxorubCollectively, 3D cell culture systems such as alginate-encapsulated cells more closely mimic the key factors of the natural tumor microenvironment such as molecular concentration gradients and important cellular interactions including cell-cell and cell-matrix communications. However, additional cellular investigations such as xenograft tumors are required to promote the predictive value of 3D cell culture in the response of ATC cells to anticancer drugs in vivo.In this study, the cytotoxicity data indicated that 3D in vitro models display more resistance to chemotherapy agents. ATC tumors are unresponsive to the current anticancer drugs. Having to do with 3D in vitro ATC models designed by the current research, it would be feasible to analyze the effect of different anticancer drugs and diverse signaling pathways affected by the chemotherapy agents and formulations. In terms of the future application of the proposed model, this culture system can be modified to support the multicellular 3D cell culture. Moreover, the attachment of different peptides can improve alginate scaffold according to extracellular matrix properties.


While the treatment of squamous cell carcinoma of the head and neck (HNSCC) with radiotherapy (RT) is complemented more and more by immunotherapy in clinical trials, little is known about the impact of the human papillomavirus (HPV) status or the applied RT scheme on the immune phenotype of the tumor cells. Therefore, we aimed to examine the impact of the HPV status of four human HNSCC cell lines on cell death and the expression of immune checkpoint molecules (ICMs) after RT with either hypofractionation irradiation (5x3.0Gy) or a high single dose (1x19.3Gy) via multicolor flow cytometry and quantitative PCR at an early time point after therapy. In our study, 5x3.0Gy RT induced high numbers of early and late apoptotic cells independent of the HPV status, but necrosis was only increased in the HPV-positive UM-Scc-47 cells. Generally, the immune stimulatory ICMs  were less affected by RT compared to the immune suppressive ones ). A significant higher surface expression of the analyzed ICMs was found after hypofractionated RT compared to a single high dose; however, regardless of the HPV status, with the exception of ICOS-L. Here, HPV-positive HNSCC tumor cells showed a stronger response to 5x3.0Gy than HPV-negative ones. On the RNA level, only minor alterations of ICMs were observed following RT, with the exception of the HPV negative cell line CAL33 treated with 5x3.0Gy, where PD-L2, HVEM and CD70 were significantly increased. We conclude that the HPV status may not distinctly predict immunological responses following RT, and thus cannot be used as a single predictive marker for therapy responses in HNSCC. In contrast, the patient-specific individual expression of ICMs following RT is preferable for the targeted patient selection for immune therapy directed against distinct ICM. Squamous cell carcinomas of the head and neck (HNSCCs) are characterized as the sixth most common cancer worldwide . In GermHPV-positive tumors have been shown to be more radiosensitive regarding the clonogenic survival compared to HPV-negative tumors . One of The immune system plays an important role in both, the development of cancer and the eradication of tumors. In a healthy system, it protects the human body from cancer cells, as these express other proteins due to their altered genome, which are recognized as tumor antigens and trigger an immune response . In ordeIt has been demonstrated that RT increases the expression of PD-L1 on tumor cells and thereby inhibits T cell-mediated anti-tumor responses. RT has immune suppressive properties, but also immune stimulatory ones, as, e.g., the induction of the release of danger signals . The latIn a clinical context, the two anti-PD-1 monoclonal antibodies nivolumab and pembrolizumab are approved for the treatment of recurrent HNSCC in the USA since 2016 and in the European Union since 2017 . These dIn order to identify/select patients who are likely to respond positively to therapy with immune checkpoint inhibitors, research has moved specifically towards prognostic biomarkers in recent years . We justOne characteristic biomarker that often applies to a large proportion of HNSCC patients is an HPV infection. HPV infections, the most common sexually transmitted disease in the United States, heal successfully in the vast majority of infected individuals . In the Regarding the influence of the HPV status, accelerated RT fractionation schemes, aiming towards achieving a reduced proliferation capability of tumor cells, and thus reducing the likelihood of a relapse, have also been under investigation for several years ,37. In rTo sum up, HPV-associated HNSCC tumors generally respond better to anticancer treatments. It is assumed that this is due to a better response to RT, but data about the role of the immune phenotype of the tumor cells in this setting are scarce . We therA significant increase in (early and late) apoptosis independently of the HPV status, but dependent on the fractionation scheme, was observed. In all examined cell lines, hypofractionated RT(5x3.0Gy) resulted in a significant increase in apoptotic cells in comparison to untreated and 1x19.3Gy irradiated cells. The HPV-positive cell line UD-Scc-2 however, not only showed the highest rate of apoptotic cells, but also a significant increase in apoptotic cells after 1x19.3 in comparison to untreated controls. Primary necrosis  remained stable across all treatments, with the exception of HPV-positive UM-Scc-47, where a significant increase in necrotic cell numbers was found after 5x3.0Gy in comparison to untreated controls and 1x19.3Gy single dose RT b.We then investigated the expression of various immune suppressive  checkpoint molecules on the cell surface:As shown in The immune stimulatory ICMs were less affected by RT compared to the immune suppressive ones . CD70 waIn the clinic, HPV-positive HNSCC tumors are generally associated with a better prognosis, and thus improved therapy outcome . They ofOur studies regarding the cell death of HNSCC cells after hypofractionated (5x3.0Gy) or single high dose (1x19.3Gy) RT showed that the applied RT scheme has a bigger impact on cell death than the HPV status in the four cell lines we have looked at . HypofraAs apoptosis is generally considered to be a rather immunosuppressive and necrosis is considered to be an immune-activating form of cell death, necrosis is therefore the preferred outcome in cancer therapy, as it is expected to induce a better anti-tumor response in treated patients ,47. WhenAs mentioned above, ICMs are key regulators of T cell-mediated anti-tumor immune responses. However, a large number of patients do not respond properly to immune therapies targeting such ICMs. Therefore, improving existing immunotherapies with optimized RT schemes and identifying other RT-regulated ICMs besides PD-L1 is an important step to increase response rates to radio-immunotherapies ,53.The integration of the HPV genome into the cellular genome of the host can lead to an increased expression of the two oncoproteins E6 and E7 ,55. HoweWe found that, in general, irradiation with 5x3.0Gy led to an increase in the surface expression of the immune inhibitory checkpoint molecules PD-L1, PD-L2 and HVEM . A signiIrradiation of HPV-positive UD-Scc-2 with 5x3Gy resulted in a significant upregulation of PD-L1 and PD-L2 a,b, whilWe thus conclude that while individual tumor cell lines show individual responses in terms of intensity and duration of ICM expression, RT with 5x3.0Gy has an overall stimulatory effect regarding the expression of suppressive ICMs, independent of cell line and HPV status. While PD-L1 expression is generally enhanced in HPV-positive HNSCCs , it is nWhen looking specifically at immune stimulatory checkpoint molecules, 5x3.0Gy again had the overall highest impact on regulation of checkpoint molecules while 1x19.3Gy resulted in expression levels that were close to those of controls . SimilarFor CD70 surface expression a, we fouHowever, when looking at ICOS-L, only the HPV-positive cell lines were characterized by a significantly increased ICOS-L surface expression after RT with 5x3.0Gy levels . As ICOSThe cumulative work of the last few years suggests that HPV-positive HNSCC tumors have a better prognosis and responsiveness to radiotherapy treatment. In addition, HPV-positive HNSCCs are also often described to be more homogenous in their behavior than HPV-negative ones ,43,45,48Notably, a hypofractionated RT scheme of 5x3.0Gy was very effective in terms of inducing cell death and modulating immune checkpoint molecules on the surface of HNSCC cells, and partly also on a mRNA level. This should be considered when designing new multimodal tumor therapies with adapted RT protocols . HoweverTaken together, our findings suggest a holistic approach to be the most promising one for optimizing multimodal treatments for HNSCC instead of particularly focusing on HPV status, one RT fractionation scheme, or single ICM inhibition. For a large cohort of patients, examining immune matrices in individual tumors depending on distinct RT protocols might add additional value in the future.2 and 95% humidity).Four different human cancer cell lines from head and neck tumors  were used, each kindly provided by Dr. T. Rieckmann . The two cell lines UM-Scc-47 and UD-Scc-2 are HPV-positive, whereas the cell lines HSC-4 and CAL33 are HPV-negative ones. All cell lines were cultured in modified D10 medium  supplemented with 10% fetal bovine serum  and 100 U/mL of Pen/Strep  under standard culture conditions . After they had grown for 24 h, the respective cell culture flasks were exposed to irradiation using a hypofractionated scheme with a single dose per fraction of 3.0Gy of X-rays  for 5 days and a scheme with a single dose of 19.3Gy  on d6, using a Isovolt Titan series-GE Technologies  X-ray machine. Non-irradiated controls were seeded in parallel for each scheme. On experimental day 7, an evaluation of all conditions, i.e., treatment and control, was performed using a CytoFLEX S flow cytometer  and samples were collected for subsequent RNA analyses. The complete procedure of the experimental set-up is shown in For the experiments, the cell lines were seeded and cultivated under standard conditions /propidium iodide (PI) staining solution (1.0 µg/mL of PI and 0.5 µg/mL of FITC-labeled AxV). Subsequent analysis was performed using a CytoFLEX S flow cytometer. The gating strategy is shown in On day 7 of the experiment, multicolor flow cytometry was used to analyse the different forms of cell death. For this purpose, tumor cells were harvested with trypsin and 1 × 10The expression of immune checkpoint molecules on the surface of the tumor cells was analyzed on day 7. For this purpose, the harvested tumor cells were incubated with the antibodies listed in Isolation of RNA was done via phenol/chloroform extraction in TriFast  according to the manufacturer’s protocol and purity of RNA was determined using a P330 nanophotometer . Pre-made Prime PCR primers from Bio-Rad . For all data, a two-sided Mann–Whitney U test was performed for comparisons of treatments within one cell line. Significances were considered as follows: *


Cancer is the third leading cause of death in the United Arab Emirates (UAE), after cardiovascular diseases and accidents. In the UAE, colorectal cancer (CRC) is the first and fourth most common cancer in males and females, respectively. Several treatment modalities have been employed for cancer treatment, such as surgery, radiotherapy, chemotherapy, hormone replacement therapy, and immunotherapy. These treatment modalities often elicit adverse effects on normal cells, causing toxic side effects. To circumvent these toxicities, there has been an increased impetus towards the identification of alternate treatment strategies. Animal venoms are rich sources of pharmacologically active polypeptides and proteins.In this proof-of-concept study, we will apply a high-throughput venomics strategy to identify and characterize anticancer bioactive peptides (BAPs) from 20 different animal venoms, specifically targeting CRC. We chose to focus on CRC because it is one of the foremost health issues in the UAE.In the initial study, we will screen 2500 different peptides derived from 20 different animal venoms for anticancer activity specifically directed against 3 CRC cell lines and two control cell lines employing the 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay for cytotoxicity. Of the 20 venoms, 3 that exhibit specific and potent anticancer activity directed against the 3 CRC cell lines will be selected; and from these 3 venoms, the specific peptides with anti-CRC activity will be isolated and characterized.This study is at the protocol development stage only, and as such, no results are available. However, we have initiated the groundwork required to disseminate the proposed study, which includes culturing of colorectal cancer cell lines and preparation of venom screens.In summary, the proposed study will generate therapeutic leads to manage and treat one of the leading health issues in the UAE, namely, CRC.PRR1-10.2196/31128 Cancer is the third leading cause of death in the United Arab Emirates (UAE) after cardiovascular diseases and accidents. In the UAE, colorectal cancer (CRC) is the first and fourth most common cancer in males and females, respectively . NumerouAgkistrodon contortrix contortrix (southern copperhead); although it is not cytotoxic to breast cancer cells, it inhibits angiogenesis induced by breast cancer in vivo [Conus inscriptus have been shown to possess prospective anticancer activity, specifically against cervical cancer [Animal venoms are veritable gold mines of pharmacologically active polypeptides and proteins. In fact, proteins and peptides with anticancer properties have been identified and characterized from venoms of snakes, bees, scorpions, wasps, ants, spiders, and caterpillars ,5. One c in vivo . Similar in vivo . Furtherl cancer .In summary, animal venoms comprise a mix of bioactive peptides (BAPs), many of which exhibit anticancer activity against specific types of cancer mediated by one of the following four mechanisms: (1) induction of cell cycle arrest, growth inhibition, and apoptosis; (2) inhibition of angiogenesis; (3) inhibition of invasion and metastasis; and (4) blocking of specific transmembrane channels .In this proof-of-concept study, we will apply a high-throughput venomics strategy to identify and characterize anticancer peptides from 20 different animal venoms, specifically targeting colorectal cancer. We chose to focus on colorectal cancer because it is one of the foremost health issues in the UAE.The proof-of-concept study will address two specific aims.In Aim 1, we will screen 2500 different peptides derived from 20 different animal venoms for anticancer activity specifically directed against 3 CRC cell lines and 2 control cell lines employing the 3--2,5-diphenyltetra- zolium bromide (MTT) colorimetric assay for cytotoxicity.In Aim 2, 3 venoms of the 20 screened in Aim 1 that exhibit specific and potent anticancer activity directed against the 3 CRC cell lines will be selected; from these 3 venoms, the specific peptides with anti-CRC activity will be isolated and characterized.The dissemination plan for the proposed research corresponding to Aim 1 and Aim 2 is shown in Aim 1 of the study is directed to the identification of three animal venoms which exhibit potent and specific cytotoxic activity on CRC cell lines  and normal colon cell lines (CCD18 and NCM-460).This step of the project will be pursued in collaboration with Latoxan , a leading producer of animal venoms, with whom we have engaged in previous research collaborations. Briefly, a library of venom peptides will be created in 96-well plates. Venoms will be first cleared from molecules over 8500 Da (cytolytic enzymes). On the basis of high-performance liquid chromatography (HPLC), each venom will be split into 20 fractions. Each fraction is expected to contain 5 to 10 peptides at a 10 μmolar concentration in a volume of 100 μl. The strength of this strategy is that the separation of peptides using HPLC facilitates the identification of the active peptide once a hit (specific cytotoxic activity on CRC cell lines) is detected. Each 96-well plate will be filled with the fractions derived from 4 venoms, equaling 80 fractions and approximately 500 different peptides in total. Therefore, the entire screen contains 2500 venom-derived peptides separated in 400 lyophilized fractions from 20 different animal venoms  solution (Sigma-Aldrich).Human colorectal cancer cell lines  and normal colon cell lines (CCD18Co and NCM-460) will be purchased from American Type Culture Collection. For cell culturing, RPMI 1640 (Sigma-Aldrich) medium will be used, supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin antibiotic cocktail (Sigma-Aldrich). The culture flasks (T25/T75) will be incubated in a 5% CO2 humidiﬁed 37 ºC incubator. The purple formazan formed will be dissolved in DMSO (Sigma-Aldrich), and the plates will be quantified by measuring the absorbance at 570 nm using a microplate reader (Hidex). The percent viability of the cells will be calculated as:High-throughput screening of 400 lyophilized venom fractions from 20 different animal species will be executed by measuring the cytotoxic activity of the fractions on CRC cell lines  and normal colon cell lines (CCD18 and NCM-460) using the [3--2,5-diphenyltetrazolium bromide (MTT) assay (Merck Millipore) using the methods depicted in ,11. Firs50 values and statistical significance will be determined using one-way analysis of variance.ICFor 3 animal venoms, fractions (containing peptides) that exhibit specific and potent cytotoxic activity on CRC cell lines will be selected and used for subsequent studies, as depicted in Aim 2. We aim to focus on peptides; therefore, during the preparation of the screen, we will eliminate the cytolytic enzymes in the venoms.The 3 venoms identified in Aim 1 will be procured in bulk from Latoxan. BAPs with CRC cell line–specific cytotoxic activity will be purified from the venoms using a combination of size exclusion, ion exchange, and semipreparatory and analytical reverse-phase high-performance liquid chromatography on an AKTA Avant multidimensional chromatography system  that is available in our laboratory. The purification strategy will be similar to that described by Banerjee et al ,13. At eUnder Aim 1, once we have screened all the venom fractions, we will analyze the hits that we have achieved. For this part of the project, we will be focusing on the hits for which cytotoxic activity is specifically observed against HT-29 cells. We will select the 3 venoms corresponding to the peptides that exhibit the most potent cytotoxic activity specifically against HT-29 cells. The remaining information corresponding to the peptides that exhibit cytotoxic activity will be used to create a biobank of active peptides, which will be the focus of later studies.Therefore, under Aim 2 of the study, we will focus on purifying BAPs from the 3 venoms that we have selected following the screening process. These BAPs will be purified to homogeneity using a combination of chromatographic techniques (as indicated above). These purified peptides will be further characterized to identify the molecular mechanism by which they mediate the anticancer activity. Because the characterization phase will require BAPs in significant quantities, we also will use fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis for bulk synthesis of the BAPs. In a later phase of the study, we will establish a bacterial expression system for the mass production of the BAPs identified and characterized in Aim 2 using the methodology depicted in .The homogeneity and molecular weight of the BAPs will be determined by electrospray ionization mass spectrometry using a PerkinElmer Life Sciences API-300 liquid chromatography/tandem mass spectrometry (MS) system at the MS facility at SGS . Typically, reverse-phase HPLC fractions will be directly used for analysis. The ion spray, orifice, and ring voltages will be standardized for analysis. Nitrogen will be used as a nebulizer and curtain gas. A LC-10AD pump (Shimadzu) will be used for solvent delivery (40% acetonitrile in 0.1% trifluoroacetic acid) at a flow rate of 50 μl/min. BioMultiview software (PerkinElmer Life Sciences) will be used to analyze and deconvolute the raw mass spectra.The purified BAPs will be reduced and pyridylethylated using procedures described previously. Briefly, BAPs (0.5 mg) will be dissolved in 500 μl of denaturant buffer . After the addition of 10 μl of β-mercaptoethanol, the mixture will be incubated under vacuum for 2 hours at 37 °C. 4-Vinylpyridine (50 μl) will be added to the mixture, and it will be maintained at room temperature for 2 hours. The pyridylethylated BAPs will be purified on a Jupiter C18 analytical column (4.6×250 mm) using a gradient of acetonitrile in 0.1% (volume/volume) trifluoroacetic acid at a flow rate of 0.5 ml/min.N-terminal sequencing of the native and S-pyridylethylated BAPs will be performed by automated Edman degradation using a pulsed liquid-phase sequencer  with an online Model 785A phenylthiohydantoin derivative analyzer (Applied Biosystems).Using a combination of different in vitro experiments, the anticancer properties of the BAPs will be characterized. The experimental protocols and the rationale for pursuing these experiments are indicated below.Cell motility is a very important parameter in determining the survival and progression of cancer. Augmented cancer cell motility is the root cause of end-stage organ damage causing mortality . The celCancer metastasis involves a slight elevation in the production of reactive oxygen species (ROS). Cancer cells appear to thrive on high levels of ROS compared to their normal counterparts, as cancer cells have developed augmented antioxidant systems . ROS gen2 humidiﬁed incubator. After 24 hours, the cells will be treated with benzopyrene and 5-fluorouracil (positive control) for 24 hours. Cells will be stained with annexin V and 7-AAD in the dark for 20 minutes according to the manufacturer’s protocols. FlowJo 10.7.1 software (BD Biosciences) will be used to analyze the data.The extent of apoptosis caused by BAP will be evaluated on a FACSAria III flow cytometer (BD Biosciences) using an annexin V and 7-aminoactinomycin D (7-AAD) apoptosis detection kit (BioLegend). Cells at a concentration of 0.3 M will be seeded in a 6-well plate and incubated in a 37 ºC 5% CO2 humidiﬁed incubator for 6 hours. Purified BAPs will be introduced to the cells, followed by incubation for 24 hours. After 24 hours, the cell culture media will be collected and centrifuged to remove the cell debris. The adherent cells will be collected after trypsinization and will be counted to normalize the cytokine concentration. The assay will be executed as per the manufacturer’s guideline on enzyme-linked immunosorbent assay (ELISA) plates (ExtraGene) precoated with human IL-6 and IL-8. The colorimetric intensity will be measured on a microplate reader (Hidex) at 450 nm.Elevated levels of vascular endothelial growth factor, IL-6, and IL-8 are proven biological markers indicating cancer progression ,19. TakiRhoC is a metastatic protein that is found to be constitutively active in many types of cancers, including CRC . We willt test in Stata (StataCorp LLC). The mean values will be reported with standard deviations. P values ≤.05 will be considered significant for differences.Statistical analyses will be accomplished using the Student This study is at the protocol development stage, and as such, no results are available. Experimental procedures in this study will be conducted in vitro and will not involve the use of animal models or samples, patient samples or data, or recruitment of human subjects. Therefore, research conducted as part of this study poses minimal risk and fits one of the exempt review categories as defined by institutional review board (IRB) regulations at Mohammed Bin Rashid University (MBRU). Further clarification and information can be obtained from the MBRU IRB at irb@mbru.ac.ae. We received funding for this study following review of our proposal. The funding ID for our research is MBRU-CM-RG2021-08. Additionally, we have initiated the groundwork for this study, which includes the purchase of CRC cell lines and preparation of the venom screen kit in collaboration with Latoxan.Animal venoms are cocktails of pharmacologically active polypeptides and proteins. Therapeutic leads from venom have been successfully developed into drugs, such as the following examples.Bothrops jararaca). Captopril is a potent hypotensive agent used to manage/treat hypertension.This drug  was deveEchis ocellatus). Tirofiban is commonly prescribed to patients recovering from heart attacks or experiencing angina.This peptidomimetic drug  was firsConus magus. Ziconotide is injected into spinal fluid to prevent pain signals from reaching the brain.Ziconotide  is a pepOur studies will identify novel BAPs with anti-CRC activity. These BAPs will act as therapeutic leads for the development of peptidomimetics; in the initial stages, we will apply a similar strategy to that depicted by Al-Amri et al  for the Androctonus bicolor, Androctonus crassicauda, and Leiurus quinquestriatus, were assessed, but the study fell short of identifying the specific BAPs that mediated the anticancer activity. Although such studies are of biochemical research interest, they are not therapeutically viable, as whole venom cannot be administered for treating patients with CRC. Our study, on the other hand, presents a rational approach in which we will first screen for anti-CRC of specific venom, followed by isolation and characterization of those BAPs that exhibit these activities with high potency. Therefore, the strategy applied in this study can also be adopted for similar studies intending to isolate and characterize BAPs from different animal venoms.Several studies are available in the literature in which the anticancer properties of animal venoms specifically targeting CRC have been investigated. However, these studies mostly focus on the anticancer properties of the whole venom rather than on the isolation and characterization of BAPs mediating the anticancer effect. For example, in the study by Al-Asmari et al , the ant


Type II testicular germ cell tumors are a severe type of cancer in young men demanding alternative treatment options to conventional chemotherapy with less side effects. In particular, patients with chemotherapy-resistant tumors face a bad prognosis and low survival rates. In other tumor entities, transcriptional cyclin-dependent kinases (7/8/9/12/13) have been demonstrated to be effective targets. Here, we studied the effects of transcriptional cyclin-dependent kinase inhibitors on a cellular and molecular level. We found several inhibitors to be highly cytotoxic for certain testicular germ cell tumor cell lines while leaving a somatic (fibroblast) control cell line unaffected. This opens up a novel field for effective and specified treatment of type II testicular germ cell tumors.Type II testicular germ cell tumors (TGCT) are the most frequently diagnosed solid malignancy in young men. Up to 15% of patients with metastatic non-seminomas show cisplatin resistance and a very poor survival rate due to lacking treatment options. Transcriptional cyclin-dependent kinases (CDK) have been shown to be effective targets in the treatment of different types of cancer. Here, we investigated the effects of the CDK inhibitors dinaciclib, flavopiridol, YKL-5-124, THZ1, NVP2, SY0351 and THZ531. An XTT viability assay revealed a strong cytotoxic impact of CDK7/12/13 inhibitor SY0351 and CDK9 inhibitor NVP2 on the TGCT wild-type cell lines  and the cisplatin-resistant cell lines . The CDK7 inhibitor YKL-5-124 showed a strong impact on 2102EP, 2102EP-R, NCCIT and NCCIT-R cell lines, leaving the MPAF control cell line mostly unaffected. FACS-based analysis revealed mild effects on the cell cycle of 2102EP and TCam2 cells after SY0351, YKL-5-124 or NVP2 treatment. Molecular analysis showed a cell-line-specific response for SY0351 and NVP2 inhibition while YKL-5-124 induced similar molecular changes in 2102EP, TCam2 and MPAF cells. Thus, after TGCT subtype determination, CDK inhibitors might be a potential alternative for optimized and individualized therapy independent of chemotherapy sensitivity. Type II testicular germ cell tumors (TGCT) are the most prevalent malignancies in young men aged 18 to 35 years . TGCTs aCyclin-dependent kinases (CDKs) are a promising target for cancer treatment. Cell-cycle-associated kinases (CDK1/2/4/6) directly regulating cell cycle progression have already been extensively studied in preclinical and clinical trials using different CDK inhibitors in TGCTs and other types of cancer ,13,14. IFlavopiridol, a pan-CDK inhibitor (CDK1/2/4/6/7/9), has been analyzed in various types of tumors . ApoptosRecent advances resulted in a toolbox of highly potent and selective inhibitors towards transcriptional CDKs, allowing for the analysis of their contribution to transcriptional regulation and, at the same time, opening new avenues for cancer treatment. In this regard, covalent inhibition of tCDKs7/12/13 by THZ1, initially launched as a CDK7 selective covalent inhibitor, has been a major breakthrough since it potently induces apoptosis across a wide variety of cancer cell lines ,26. BaseDue to the lack of selectivity of THZ1-based compounds for CDK7 targeting, the laboratory of Nathanael Gray combined a PAK4 inhibitor scaffold with the covalent THZ1 linker, resulting in a highly selective CDK7 inhibitor without cross-reactivity to CDK12/13. CDK7 inhibition by YKL-5-124 alone is not able to induce apoptosis in HAP1 but does so when combined with CDK12/13 inhibition . MoreoveNVP2 is a selective ATP-competitive CDK9 inhibitor . CDK9 inIn this study, we analyzed the effects of flavopiridol, dinaciclib, THZ1, YKL-5-124, SY0351, THZ531, NVP2 and THAL-SNS-032 on different TGCT cell lines to identify and characterize novel specific treatment opportunities for TGCTs, aiming for reduced side effects compared to platinum-based treatment and to open up a new therapeutic option for patients with cisplatin-resistant or refractory TGCTs. We found NVP2 and SY0351 to be most effective at inducing apoptosis in seminomas and EC cells applying low nanomolar concentrations, suggesting that inhibition of CDK9 and CDK7/12/13 might be an alternative treatment option for TCGT.L-glutamine. FS1 cells were grown in DMEM supplemented with 20% FSB, 1% penicillin–streptomycin, 200 mM L-glutamine and 1% nonessential amino acids (NEAA). MPAF cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin–streptomycin, 200 mM L-glutamine and 1% NEAA. TCam2 cell line was cultured in RPMI medium supplemented with 10% FBS, 1% penicillin–streptomycin and 200 mM L-glutamine. All cell lines were kept at 37 °C and 7.5% CO2. 2102EP and NCCIT were a kind gift from Prof. Dr. L. Looijenga . Cisplatin-resistant lines 2102EP-R and NCCIT-R were obtained from PD Dr. F. Honecker . MPAFs were a kind gift from PD Dr. M. Peitz . FS1 cells were obtained from Prof. Dr. V. Schumacher .The following cell lines were used 2102EP, 2102EP-R, NCCIT, NCCIT-R , TCam2 (seminoma cell line), FS1 (Sertoli cell line) and MPAF (human adult fibroblast cell line). Cells were cultivated as described previously ,39. In b2. For readout, absorption was measured at 450 nm and 650 nm. Specific XTT signal (450 nm) was subtracted by nonspecific absorbance (650) and blank (cell culture medium). Averages of technical triplicates were calculated, and treated sample was referenced to the corresponding dimethyl sufoxide (DMSO) control. Experiment was repeated several times to obtain biological replicates. For statistical analysis, two-tailed Student’s t-test was applied. Datapoints with p-value < 0.05 indicated significantly changed viability of treated compared to control group and labeled with an asterisk.The assay was performed based on the reduction in XTT tetrazolium salt as described previously . In summg at 4 °C for 15 min. Protein concentration in the clear supernatant was determined by Pierce BCA protein assay kit  according to manufacturer’s manual. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed as described previously [® Turbo™ Transfer System  for 30 min at 2.5 ampere and 25 volts. Target-specific primary and species-specific HRP-linked secondary antibodies were diluted according to Protein extraction was performed on untreated cells with RIPA buffer  supplemented with cOmplete ULTRA Tablets protease inhibitor . After sonication, the lysate was centrifuged at 13,000× eviously . In summ5 cells/well). The next day, cells were treated with a CDK inhibitor  or DMSO (0.0002%). For cell cycle analysis, cells were harvested after 20 h of treatment and resuspended in PBS. Ice-cold methanol was added dropwise while shaking until 70% methanol v/v was reached. Cells were incubated for 2 h at 4 °C and subsequently washed twice with cold PBS. Staining  was performed for 30 min at 37 °C. The cell cycle distribution was measured in an FACS Canto  and analyzed using FlowJo™ v10.8 Software . For apoptosis analysis, PE AnnexinV Apoptosis Detection Kit with 7-AAD  was used according to the manufacturer’s manual. Measurement was performed in an FACS Canto while analysis was performed in the corresponding BD FACSDiva softwareTM . Two-tailed Student’s t-test was applied for calculation of significance.Cells were seeded in 6-well cell culture plates (1.5 × 105 cells/well). Treatment was started the next day for 1 h or 24 h with 10 nM NVP2, 10 nM SY0351, 100 nM YKL-5-124, 100 nM THZ531 or DMSO. RNA was isolated using the RNeasy Mini Kit . To determine RNA integrity (RIN), Nano 6000 Assay kit with the Agilent Bioanalyzer 2100 system  was used. Only samples with RIN > 7 were used for RNA sequencing analysis. RNA quality control, library preparation ) and RNA sequencing were performed by the Core Facility Next Generation Sequencing (University of Bonn). An Illumina HiSeq 2500 V4 device  was used. Raw data quality was initially investigated using FastQC [p value < 0.05 (Benjamini–Hochberg method). The volcano plots were prepared based on the differential expression data using ggplot2 3.3.3 [Cells were seeded into 6-well cell culture plates  and one CDK degrader (THAL-SNS-032) on the viability of three different parental TGCT cell lines , two cisplatin-resistant subclone lines  and two control cell lines  via an XTT assay . The CDKThe EC cell lines  seemed to be highly sensitive to dinaciclib and flavopiridol, indicated by a strong decrease in viability at concentrations of 1 and 50 nM, respectively. While TCam2 and FS1 cells showed only a moderate sensitivity, MPAF cells were almost resistant to these drugs.Interestingly, the CDK7 inhibitor YKL-5-124 reduced cell viability in 2102EP cells selectively while all other cell lines showed lower sensitivity towards this compound. MPAF cells were especially unaffected by YKL-5-124 even at the highest concentration tested (500 nM). THZ1 and SY0351, both inhibiting CDK7, led to decreased viability while SY0351 caused higher cytotoxicity at only 10 nM compared to THZ1. THZ531 was only effective at high concentrations (100 and 500 nM).To further analyze these findings, the IC50 values were calculated for each time point, each cell line and each inhibitor , Table 2The lowest IC50 values were observed for dinaciclib treatment of tumor cell lines at 72 h between 0.8 and 4.3 nM, indicating a very high potency while TGCT cell lines appeared to be less sensitive towards flavopiridol (IC50 at 72 h between 7.5 and 273 nM). Based on these data, SY0351 (IC50 at 72 h 6.4 to 11.6 nM) has a much stronger effect on the tumor cells compared to THZ1 (IC50 at 72 h 6.5 to 847 nM). IC50 values for YKL-5-124 are low for 2102EP/2102EP-R cells (72 h 17.2 nM 2102EP and 23.6 nM 2102EP-R) and clearly higher for all other cell lines. NVP2 is also considered highly effective with IC50 values ranging from 6.1 to 16.1 nM for the tumor cell lines (72 h). The CDK9 degrader THAL-SNS-032 showed higher IC50 values . The highest IC50 values were observed for THZ531 (from 74.5 to greater than 1000 nM at 72 h for tumor cell lines). Of note, the fibroblast control cell line MPAF tolerated high levels of these CDK-inhibitors, showing IC50 values above 1000 nM independent of the inhibitor and the time. These results indicate that a treatment using CDK inhibitors might specifically impinge on germ cell tumors, leaving somatic cells unaffected.For investigation of the cellular and molecular effects, we decided to focus on the four inhibitors NVP2, SY0351, YKL-5-124 and THZ531. We determined changes in cell cycle progression by Hoechst staining using fluorescence-activated cell sorting (FACS) after 20 h of inhibitor treatment . In geneExposure to NVP2 resulted in elevated G1-phase populations in the EC cell lines  and increased the fraction of cells in the G1 and G2/M phases for the TCam2 cell line with a reduced number of cells in the S phase, accordingly. In the control cell line FS1, only a small fraction of cells was shifted to the G1 and G2/M phases. Similar effects were observed after SY0351 treatment, i.e., reduction in cells in the S phase, accumulation of cells in the G1 phase in the EC cell lines and G1- and G2/M-phase enrichment of TCam2 cells. The cell cycle of FS1 cells was hardly affected while MPAF cells showed reduced fraction of cells in the G1 phase. Treatment with YKL-5-124 resulted in reduction in 2102EP and TCam2 cells in the S phase but induced a strong increase in the S phase for NCCIT. Further, slightly elevated number of NCCIT cells were found in the G2/M phase, indicating a G2/M-phase cell cycle arrest with an overrepresentation of cells in the S phase which had not reached the G2/M phase after 20 h of treatment. For 2102EP and TCam2 cells, an elevated number of cells were observed in the G2/M phase and G1 phase, respectively. Of note, data from FS1 and MPAFs suggest cell cycle progression. THZ531 led to a highly increased number of cells in the G2/M phase for the 2102EP and NCCIT cell lines. TCam2 and MPAF cells showed hardly any changes in terms of cell cycle distribution, which might originate from their longer doubling times .Next, we measured the effect of NVP2, SY0351, YKL-5-124 and THZ531 on apoptosis by 7AAD/AnnexinV FACS analysis after 24 and 48 h inhibitor treatment . NVP2 trEGR1 or transcription factor 1 subunit JUN were found to be deregulated in TCam2 cells after 1 h of YKL-5-124 treatment , suggesting cell cycle arrest after more than 24 h of treatment  activity to be upregulated , supportRNF7, ASB6, UBE2E1, NAE1), which suggests an increase in proteasomal degradation  and apoptosis induction was upregulated . In TCam2 cells, after 1 h of SY0351 treatment, STRING interaction analysis revealed initial upregulation of apoptosis and downregulation of translation factor activity  and eukaryotic translation initiation factors , respectively. Mild effects in MPAF cells were observed only after 24 h SY0351 treatment , a response to the drug appeared after only 1 h of treatment. In 2102EP cells, functional analysis of STRING interaction networks based on mRNA sequencing data revealed upregulation of the ubiquitin conjugation pathway , TRIM protein  and PRAME  family to be upregulated. This suggests increased DNA-methylation-dependent heterochromatin assembly, ubiquitination activity and negative regulation of transcription, respectively  contributed to RNA splicing or RNA processing, which was obviously downregulated by CDK9 inhibition  in 2102EP cells 1 h after treatment, which is in accordance with the high rate of apoptosis induced by NVP2 . InteresPAN2, BTG2, PABC1 and PARN expression responsible for RNA degradation and poly(A)-specific ribonuclease activity  and further proteasome-associated transcripts . The peptide microarray analysis  and cisplatin-resistant  TGCT cell lines representing seminomas (TCam2) and embryonal carcinomas . Application of the CDK inhibitors/degrader revealed a strong decrease in viability for cisplatin-sensitive and cisplatin-resistant cell lines. 2102EP and TCam2 cells treated with YKL-5-124, SY0351 and NVP2 showed a disturbed cell cycle and apoptosis induction, indicating a potent effect of the inhibitors towards the TGCT cell lines. THZ531 caused cell cycle deregulation only in the 2102EP cells and weak apoptosis induction in 2102EP and TCam2 cells. Analysis of molecular mechanisms demonstrated cell-line-specific responses for NVP2, SY0351 and THZ531 treatment and a general response to YKL-5-124 treatment. We believe that such cell-line (and therefore tumor-entity)-specific responses open up new possibilities of precise treatment of TGCT sub-entities independent of cisplatin resistance.Interestingly, the Sertoli (FS1) and the fibroblast (MPAF) control cell lines showed strong to moderate and low sensitivity towards the applied compounds, respectively. Taking this into consideration, the CDK inhibitors/degrader might have small to severe side effects on the healthy testis tissue. However, the results suggest that there will be only very small to no effects on fibroblast cells. It has been shown that application of the CDK7/12/13 inhibitor THZ1 led to impaired spermiogenesis in mice after application to testes . HoweverTo our surprise, the molecular response to THZ531, SY0351 and NVP2 treatment varied between 2102EP, TCam2 and MPAF cell lines . For exaIn 2102EP cells, the CDK7/12/13 inhibitor SY0351 induced ubiquitin pathway upregulation and prompted stress response after 1 h of treatment, confirming the strong impact on cell viability observed in the XTT assays. Further, inhibition of CDK7 resulted in a lack of CDK1/2/4 phosphorylation, leading to G1- or G2/M-phase cell cycle arrest . Indeed,YKL-5-124 treatment (1 h) caused downregulation of RNA-Pol-II-mediated transcription and immediate early gene transcription in TCam2 cells. Olson et al. found that YKL-5-124 has only a very limited effect on RNA Pol II phosphorylation and activity . Since tGO analysis of RNA sequencing data from 2102EP, TCam2 and MPAF cells after 24 h of treatment with the CDK7-specific inhibitor YKL-5-124 revealed a similar reaction, i.e., the general downregulation of RNA splicing. CDK7 can be regarded as a master regulator of transcription, by virtue of being part of the general transcription factor TFIIH and as part of CAK activating CDKs 9/12/13 by phosphorylation . Rimel eSurprisingly, YKL-5-124 elicited moderate to strong apoptosis in the seminoma cell line TCam2 and in EC cell lines , respectively. In other studies, it has been shown that CDK7 inhibition alone leads to cell cycle arrest but does not induce apoptosis ,32. AccoThe role of CDK9 activity was investigated by CDK9 inhibition (NVP2) and CDK9 degradation (THAL-SNS-032). Although NVP2 and THAL-SNS-032 are characterized by a different mode of action, both compounds revealed highly decreased viability in TGCT cell lines, underlining the relevance of CDK9 for cellular survival. NVP2 treatment  seemed to induce a prompt stress response of 2102EP and TCam2 cells indicated by upregulation of the cell cycle inhibitor p21 and downregulation of RNA-Pol-II-mediated transcription and immediate early gene transcription . Lower RNA-Pol-II-mediated expression of immediate early genes is an expected finding after CDK9 inhibition, which is the catalytic subunit of P-TEFb necessary for transcription elongation . P21 medAfter 24 h of NVP2 treatment, 2102EP cells showed upregulation of PRAME, TRIM and MBD3L family members. MBD3L2/3/5 are responsible for introducing epigenetic changes by DNA-methylation-dependent heterochromatin formation. PRAME family members in combination with MBD3L2/3 and 5 might induce negative regulation of transcription. TRIM proteins are considered to be involved in modulation of ubiquitin protein ligase activity, indicating upregulated proteasomal degradation. However, it remains elusive how the NVP2-mediated inhibition of CDK9, resulting in alteration of TRIM, PRAME and MBD3L2, 3, 5 levels, contributes to apoptosis induction.Interestingly, 1 h of NVP2 treatment of TCam2 and 2102EP cells led to upregulation of CDK15 (PFTAIRE2) phosphorylation activity. RNA sequencing analysis revealed significantly increased corresponding cyclin Y expression in 2102EP cells. Park et al. suggested that CDK15 acts as an antagonist of TNF-related apoptosis-inducing ligands . FurtherTo sum up the effects caused by NVP2 treatment, we observed cell cycle deregulation and strong apoptosis for TCam2 and 2102EP cells induced after downregulation of RNA splicing and deregulation of chromatin organization, respectively.In this study, we investigated the impact and molecular effects of different CDK inhibitors on TGCTs. SY0351 and NVP2 appear to be candidates for a broad application in TGCT treatment since we observed high potency of both compounds towards 2102EP (EC) and TCam2 (seminoma) cell lines. YKL-5-124 turned out to be a highly selective treatment option because the strongest effects were observed for 2102EP cells. Importantly, SY0351, NVP2 and YKL-5-124 displayed a similar cytotoxic effect in wild-type and cisplatin-resistant cell lines, supporting an application which is independent of cisplatin resistance. Unique responsiveness of different cell lines representing different tumor entities shows varying effects on cellular and molecular levels todifferent CDK inhibitors. Thus, exact determination and characterization of tumor composition  and molecular features are crucial for personalized, precise and effective therapy. All in all, CDK inhibitors show a great potential for an alternative and individualized treatment strategy for TGCTs.


DNA damage activates cell cycle checkpoint proteins ATR and CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of ATR (ATRi) and CHK1 (CHK1i) enhance damage-induced cell death. Intriguingly, both CHK1i and ATRi alone elicit cytotoxicity in some cell lines. Sensitivity has been attributed to endogenous replications stress, but many more cell lines are sensitive to ATRi than CHK1i. Endogenous activation of the DNA damage response also did not correlate with drug sensitivity. Sensitivity correlated with the appearance of γH2AX, a marker of DNA damage, but without phosphorylation of mitotic markers, contradicting suggestions that the damage is due to premature mitosis. Sensitivity to ATRi has been associated with ATM mutations, but dysfunction in ATM signaling did not correlate with sensitivity. CHK1i and ATRi circumvent replication stress by reactivating stalled replicons, a process requiring a low threshold activity of CDK2. In contrast, γH2AX induced by single agent ATRi and CHK1i requires a high threshold activity CDK2. Hence, phosphorylation of different CDK2 substrates is required for cytotoxicity induced by replication stress plus ATRi/CHK1i as compared to their single agent activity. In summary, sensitivity to ATRi and CHK1i as single agents is elicited by premature hyper-activation of CDK2. The underlying mechanism of genomic instability  can provide an Achilles heel through which novel and selective therapeutic strategies can be designed. Inhibitors of poly(ADP-ribose) polymerase (PARP) have pioneered this chemical-genetic synthetic lethal approach because they selectively inhibit tumors deficient in BRCA1, BRCA2 or other proteins involved in homologous recombination repair2 phase. CHK1 inhibitors (CHK1i) were initially developed as a means to circumvent this arrest, force premature cell cycle progression and enhance the cytotoxicity of DNA damaging therapies4.DNA damage elicits two parallel and interacting pathways to elicit cell cycle arrest and repair. Stalled replication forks generate single-strand regions of DNA (ssDNA) that activate ATR and subsequently CHK1. DNA double-strand breaks (DSB) recruit the MRN complex , which in turn recruits and activates ATM and subsequently CHK2. Mre11 possesses a nuclease activity that contributes to the generation of single-strand regions at DSB which in turn recruit and activate ATR/CHK1. The consequence of activating CHK1/2 is that they phosphorylate and inhibit CDC25 phosphatases thereby preventing activation of CDK1/2 resulting in cell cycle arrest at either S or G5. MK-8776 still inhibited CHK1 in the resistant cells, but they could tolerate this inhibition for at least 7 days while continuing to proliferate. If 15% of human tumors are also hypersensitive to CHK1i, this likely represents a chemical-genetic synthetic lethal interaction that can be therapeutically targeted. Identification of the defects that sensitize tumor cells to CHK1i would facilitate patient stratification and provide precision medicine for patients with such tumors.Intriguingly, some cell lines are very sensitive to CHK1i as monotherapy. We recently published a study of the response of 65 human cell lines to the CHK1 inhibitor MK-8776; about 15% of these cell lines are hypersensitive to MK-8776, resulting in ssDNA and DSB that occur after only a short incubation time (6 h)5. Furthermore, this sensitivity is dependent on cyclin A rather than cyclin E5. In an attempt to further understand the mechanism of sensitivity to CHK1i, we have now performed a comparative analysis of the sensitivity to inhibition of the upstream regulator of CHK1, namely ATR. It has been proposed that sensitivity to both CHK1i and ATR inhibitors (ATRi) depends on endogenous replicative stress10, yet this is inconsistent with the very different pattern of sensitivity we observe for these two drugs. Replication stress has been defined as slowing or stalling of replication fork progression that usually results in stretches of ssDNA, and is often observed as constitutive activation of the DNA damage response11. Here, we survey our panel of cell lines for constitutive activation of the DNA damage response and find this also does not correlate with sensitivity to either CHK1i or ATRi. Loss of function of ATM has often been associated with sensitivity to ATRi15, and we find this to be true in an isogenic background. However, many ATM mutations do not appear to generate ATM dysfunction, while some cell lines are sensitive to ATRi with no known defect in the ATM pathway. Of particular note, cells with low levels of ATM may still be fully functional for ATM signaling, and therefore assessment of ATM levels may mislead attempts to stratify patients for those who may respond to treatment with an ATRi. Finally, we establish that sensitivity to ATRi, like sensitivity to CHK1i, depends on hyperactivation of CDK2.We recently demonstrated that sensitivity to CHK1i resulted from aberrant hyper-activation of CDK2 in early S phase, followed by the phosphorylation of H2AX (γH2AX)5, this study began with a parallel assessment of the sensitivity of the cells to the ATRi AZD6738. Three different exposure times with drug were used: 24 h, 48 h or 7 days. After the shorter incubations, drug was removed and cells incubated in complete medium until almost confluent . The different incubation times provide information on the exposure time required for growth inhibition, and the potential for recovery following brief drug treatment. The short drug exposure times are also more relevant to the administration schedule used in patients. The concentration that inhibited growth by 50% (GI50) was then calculated and compared to the sensitivity to CHK1i . This lack of correlation between γH2AX and pHH3 contradicts the prior suggestion that premature mitosis induced by either CHK1i or ATRi leads to γH2AX18.It is apparent that many more cell lines are sensitive to ATRi than CHK1i  that only appeared to arrest in G2 suggesting a checkpoint defect, but CAKI1 and H322M were fully competent for S phase arrest19. These data provide interesting cell lines for study of the impact of specific defects in DNA damage response, but there is no correlation with response to CHK1i or ATRi.The concurrent analysis of SN38-induced phosphorylation suggested that some cell lines are defective for eliciting a DNA damage response (dark blue). The most obvious in this regard is TK10, while CAKI1 and H322M also exhibited limited response. One potential cause for this apparent defect is a lack of induction of DNA damage by SN38 in these cell lines, albeit we have previously reported that all these cell lines arrest in G2 at or below the concentration of SN38 used here, and most arrest in S at this concentration14. In Fig. Dysfunctional ATM has frequently been reported to explain sensitivity to ATRi, and cells with mutant ATM are thought to have low levels of ATMTo generate a complete ATM knockout, we engineered a second CRISPR guide targeting exon 6 and transfected it into the 231 ATM(1) cells. This resulted in a clone termed 231 ATM(0) that expressed no detectable ATM protein. Sequencing of exon 6 revealed 4 unique alleles, three of which contained insertions or deletions resulting in frameshift mutations, and one which contained an in-frame insertion  cells with partial deletion of ATM were no more sensitive to ATRi  are relatively insensitive to ATRi, but major limitations of this analysis are that (a) some mutations are heterozygous and may not be dominant; (b) not all ATM mutations may be dysfunctional; and (c) there may be other defects in the ATM pathway that elicit sensitivity to ATRi. We therefore assayed for ATM function across these cell lines.21, and this is consistent with the role of ATM in regulating removal of potentially lethal DNA-bound topoisomerase I complexes22. Cells deficient for ATM are expected to be sensitive to the topoisomerase I inhibitor SN38, and show no additional sensitivity when ATM is inhibited. This effect was observed when we compared MDA-MB-231 cells that were competent or deleted for ATM , however 90% is reportedly mutated at D1853N. These cells were very sensitive to SN38 with no further sensitization by ATMi. The D1853N mutation occurred in several other cell lines, but was always heterozygous and did not elicit ATM dysfunction. Hence, D1853N appears dysfunctional only if homozygous.19, and it may be this latter modification that renders them sensitive to SN38 and only slightly more sensitive to the addition of ATMi.HCT116 (resistant to ATRi) and U251 cells (intermediate sensitivity) are also reported to have a heterozygous D1853N mutation, and we have previously reported that both have very low MRE11 proteinNCI H23 cells are homozygous at Q1919P (significant Polyphen score), sensitive to SN38 and only marginally impacted by ATMi. They are also very sensitive to ATRi.TK10 cells (sensitive to ATRi) are homozygous for P1054R mutation (significant SIFT and Poyphen scores), but also have the lowest level of ATM mRNA. While they are relatively resistant to SN38, they are insensitive to addition of ATMi. This suggests this mutation is dysfunctional.NCI H522 (fairly resistant to ATRi) have the same P1054R mutation but only at 27%, yet are very sensitive to SN38 and insensitive to ATMi, suggesting this mutation may be dominant. However, UACC62 cells (resistant to ATRi) express this mutation at 70% but are only slightly sensitive to SN38 and are still sensitive to ATMi. Hence, it appears that the lack of impact of ATMi in both H522 and TK10 cells more likely reflects an alternate defect in the ATM pathway.OVCAR8 (resistant to ATRi) have two heterozygous mutations in ATM (V613L and D1853N) and are very sensitive to SN38 and almost insensitive to ATMi. Whether one of these alleles is dominant or they are both dysfunctional but in different alleles is unknown.IGROV (resistant to ATRi) have a R248Q mutation with significant SIFT and Polyphen scores but the mutation is heterozygous and the cells are not very sensitive to SN38 and they are sensitized by ATMi.23; however, these cells were still relatively resistant to ATRi.Several other cell lines were also insensitive to ATMi, but with no know mutation in ATM, suggesting alternate mutations in this damage response pathway may exist. This was true of CAPAN1 cells that are defective for BRCA2, sensitive to SN38 and unaffected by ATMi. OVCAR4 were also very sensitive to SN38 and insensitive to ATMi but despite the lack of ATM, they exhibit no known mutations in these pathwaysWe used this approach to assess the possible dysfunction of ATM in many of our cell lines and compared it to the cytotoxicity Fig.  and prot25. Our data clearly indicate that this approach is not justified, as cell lines that have dysfunctional mutations have highly variable levels of ATM protein. Further, when we investigated potential ATM dysfunction in 5 of the lowest ATM expressors, we found that several appeared fully functional for ATM  to assess ATM levels with the expectation that patients whose tumor has low ATM would respond to ATRi. In this regard, it has been variously reported that 9% or 41% of non-small cell lung cancers (NSCLC) are negative for ATMtry Fig. A. While We also scored 21 human NSCLC tumors and found variable response Fig. B. Five t27. At higher concentrations, CVT313 also prevents CHK1i-mediated S phase progression, γH2AX and replication catastrophe following incubation with DNA damaging agents27. This has led to the conclusion that different threshold activities of CDK2 are required for single agent activity versus its use in drug combinations4. These threshold activities likely depend on the particular cyclin partner, as suppression of cyclin A, but not cyclin E, protects cells from CHK1i5.The sensitivity of cells to CHK1i as a single agent can be prevented by addition of low concentrations of the CDK2 inhibitor CVT313. This was seen both as inhibition of γH2AX and recovery from growth inhibition and cell death5.Here, we confirm that low concentrations of CVT313 1.2–2.5 µM) also prevent γH2AX induced by ATRi in AsPC-1 cells Fig. A. Low co µM also 27. Replication stress also involves stalled replication forks that require a low threshold activity of CDK2 to restart replication (inhibited by high concentrations of CVT313) whereas the single agent activity of ATRi, like CHK1i, depends on high threshold activity of CDK2 (inhibited by low concentrations of CVT313). These observations further support our conclusion that endogenous replication stress does not explain the sensitivity of cells to either CHK1i or ATRi as single agents.In parallel, we compared the concentrations of CVT313 that prevented S phase progression when ATRi is added to damaged cells. This was performed in resistant cell lines as the DNA damage induced by single agent activity of ATRi exacerbates the S phase progression in sensitive cells. As discussed above Fig. B. Simila10. Oncogene-induced stress is also frequently implicated in sensitivity to these drugs, yet this also involves endogenous replication stress29. However, while replication stress is a consequence of inhibition of CHK1 or ATR, our results do not support the contention that endogenous replication stress is a pre-requisite for sensitivity to checkpoint inhibitors. First, we established that there exists a very different profile of sensitivity to CHK1i and ATRi, hence replication stress cannot be the explanation for sensitivity to both drugs. Second, DNA damaging agents such as hydroxyurea or gemcitabine that induce replication stress are further sensitized by CHK1i or ATRi, and it has been assumed that this is the same mechanism for the single agent activity of CHK1i and ATRi. When used in combination with hydroxyurea or gemcitabine, sensitivity to CHK1i or ATRi results from reactivation of stalled replication forks, a process that requires only a low activity threshold of CDK226. In this drug combination, the addition of CHK1i does not induce S phase progression because of the lack of dNTPs as a result of the inhibition of ribonucleotide reductase. However, when cells are arrested in S phase with the topoisomerase inhibitor SN38, the addition of CHK1i does induce S phase progression, and this also requires only a low level activity of CDK227. We demonstrated a similar phenomenon here whereby ATRi-induced S phase progression after DNA damage by SN38 is inhibited only at high concentrations of CDK2i . In contrast, we previously established that the single agent activity of CHK1i is inhibited by low concentrations of CDK2i (i.e. high activity CDK2)26, and that is reiterated here for single agent activity of ATRi. In addition, we confirm that suppression of cyclin A protects cells from γH2AX induced by ATRi in sensitive cells, as previously reported for CHK1i5. Hence, the mechanism of single agent activity for both CHK1i and ATRi differs from the combination with hydroxyurea or SN38, and consequently does not require replication stress.Variable sensitivity of cell lines to both CHK1i and ATRi has been frequently reported, and this has led to investigations into the underlying mechanisms with the goal of stratifying cancer patients most likely to respond to these drugs. Probably, the most frequently implicated reason for this sensitivity is the presence of endogenous replication stress18, yet if this were the case, there would be a strong correlation between the appearance of γH2AX and pHH3. In all the experiments reported here, we observed pHH3 only in the G2/M phase of the cell cycle, and it was not associated with the cells positive for γH2AX which were primarily in S phase. Premature mitosis can result when CDK1 is activated in S phase. That this did not occur concurrent with γH2AX is consistent with our observations that CDK2 is preferentially activated when cells are incubated with CHK1i27. Premature mitosis may be more relevant to incubation with a WEE1 inhibitor which may prematurely activate CDK1 in S phase cells18.It has also been reported that sensitivity to both drugs results in premature mitosis16 leading to the assumption in subsequent papers that inhibition of CHK1 is more deleterious to cycling cells than inhibition of ATR31. This conclusion derived from an experiment in U2OS that compared only a single concentration of each drug, and a failure to realize that U2OS cells are one of few cell lines that are very sensitive to CHK1i as a single agent. Furthermore, UCN-01 has numerous off-target effects that might impact γH2AX. From our analysis of 65 cell lines, we confirm that U2OS cells are very sensitive to CHK1i (MK-8776), and only slightly less sensitive to ATRi (AZD6738)  and H23 cells that lack functional ATM and are very sensitive to ATRi  rather than ATRi12. In summary, some defects may elicit sensitivity in rare tumors, but no single defect appears to predict the sensitivity or resistance to ATRi or CHK1i in a significant number of tumors, so the current knowledge is of little value in stratifying patients for therapy. In contrast, we have demonstrated the importance of aberrant hyper-activation of CDK2 in eliciting sensitivity to both drugs and suggest that resolving the underlying mechanism may provide a better predictive prognostic marker.Many other gene dysfunctions have been implicated in sensitivity to ATRi, using isogenic cell lines or genome-wide screensThe majority of cell lines were obtained from the Developmental Therapeutics Program of the National Cancer Institute as part of the NCI60 cell line panel. Other cell lines were obtained from the American Type Culture Collection. Cells were expanded and stored at low passage number and experimental cultures were replaced approximately every 3 months. Cells were maintained in RPMI1640 (Corning/Mediatech) plus 10% fetal bovine serum , and 1% antibiotic/antimycotic . Cell lines were confirmed negative for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza).MK-8776 , AZD6738  and CVT313 (Sigma) were stored as a 10 mM solution in DMSO. No apparent change in potency of the stock solutions was observed over the course of this study.37. Fluorescence was read on a microplate spectrofluorometer. Results were expressed as the concentration that inhibited growth by 50% (GI50). The results for MK-8776 are derived from our prior publication with a few additions and deletions5. While it was impractical to repeat this screen in all cell lines, those selected for further analysis were reconfirmed in replicate experiments. As the starting cell number in this assay is very low, growth inhibition of 100% cannot discriminate between no growth (cytostasis) versus cell death. To assess cell death, cells were plated at 10,000 cells/well and harvested every 2 days for 8 days. This facilitated assessment of the decrease in cell number below the starting inoculum which equates to cell death38.Cells were plated at 500–2000/cells per 100 µl in each well of a 96-well plate (depending on growth rate). The following day, drugs were added at twofold dilutions (8 wells per concentration). After 24 or 48 h, drug was removed, wells washed with phosphate buffered saline (PBS), and fresh media added. Alternately, drugs were left on the cells until harvest. Cells were harvested on day 6 or 7 when approaching confluence. Cells were lysed and DNA was stained with Hoechst 3325839. As required, cells were also labeled concurrently with Alexa 488-conjugated γH2AX and Alexa 647-conjugated pHH3 . Cells were analyzed on a Becton Dickinson Gallios flow cytometer. Data was analyzed using FlowLogic. All experiments were repeated in multiple cell lines to confirm that any particular phenotype was not unique to one cell line.Cell cycle analysis was conducted by flow cytometry using propidium iodide as described previouslyCyclin A siRNA was Dharmacon On-Target Plus SMARTpool CCNA2 (L-003205-00-0005); cyclin E siRNA was Ambion Silencer Select CCNE1 (s2526); and the control siRNA was Ambion Silencer Select Negative Control #1 (4390843). Cells were transfected with 25 pmol siRNA using Invitrogen Lipofectamine RNAiMax transfection reagent according to manufacturer’s protocol. After 24 h, media was replaced and cells incubated in complete media with or without 3 µM AZD6738 for 24 h.Cells were rinsed in PBS, lysed in Laemmli lysis buffer, and boiled for 5 min. Protein levels in the lysates were determined by Pierce 660 nm Protein Assay Reagent with added Ionic Detergent Compatibility Reagent, and 40 µg was added to each lane, separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were sectioned for appropriate regions such that multiple antigens of different sizes could be analyzed from a single blot. Western blotting was performed with the following primary antibodies: Cell Signaling Technology: pS1981-ATM (13050S), pT1989-ATR (58014S), pS345-CHK1 (2348S), pT68-CHK2 (2197S), γH2AX (9718S), PARP (9532S). Santa Cruz Biotechnology: CHK1 (sc8408), ATM (sc135663), ATR (sc15173). EMD Millipore: CHK2 (05-649). The following secondary antibodies were obtained from Cell Signaling technology mouse IgG-DyLight 800 (5257), rabbit IgG-DyLight 800 (5151), mouse IgG-DyLight 680 (5470). Fluorescent images of the desired region were captured using a Licor Odyssey imager and processed using Image Studio Lite. This approach provides a wide dynamic range that is directly quantified by the fluorescent scanner. The entire image captured is presented in each figure. PARP was used as a loading control while simultaneously confirming that apoptosis had not occurred. Given the large number of western blots in this screen, it was prohibitive to replicate all of them, but in cases where some unusual event was observed such as defective DNA damage signaling, no CHK2, no expression or over-expression of ATM, a new sample was analyzed to confirm the observation.https://media.addgene.org/cms/filer_public/4f/ab/4fabc269-56e2-4ba5-92bd-09dc89c1e862/zhang_lenticrisprv2_and_lentiguide_oligo_cloning_protocol_1.pdf), ATM EX3 sgRNAs were inserted into the lentiCRISPRv2 puro vector (Addgene #98290), while ATM EX6 sgRNAs were inserted into the lentiCRISPRv2 blast vector (Addgene #98293).Optimal single guide RNAs (sgRNAs) targeting ATM exons 3 and 6 were selected using the online tool Benchling and obtained from Integrated DNA Technologies , and 2 µg pMD2.G envelope plasmid (Addgene #12259). The following day, medium was replaced with IMDM containing 2% serum and 2 mM caffeine. After 24 h, viral supernatant was harvested, filtered, and stored at − 80 °C.Twenty-four h prior to transduction, MDA-MB-231 cells were seeded at 50% confluency in complete RPMI1640. Transduction was performed by replacing medium with 5 mL complete RPMI1640, 5 mL lentiCRISPRv2 puro ATMEX3 viral supernatant, and 1:1000 polybrene. Viral medium was replaced with complete RPMI1640 after 24 h. Cells were allowed to recover for 6 days, then selected for antibiotic resistance for 4 days in complete RPMI1640 supplemented with 2 µg/mL puromycin. Clonal cell lines were obtained using glass cloning rings. CRISPR efficacy was determined via Sanger sequencing of ATM exon 3 and western blotting for total ATM protein. A clone harboring one apparent functional copy of ATM exon 3 was maintained as the 231 ATM(1) cell line.The 231 ATM(1) cells were further transduced with lentiCRISPRv2 blast ATMEX6 viral supernatant as described above and selected for 10 days in complete RPMI1640 with 10 µg/mL blasticidin. Clones were isolated as described above, and CRISPR efficacy was determined via Sanger sequencing of ATM exon 6 and western blotting for total protein. A clone expressing no detectable ATM protein was maintained as the 231 ATM(0) cell line.-/- mice were obtained from the Norris Cotton Cancer Center Mouse Modeling Shared Resource. Six mice  were injected subcutaneously with 5 × 106 MDA-MB-231, 231 ATM(1), or 231 ATM(0) cells. Tumor dimensions were measured twice weekly using calipers (volume = [length2 × width]/2). When tumors reached ~ 400 mm3, they were harvested, fixed in formalin, and embedded in paraffin.Animal studies were reviewed and approved by the Dartmouth College Animal Care and Use Committee (IACUC) and conducted in accordance with all applicable federal regulatory requirements, and in compliance with the recommended ARRIVE guidelines. Female NOD-scid/IL2RgHuman non-small cell lung cancer specimens were obtained from archived tumor samples in the Research Pathology biospecimen bank, and in accordance with a protocol approved by the Dartmouth College Institutional Review Board. The tumor DNA had been analyzed by partial exon sequencing as part of standard molecular genotyping, albeit only 16 of 66 exons of ATM had been sequenced.Tumor sections were cut at 4 µm, mounted on slides, and air dried at room temperature for at least 30 min. The slides were loaded on to a Leica Bond automated immunostainer, baked, dewaxed and incubated with BOND epitope retrieval solution 2  for 20 min at 100 °C. Primary antibody binding used the Bond Polymer Refine Detection Kit (DS9800) and anti-ATM antibody (Abcam: ab32420), and incubated for 30 min at 1:150 dilution (0.8 µg/mL). Primary antibody binding was visualized using Leica Bond Refine Detection kit with DAB chromogen and hematoxylin counterstain.The large cell line screens presented here were performed to identify differences in drug sensitivity or protein expression. For the candidate cell lines selected for further study, their sensitivity or resistance was confirmed. Similarly, outlier responses in the DNA damage response pathway were also confirmed by replicate western blots. The results of the protein analyses were further compared statistically for correlations. GraphPad Prism 8 was used to generate box-and-whisker plots of SN38-induced fold change of each antigen in ASPC-1 cells (n = 18). Whiskers represent minimum and maximum values, boxes represent 25th–75th percentiles, and lines represent the median for each antigen Fig. B. For SuSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.