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.


Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB.Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET  protein inhibitors , and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses.Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy.Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.The online version contains supplementary material available at 10.1186/s12885-021-08782-9. MYC (MYCN) oncogene, which occurs in 20–30% of all NB tumors and nearly 50% of the high- risk cases, remains a key predictor of poor outcomes. MYCN-amplified NB tumors typically exhibit high malignancy, metastatic properties, and treatment resistance [Neuroblastoma (NB) is the most common extracranial pediatric solid tumor of neural crest origin and accounts for approximately 10% of childhood cancers and 15% of cancer-related deaths in children. Approximately 50% of NB patients are diagnosed with high-risk disease, and despite intensive multimodal therapy options , effective treatment for these patients remains elusive . Particusistance . TherefoOne of the most frequently deregulated oncogenic pathways in cancers, is the protein synthesis (translation) pathway that drives increased cell proliferation and cancer progression/resistance , 6. SimimTOR signaling is another key regulator of protein synthesis, which is frequently deregulated in cancers including NB –20. MTORWe hypothesize that combined inhibition of transcription (by BET-protein inhibition) and translation (by mTOR inhibition) will synergistically blockade global protein synthesis and proliferation in MYC-driven NB tumor cells. Using small molecule/pharmacologic approaches, we tested this hypothesis by targeting BET with JQ1 or OTX-015 and mTOR with temsirolimus, in NB cell lines.Non-MYCN-amplified NB cell lines  and MYCN-amplified NB cell lines  were purchased from American Type Culture Collection (USA). Non-MYCN-amplified NB cell line CHLA-255 was provided by Dr. Kishore Challagundla (UNMC). The identity of cell lines was confirmed by their respective cell bank using STR analyses. Cell lines were also verified for mycoplasma-free condition using the MycoSensor-PCR assay kit . Cell lines were cultured in Eagle’s Minimal Essential Medium (EMEM) or Roswell Park Memorial Institute (RPMI)-1640 media containing 10% fetal bovine serum and 1% penicillin-streptomycin . Experiments were performed under 8–10 passages for each cell line. Small molecule inhibitors  and cisplatin (a chemotherapeutic drug) were purchased from Sellekchem LLC (USA).Effects of inhibitors on NB Cell growth/viability were assessed using the MTT assay as previously described , 23.Effects of inhibitors on NB spheres were performed using the neurosphere/sphere assay as previously described .Analysis of cell cycle distribution in NB cells was performed using a propidium iodide (PI staining) flow cytometry kit  according to manufacturer’s instructions. Apoptosis in NB cells was assessed using an Annexin-V flow-cytometry assay kit  following the manufacturer’s instructions. The flow cytometry analysis of Annexin-V/PI stained cells was determined using the FACSCalibur cell sorter system .4 cells/well) for 24 h. After treatment, culture media was replaced with fresh media containing O-propargyl-puromycin (OPP) and incubated for 30 min at 37 °C to be incorporated in translating polypeptide chains. Cells were then fluorescently stained with 5-FAM-Azide. The detection of fluorescent-labelled OPP was performed using the Protein Synthesis Assay Kit , according to the manufacturer’s instructions.NB cells were treated with inhibitors alone or in combination in 96-well plates  was analyzed using two-tailed Student’s t-test or analysis of variance (ANOVA) and p-values > 0.05 considered significant. GraphPad Prism-V6 software was used to determine IC50 values and dose-response curves of inhibitors in NB cell lines. The Chou-Talalay combination index (CI) method was used to analyze synergy/interaction between inhibitors by using CalcuSyn software . CI < 0.9 indicates synergism, 0.9–1.1 additivity and > 1.1 antagonism.Each experiment was repeated at last an additional three times and the mean ± standard error values calculated. Statistical significance  to target MYCN transcription (BET proteins) and mTOR signaling, respectively, , 25 in NWe next tested the efficacy of combined JQ1/TEM to explore potential for synergistic growth inhibition on NB cells. NB cell lines, including three MYCN-amplified and two non-MYCN-amplified NB cell lines, were treated with increasing concentrations of JQ1 and TEM alone or in combination for 72 h. As shown in Fig. To determine combined effects of JQ1 and TEM on cell cycle and apoptosis, two highly MYCN-amplified NB SK-N-BE2, SK-N-DZ) cell lines were treated with a suboptimal concentration of each inhibitor alone or in combination and subjected to cell cycle and apoptosis analyses using propidium-iodide and Annexin-V staining, respectively, followed by flow cytometry. The cell cycle analyses in both MYCN-amplified cell lines revealed that JQ1 and TEM alone slightly caused cell cycle arrest in G1 phase, while co-treatment with JQ1 and TEM drastically arrested the cells in G1 phase . Co-treatment with JQ1 and TEM further downregulated the expression of the above mentioned mTOR signaling components, as well as MYCN expression, compared with single agent treatments. These data suggest that concurrent inhibition of MYCN transcription and mTOR signaling cooperatively suppresses the protein synthesis pathway, justifying why this combined inhibition exerts the greatest antitumor effects in MYCN-amplified NB.To establish the molecular mechanism(s) associated with JQ1/TEM anti-NB activity, we examined the expression/activation of key components of MYCN and mTOR signaling pathways by western blotting in SK-N-BE2 and SK-N-DZ cell lines. Single agent treatments of MYCN-amplified cells with JQ1 or TEM significantly suppressed expression of MYCN and BET protein BRD4 and downregulated the levels of phosphorylated/activated signaling proteins  of the mTOR (translation) pathway Fig. a and b. Because of the key role of MYCN/mTOR signaling in controlling global protein synthesis, we further tested whether the inhibition of MYCN/mTOR represented a global blockage of protein synthesis. To this end, we performed protein synthesis assay using a robust chemical method based on a cell permeable analogue of puromycin, O-Propargyl-puromycin (OPP), in SK-N-BE2 and SK-N-DZ cell lines treated with JQ1 and TEM alone or in combination. Incorporation of OPP to nascent polypeptide chains can be labeled via copper catalyzed click-chemistry using 5-FAMAzide in order to quantify total protein synthesis. In this assay, we used cycloheximide as the positive control for protein synthesis inhibition. This fluorescence-based assay displayed high protein synthesis in control solvent (DMSO)-treated cells and strong inhibition of protein synthesis when blocked with cycloheximide Fig. c. AlthouWe next investigated the effect of JQ1 and TEM, alone or combined, in a neurosphere model of MYCN-amplified NB cells. Figure Given the limited success of current therapies, we next sought to determine whether JQ1 or TEM could enhance the anti-NB efficacy of chemotherapy by sensitizing NB cells. Cisplatin is one of the most common chemotherapeutic drugs in the treatment of NB patients. To evaluate the enhanced efficacy of inhibitors on cisplatin-mediated NB cytotoxicity, we treated NB  cell lines with cisplatin and either JQ1, TEM, or the combination cisplatin alone or in combination, in stepwise doses. At 72 h we determined cell growth using the MTT assay. Results shown in Fig. In previous experiments, the rationale for using JQ1 was its advantages over other BET inhibitors in preclinical cancer studies. Preclinical studies with JQ1 offered a great opportunity to better understand the biology of BET proteins and validate these proteins as the anti-cancer targets , 24. In Despite the availability of intensive multimodal therapy, the prognosis for patients with MYCN-amplified NB remains extremely poor . Since MBET protein inhibitors exert biological effects by competitively binding to the acetyl-lysine pockets and displacing BET proteins from chromatin, leading to the repression of oncogene transcription. BET protein inhibitors have been shown to target MYC/MYCN transcription in several cancers, including NB, demonstrating their potential as preclinical anticancer agents –17. In cIn addition, our data provide insights into the molecular mechanism(s) underlying the antitumor efficacy of JQ1/TEM we observed in NB. We show that co-treatment with JQ1 and TEM synergistically reduce the expression of key component proteins of the activated mTOR translation pathway, along with suppression of MYCN and BRD4 (a key member of BET family proteins) protein expression in NB cells. We further show that this co-inhibition of MYCN/mTOR by JQ1/TEM results in significant blockade of global protein synthesis in NB cells. These observations in NB are consistent with our previous study in medulloblastoma where we have shown similar combined activity of JQ1/TEM on MYC/mTOR targets, leading to inhibition of global protein synthesis. Overall, these findings suggest a synergistic and cooperative activity of JQ1/TEM on MYCN and mTOR pathways in aggressive NB.While our data show that although individual or concomitant inhibition of BET/mTOR has greater antitumor efficacies against MYCN-amplified NB cells, the combination also efficiently inhibits cell growth and induces apoptosis in non-MYCN-amplified NB cells, pointing to a broader relevance of our combined approach in NB. Because non-MYCN-amplified NB cell lines, including SK-N-AS, express MYC  which isIn NB and other cancers, MYCN and mTOR signaling have been shown to play key roles in cancer stem cells and contribute to relapse and drug-resistance. NB cells express neural stem cell markers such as CD133 and Nestin and have the ability to form neurospheres –33. Our Since overexpression or amplification of MYC/MYCN and activation of mTOR signaling are often associated with chemoresistance in many cancers including NB –38, we aIn clinical trials studies, TEM alone or in combination with other clinical agents, such as chemotherapeutic drugs temzilomide and irrinotican, has shown no significant benefit for relapsed NB patients , 43. ThiOur study demonstrates that targeting dysregulated protein synthesis pathway by pharmacologic dual-inhibition of MYCN transcription (by BET protein inhibition) and mTOR signaling has significant preclinical anti-NB efficacies in inducing cell growth inhibition, cell cycle arrest and apoptosis in vitro. Combination of JQ1 or OTX with TEM synergistically inhibited global protein synthesis by downregulating the key components and downstream targets of MYCN/mTOR signaling. Thus, this study is the first to demonstrate synergy in the combined inhibition of BET protein and mTOR signaling in NB. Our study also revealed that inhibition of MYCN/mTOR not only inhibits cell growth and survival in NB cells, but also chemosensitizes NB cells. Beyond NB, this therapeutic approach can be of broader relevance for therapy of MYC/MYCN-addicted cancers, as we have previously shown a synergistic antitumor efficacy of BET/mTOR inhibitors in MYC-driven medulloblastoma . While fAdditional file 1.