Furthermore, copper complexes of DDC, Pyr and 8-HQ had greater therapeutic activity compared to the copper-free ligands in all cell lines; whereas the copper-dependent activities of Plum and CQ were cell-line specific. Four of the copper complexes (Cu(DDC)2, Cu(Pyr)2, Cu(Plum)2 and Cu(8-HQ)2) showed IC50 values less than that of cisplatin in all tested cell lines. The complex copper DDC (Cu(DDC)2) was selected for in vivo evaluation due to its low nano-molar range activity in vitro and the availability of an injectable liposomal formulation. Liposomal (Cu(DDC)2) was tested in a fast-growing platinum-resistant A2780-CP ovarian xenograft model and was found to achieve a statistically significant reduction  in tumour size. This work supports the potential use of copper-based therapeutics to treat cancers that are insensitive to platinum drugs.Insensitivity to platinum, either through inherent or acquired resistance, is a major clinical problem in the treatment of many solid tumors. Here, we explored the therapeutic potential of diethyldithiocarbamate (DDC), pyrithione (Pyr), plumbagin (Plum), 8-hydroxyquinoline (8-HQ), clioquinol (CQ) copper complexes in a panel of cancer cell lines that differ in their sensitivity to platins (cisplatin/carboplatin) using a high-content imaging system. Our data suggest that the copper complexes were effective against both platinum sensitive (IC Platinum (Pt) drugs are the most successful class of inorganic medicinal compounds used to treat cancer , 2. They2) in animals bearing a xenograft tumours [While Pt drugs have been successful for many patients, they also produce serious side effects including nephrotoxicity, neurotoxicity and ototoxicity . To addr tumours .2) was active in animals bearing Pt-resistant A2780-CP xenograft tumours. The results highlight the potential of the copper-based therapeutics as candidates to treat Pt resistant cancers.In these studies, we selected five bidentate copper ligands Scheme  and screPlumbago indica. Pyrithione , 8-Hydroxyquinoline, Sodium Diethyldithiocarbamate trihydrate, Copper Sulfate, Clioquinol (5-Chloro-7-iodo-8-quinolinol), and all other chemicals were purchased from Sigma Aldrich. Carboplatin (CBDCA) and Cisplatin (CDDP) were obtained from Hospira. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and Cholesterol (Chol) were purchased from Avanti Polar Lipids .Plumbagin was obtained from 2. Media for the SCC-25 cell line was also supplemented with 400 ng/mL hydrocortisone. All cell lines tested negative for mycoplasma when obtained by the lab. Prior to drug treatment, the cells were seeded into 384 well plates and incubated 24 h in media prior to treatment.The A549, FaDu, Cal-27, SCC-25, and MV-4-11 cell lines were purchased from ATCC. A2780-S and A2780-CP cell lines were obtained from Dr. Mark W. Nachtigal at the University of Manitoba  and the H1933 cells were provided by Dr. William Lockwood at the BC Cancer Agency’s Vancouver Research Centre . All cell lines were used for up to eighteen passages. A549 and H1933 cells were maintained in RPMI (Gibco). A2780-S, A2780-CP and SCC-25 were maintained in DMEM/F12 (Gibco). MV-4-11, Cal-27, and FaDu cells were maintained in IMDM (Gibco), DMEM (Gibco), and MEM (Gibco), respectively. Media for all cell lines was supplemented with 2 mM L-glutamine (Gibco) and 10% fetal bovine serum (Gibco) and maintained at 37 °C and 5% CO4 and the ligand at a fixed 2:1 ratio in DMSO (50%). The ligands DDC, CQ, 8-HQ, Plum were solubilised in DMSO and Pyr was dissolved in sterile water. CBDCA and CDDP were dissolved in sterile saline (0.9%).For in vitro testing, copper complexes were synthesized prior to cell treatment by mixing CuSOThe adherent cell lines  were exposed to the indicated compounds in triplicate wells for 72 h. Following treatment, cells were stained in situ with Hoechst 33,342 and ethidium homodimer-I to differentiate between viable (Hoechst-positive/ethidium homodimer-negative) and dead cells that had lost membrane integrity (Hoechst-positive/ethidium-positive). Cells were imaged with the IN Cell Analyzer 2200  and 4 images/well were collected. Images were analyzed with the ToolBox Developer 1.9 software  to obtain viable and dead cell counts based on differential staining of cell nuclei. The suspension cell line (MV-4-11) was treated for 72 h and viability was assessed using the Presto Blue™ assay (Life Technologies) following the manufacturer’s instructions. The viability data were normalized to vehicle control (0.5% DMSO in media) and expressed as fraction affected where value of 1 corresponded to 100% loss of cell viability relative to vehicle controls and 0 corresponds to a viability comparable to control cells in culture.3H- cholesteryl hexadecyl ether, was incorporated into the chloroform mixture and the solution was dried of chloroform using nitrogen gas and a thin film was generated using a high vacuum for 2 h. The lipid film was rehydrated using 300 mM CuSO4 and extruded for at least 10 passes through a high pressure LIPEX extruder  using 0.1 μm polycarbonate filters. The final liposome size was determined by quasi-electric light scattering with a ZetaPALS analyzer (Brookhaven). The unencapsulated copper was removed via size exclusion chromatography  and replaced with a buffer containing sucrose (300 mM), HEPES (20 mM) and EDTA (15 mM) . The resulting copper-free liposomes were dialyzed against a sucrose (300 mM) and HEPES (20 mM) buffer  and used for Cu(DDC)2 synthesis within the liposomes.For efficacy studies, copper complexes were synthesized inside liposomes, as previously described . Liposom2 synthesis inside the liposomes was performed at 25 °C in a round bottom flask. Copper-containing liposomes (20 mM) were mixed with DDC (10 mM) for 1 h. As DDC permeates across the liposomal lipid bilayer it interacts with the encapsulated copper to form Cu(DDC)2. The free, un-reacted, DDC was removed by SEC and the Cu(DDC)2 containing liposomes, collected in the excluded volume were used for in vivo tumour efficacy studies.Cu(DDC)6 cells in a volume of 50 μL using a 28-gauge needle. Treatment was initiated on day four and treatment groups included the vehicle control (SH buffer), CuSO4-liposomes , and Cu(DDC)2 liposomes (8 mg/kg Cu(DDC)2 and 50 mg/kg lipid) on a Monday, Wednesday and Friday ×2 weeks dosing schedule. The copper dose of the CuSO4 liposome control was equivalent to that of Cu(DDC)2.A2780-CP cells that were used for subcutaneous (sc) implantation were between passages 3–10 and maintained in DMEM/F12 (Gibco) at a confluence of 80–90%. NRG mice (7 per group) were inoculated sc with 1 × 102)/2. Animals were terminated by isoflorane followed by CO2 asphyxiation when animals reached a humane endpoint for these studies was defined when tumours exceeded 800 mm3 or when tumours ulcerated.Animals were monitored at least three times weekly for body weight and tumour growth which was measured with calipers and tumour volumes were calculated based on a formula of  were extrapolated from nonlinear regression (curve fit) of the cytotoxicity curves using Prism 6.0 (GraphPad software). To determine whether the cytotoxic effects of copper complexes are associated with platinum sensitivity, the IC50 values of each copper complex was plotted against the CDDP IC50 for each cell line. Each data point represents one cell line. The Pearson Correlation coefficient and corresponding two-tail p-values were then determined using Prism 6.0 (GraphPad software). Tumour volumes between different treatment groups were compared using one-way ANOVA followed by Tukey adjustments to correct for multiple comparisons using Prism 6.0. An adjusted P-value <0.05 was considered statistically significant.All data were plotted as mean ± SEM or mean ± SD, as described in the figure legends. The IC50 of CDDP and CBDCA was 3.7-and 8.5-fold greater, respectively, in the Pt resistant cells when compared to Pt sensitive cells. To determine if Pt resistance impacts activity of copper complexes, A2780-S and A2780-CP cells were treated with Cu(DDC)2. The data in Fig. 50 value of the copper complex was not different for Pt sensitive or resistant cells. These data suggest that copper complexes may be effective regardless of Pt sensitivity status.CDDP and CBDCA, two commonly used Pt-based therapeutics were tested in a pair of isogenic ovarian cancer cells: A2780-S cells are the parental cells that are sensitive to CDDP while A2780-CP cells are platinum-resistant. As shown in Fig. 50 values ±95% confidence intervals for the ligands and their corresponding copper complexes are shown in Fig. 4 as a control to ensure that copper alone did not impact viability . The results in Fig. 50 ranging from 5 to 300 μM DDC. In contrast, the IC50 for the Cu(DDC)2 ranged from 0.02–0.15 μM corresponding to differences of 90–11,000 fold 2), the IC50 decreased to 0.1–7.4 μM 2 Ranged from 0.8–3 μM in the tested cell lines. In some cell lines  there was only approximately a 2-fold difference in activity between Plum and Cu(Plum)2 2 ranged between 0.2–4.5 μM compared to 1.5-30 μM for 8-HQ); however, in A2780-S and FaDu cells, the toxicity of 8-HQ appeared to be copper independent 2 ranged from 10 to 350 μM and 20-60 μM, respectively. Here the dependence on copper complexation for CQ cytotoxicity appears to be cell line-specific: copper-dependent activity was observed in A2780-S, A2780-CP and A549 cells, whereas in MV-4-11, SCC-25 and H1933, the cytotoxicity of CQ was copper-independent. Moreover, CQ appears to be more cytotoxic than Cu(CQ)2 in Cal-27 and FaDu cells 2. DDC and CQ were the least active ligands. The Cu(CQ)2 complex did not show improvement in cytotoxicity whereas the copper complex of DDC was the most active in all cell lines. In general, except for Cu(Pyr)2, the activity of the copper complexes used in our study show no correlation with Pt CDDP sensitivity in tested cell lines 2 was synthesized inside a liposomal formulation [2 in a Pt-resistant A2780-CP subcutaneous tumour xenograft model. The data, shown in Fig. 2 engendered a statistically (p < 0.05) significant ~50% reduction in tumour burden when compared to the activity of the vehicle control. These data provide proof-of-concept results suggesting that liposomal copper complexes have the potential to be developed as a novel class of therapeutic agents for use in the treatment of Pt-resistant cancers.Cu(DDC)mulation . Here we, cell lines can be developed to replicate this effect (e.g. A2780-CP), wherein cells are treated with low dose Pt drugs such as CDDP over extended periods of time until resistance is observed through changes in IC50 values [Pt drugs are standard first-line therapies for the treatment of lung, ovarian, and head and neck cancers , 26. Whict e.g. A80-CP, wh0 values . The two0 values . Through0 values . The cop0 values , 30. Thi0 values . Identif2 in Pt sensitive and insensitive cell lines. Cu(DDC)2 was active irrespective of Pt sensitivity at equivalent doses. This Pt sensitivity-independent activity could be explained by the Cu(DDC)2 mechanism of toxicity which overcomes resistance mechanisms commonly seen in Pt-resistant cancers. Cu(DDC)2 is able to accumulate in cancer cells irrespective of CTR1 expression [The ovarian cancer cell line A2780-CP had a clear resistance to CDDP and CBDCA when compared to the activity of these drugs in A2780-S cells. In order to test the utility of copper based therapies in platinum-resistant cancer cells, preliminary studies investigated Cu(DDC)pression  and cytopression . These r50 cytotoxicity to all the ligands tested. In contrast, Plum and CQ could be used as single agents and pharmaceutical challenges associated with their solubility may be overcome using the Metaplex™ approach that we have described previously to prepare a nanomedicine formulation of Cu(DDC)2 with no loss in activity [Han et al. tested the Cu(I)-DDC complex in a subcutaneous tumour model of adenocarcinoma with ~60% reduction in tumour burden by day 25 [Five copper binding ligands  were tested in a panel of eight cancer cell lines of different origins  and differing sensitivity to Pt-based drugs exemplified by CDDP/CBDCA activity. Our in vitro data suggest that the cytotoxicity of ligands DDC, Pyr and 8-HQ appeared to be copper-dependent whereas the cytotoxicity of Plum and CQ were copper independent. This finding is important as both DSF (metabolized to DDC in vivo) + Cu-Gluconate and CQ were involved in clinical trials with no listed benefit –35. By kactivity , 15. Cu human bronchial epithelial cells [Our data support the utility of copper-based therapeutics in the treatment of Pt-insensitive cancer. To date, not a single copper-based therapeutic has received FDA approval. This is in part due to the solubility challenges associated with these complexes. All copper complexes studied as well as the ligands Plum, 8-HQ and CQ are water insoluble (<0.1 mg/mL); in contrast, DDC and Pyr are water soluble as ligands but their therapeutic activity is significantly enhanced in the presence of Cu. All copper complexes had to be dissolved in DMSO  to allow for in vitro testing. The ICH guidelines for residual solvents in a product indicate that DMSO cannot be included at concentrations above 0.5% which would be needed to administer the complexes at relevant doses . While tal cells .2 in a xenograft tumour model of A2780-CP Pt-resistant ovarian cancer. In this study, we showed that liposomal Cu(DDC)2 (8 mg/kg) but not Cu-liposomes produced a statistically significant ~50% reduction in tumour burden when compared to the vehicle control. This difference is not considered clinically relevant  but thetability . Also th taxanes ) is warr50 values below 10 μM in the eight cancer cell lines regardless of their Pt sensitivity. Additionally, we tested one of these complexes (Cu(DDC)2) in vivo in a Pt-resistant ovarian cancer xenograft model and attained a 50% reduction in tumor volume when compared to vehicle-treated control mice. These data not only provide in vivo validation that copper-based therapeutics could be used to treat Pt-resistant cancer but also provides proof-of-concept that the Metaplex™ technology could be used to develop formulations of inherently insoluble copper complexes for pre-clinical and clinical evaluations.Pt drugs have been widely successful in the treatment of cancer. The treatment of Pt-resistant cancers has been a pharmaceutical focus that requires new therapeutics with mechanisms of action differing from those invoked by Pt-based therapies. Copper-based therapeutics represents a new class of drugs that can address many of the challenges associated with Pt-resistant cancers. Our data show that four out of five screened ligands that form copper complexes reached IC


Hematopoietic cell transplant is a potentially curative procedure for many benign and malignant conditions. The efficacy of allogeneic transplant relies in part on the cytotoxicity of the conditioning regimen and the graft versus tumor effect mediated by alloreactive donor T cells; the same cells are also implicated in the development of graft versus host disease (GVHD). Selective identification and depletion of the T cells implicated in GVHD, while preserving the T cells responsible for graft versus tumor effect has been the focus of many research groups in the recent years. Here we briefly review the physiology of T cells in transplantation, and comment on a recent clinical trial published by Bleakly et al. using a novel way of graft engineered allograft via naïve T cell depletion. GVT without causing GVHD? A recent study by Bleakly et al. in the Journal of Clinical Investigation depicts another novel way of deciphering optimum outcomes in HCT [Hematopoietic cell transplant (HCT) is considered potentially curable for many conditions; however, it comes with the price of a devastating side effect: graft-versus-host disease (GVHD), which is the leading cause of non-relapse mortality and morbidity. Pioneering work done by many investigators in 1970s and 1980s indicated that T-cells mediate potent graft-versus-tumor (GVT) effects, a concept that was later consolidated by large database registry studies in which relapse was found to be higher in identical twins compared to matched donors [s in HCT .The preparative regimen for a HCT exerts direct cytotoxicity, whereas the harvested allograft, which is composed of progenitor cells CD34+), T-cells , B-cells CD19+), NK cells CD56+), and dendritic cells CD14+), can exert potent GVT or cause GVHD [4+, T-cel6+, and d+, NK cel+, can exThe paper by Bleakley et al. is the first in human trial to apply this concept with respect to naïve T-cell depletion . The autSome issues besides failure to meet the co-primary end point are worthy of mentioning. The intensity of the regimen used in this trial limits its applicability to young patients only, while the AML/MDS is mainly a disease of older populations; therefore, investigating less intense regimens with the same concept is required for external validity. For HCTs in malignancies, two newer end points—cGVHD/relapse free survival (CRFS) and GVHD/relapse or progression free survival (GRFS)—have become standard end points for clinical trials; thereby, we hope that if this concept is taken to a phase III study, the next step would be CRFS or GRFS as the primary end point. Another limitation , is that the feasibility of selective T-cell depletions by sophisticated techniques is limited to a few transplant centers; another method of T-cell depletion utilizing post-transplant cyclophosphamide is logistically easier, economic, and widely available, so not surprisingly, its use is on the rise globally. Thus, comparing these two approaches would be required eventually to test which approach would confer the optimum GVL without increasing GVHD.p < 0.001) [While the results of this trial are impressive in opening new avenues in transplantation (matched and mismatched HCT), these need to be confirmed in larger randomized trials. Ideally, one would compare two similar transplant platforms with identical conditioning regimens and GVHD prophylaxis, with or without naïve T-cell depletion. For separating out GVL from GVHD, many investigators have embarked on various avenues of graft manipulations. The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) conducted a TCD allograft approach with selective CD34+ selection, and compared the results with T-cell replete allografts plus immunosuppressive therapy (IST) with impressive results, indicating significantly less 2-year rates of cGVHD with TCD grafts than the IST group  . There w< 0.001) . Dendrit< 0.001) . “HLA-Mi< 0.001) . Lastly,We believe that the approach by Bleakly et al. and colleagues has shown that outcomes, which are clinically meaningful to both the clinicians and patients, are promising enough to further expand this avenue into a randomized trial. Although acute GVHD was not low, it was easily treatable and the mortality was not increased compared to a historical cohort of T-cell replete HCT. Most importantly, it is clear that the risk of cGVHD was much lower with naïve T-cell depleted allografts, without increasing the risk of relapses as indicated by the results of this trial. Compared to other novel strategies, this method is safe, and mature enough  to lead to a multicenter randomized clinical trial in the hope of finally resolving the issue of the holy grail of HCTs for the benefit of mankind.


HPV-negative disease necessitate development of treatment strategies tailored to tumor HPV-status. High-throughput robotic screening of 1,433 biologically and pharmacologically relevant compounds at a single dose (4 μM) was carried out against 6 HPV-positive and 20 HPV-negative HNSCC cell lines for preliminary identification of therapeutically relevant compounds. Statistical analysis was further carried out to differentiate compounds with preferential activity against cell lines stratified by the HPV-status. These analyses yielded 57 compounds with higher activity in HPV-negative cell lines, and 34 with higher-activity in HPV-positive ones. Multi-point dose-response curves were generated for six of these compounds , confirming Ryuvidine to have preferential potency against HPV-negative cell lines, and MK-1775 to have preferential potency against HPV-positive cell lines. These data comprise a valuable resource for further investigation of compounds with therapeutic potential in the HNSCC.Head and neck squamous cell carcinoma (HNSCC) is a common cancer diagnosis worldwide. Despite advances in treatment, HNSCC has very poor survival outcomes, emphasizing an ongoing need for development of improved therapeutic options. The distinct tumor characteristics of human papillomavirus (HPV)-positive  There has been an epidemic rise in oropharyngeal cancer worldwide due to increasing rates of oral infection with human papillomavirus (HPV) , 2. HPV-Cell lines are imperfect models of cancer , 6, howeThis study aimed to carry out a high-throughput drug screen of a large collection of compounds against HNSCC cell lines, with the objective of preliminary identification of potent and HPV-status selective agents suitable for further investigation.The top 20 compounds with the highest activity from the kinase inhibitor and Tocris panels are reported in Figure Thirty compounds from the kinase panel, and 27 compounds from the Tocris panel were found to be more potent against HPV-positive cell lines Figure . Among tSix compounds from the kinase inhibitor panel and 28 compounds from the Tocris panel were found to have higher activity in HPV-negative cell lines Figure . Three c50 of 1.562 μM ± 0.755 (mean ± standard deviation) in HPV-negative and 3.169 μM ± 1.305 in HPV-positive cell lines , confirming the findings in the high-throughput screen. MK-1775 showed significantly higher potency against HPV-positive cell lines with an average IC50 of 0.535 μM ± 0.4828 in HPV-positive and 3.647 μM ± 3.73 in HPV-negative cell lines , again showing compatible findings with the high-throughput drug screen. SNS-032, Flavopiridol, AZD-7762 and ARP-101 compounds did not show significantly different potency in cell lines stratified by HPV-status following validation studies across a range of concentrations (t-test p>0.05). Dose-response studies of MK-1775, Ryuvidine, AZD7762 and Flavopiridol in control non-cancerous IMR90 cell line showed higher IC50 values  compared to susceptible HNSCC cell lines, demonstrating the presence of a therapeutic window.After the generation of multi-point dose response curves, Ryuvidine was selectively potent against HPV-negative cell lines, with an average ICTo our knowledge, this is the first high-throughput drug screening study that focuses specifically on head and neck cancer cell lines. Our study of 1,433 compounds in 26 cell lines identified 57 and 34 compounds with preferential activity against HPV-negative and HPV-positive cell lines, respectively. Validation studies with six of these confirmed selective potencies in HPV-negative (Ryuvidine) and HPV-positive (MK-1775) cell lines. Given that HPV-positive and negative tumors are distinct from a molecular and prognostic perspective , this fiMultiple studies and clinical trials have identified a superior response to chemotherapy, and better survival outcomes in HPV-positive HNSCCs compared to the HPV-negative tumors , 11–14. Ryuvidine functions as an inhibitor of the CDK4 and SETD8 proteins , 18. CDKMK-1775, also known as AZD1775, is a WEE1 kinase inhibitor . WEE1 kiPreliminary dose-response curve validation studies were able to validate the selectivity of 2 of the 6 tested compounds that were derived based on our high-throughput study. This is not surprising as the high-throughput study included only a single dose of each compound, with the purpose of preliminary identification of compounds with potential for further assessment. More of the identified compounds may show HPV-status specific activity against HNSCCs following validation. Furthermore, analyses of the available HNSCC cell lines have revealed common genetic alterations other than HPV-status that are also reflected in tumor populations . These iHigh-throughput testing of large chemical compound libraries can serve as valuable resources in the context of a necessity for better chemotherapeutics in the field of head and neck oncology. In this study, we have provided the head and neck cancer research community with the results of a preliminary screen of the activity of over 1400 compounds against 26 HNSCC cell lines. Initial validation studies led to the identification of Ryuvidine and MK-1775 compounds to have preferential activity against HPV-negative and HPV-positive cell lines respectively. We hope that the provided datasets can serve as a valuable resource to lay the foundation for further investigation of compounds with therapeutic potential in the field of head and neck oncology.2 in the medium specified in Twenty-eight HNSCC cell lines were obtained from established culture collections or from collaborators as listed in A custom-made compound library from Tocris Bioscience comprised of 1,113 compounds and a kinase inhibitor library of 320 compounds were used for high throughput drug screening. The drug screen was carried out against 6 HPV-positive and 20 HPV-negative cell lines . High-thet. al. [t-tests were utilized to compare the average B score of HPV-positive cell lines to that of HPV-negative cell lines for each drug. These p-values were then controlled for local false discovery rates using the fdrtool package version 1.2.15 [ie HPV+ or HPV-) and a B score of higher than −2 in the opposite disease type, then sorted by the mean difference in the two arms of comparison (from high to low).Fluorescence intensity derived from the high-throughput screen were normalized using B scores as per recommendation of Malo et. al. . For celn 1.2.15  in the R50) value for each cell line, normalized Relative Fluorescence Units (RFUs) of the drug-treated replicates were calculated as a percentage of the mean RFU of the vehicle control (DMSO-only) treatment replicates. IC50 values, defined as the concentration at which the normalized RFU reached 50%, were calculated by non-linear regression . When a non-linear regression model could not be converged for a compound and cell line, no IC50 value was assigned and data was excluded from comparison analysis. An unpaired t-test was used to assess the significance of the differences in IC50 values between HPV-positive and HPV-negative cell lines for each of the compounds, and a p value of less than 0.05 was set as the significance limit. As a preliminary assessment of compound toxicity, we also tested 4 of the compounds  against the IMR90 non-cancerous cell line (ATCC CCL-186) over a dose range of 0.03 μM to 10 μM with the same methodology to generate dose-response curves and calculate IC50 values.Six drugs  that demonstrated high differential activity in either HPV-positive or negative cell lines and mechanisms of action that were deemed to be interesting in HNSCC were selected for further analysis. Dose response curves were generated, and drug validation studies for these compounds were tested in 8 HPV-positive and 11 HPV negative cell lines (cell lines listed in


SNCA gene encoding for alpha-synuclein protein lead to familial Parkinson’s disease (PD). The generations of libraries of familial human ALS iPSC lines have been described, and the iPSCs rapidly became useful models for studying cell autonomous and non-cell autonomous mechanisms of the disease. Here we report the generation of a comprehensive library of iPSC lines of familial PD and an associated synucleinopathy, multiple system atrophy (MSA). In addition, we provide examples of relevant neural cell types these iPSC can be differentiated into, and which could be used to further explore early disease mechanisms. These human cellular models will be a valuable resource for identifying common and divergent mechanisms leading to neurodegeneration in PD and MSA.Induced pluripotent stem cells (iPSCs) are becoming an important source of pre-clinical models for research focusing on neurodegeneration. They offer the possibility for better understanding of common and divergent pathogenic mechanisms of brain diseases. Moreover, iPSCs provide a unique opportunity to develop personalized therapeutic strategies, as well as explore early pathogenic mechanisms, since they rely on the use of patients’ own cells that are otherwise accessible only post-mortem, when neuronal death-related cellular pathways and processes are advanced and adaptive. Neurodegenerative diseases are in majority of unknown cause, but mutations in specific genes can lead to familial forms of these diseases. For example, mutations in the superoxide dismutase 1 gene lead to the motor neuron disease amyotrophic lateral sclerosis (ALS), while mutations in the  SNCA gene encodes for aSYN. When SNCA is mutated or multiplied, it leads to an early onset familial PD.2 Interestingly, while aSYN aggregates are found in neurons in dementia with Lewy bodies and PD, they are located in oligodendrocytes in MSA.3The group of synucleinopathies consists mainly of three neurodegenerative diseases: dementia with Lewy bodies, Parkinson’s disease (PD) and multiple system atrophy (MSA), the last being divided into Parkinsonian type MSA with degeneration of nigro-striatal dopamine neurons, and cerebellar type MSA with ataxic symptoms. The diseases share the same hallmark: intracellular aggregates composed in majority of a protein called alpha-synuclein (aSYN). The SNCA is expressed in neurons. A recent study from our laboratory showed that SNCA is also expressed at early stage of oligodendrocyte development.4 The role of aSYN in oligodendrocytes largely remains to be clarified. It is thought that under pathological condition, aSYN protein can aggregate in neurons and over time form Lewy bodies.5 Moreover, it was shown in experimental models that aSYN aggregates are toxic to neurons,7 and that a neuroblastoma cell line over-expressing human recombinant SNCA releases factors including aSYN, leading to glial reactivity,8 suggesting aSYN aggregates may cause neuronal injury. However, recent work evidenced that neuronal dysfunction and protein aggregation may be two independent events.9 Thus, although the progression of Lewy pathology throughout the brain may be due to a prion-like mechanism of cell-to-cell transfer of aSYN,10 it remains unclear what the initial molecular cascades leading to neuronal dysfunction are, and how they differ depending on the genetic background of the patients.Naturally, 11 has revolutionized our way to approach scientific problems related to human diseases. Importantly, this discovery offers unlimited access to patient cells, which can subsequently be differentiated into relevant cell types to study early pathogenic mechanisms of neurodegeneration.12–17 Such iPSC-based research strategies could lead to the discovery of new therapeutic targets, biomarkers, and the development of humanized high-throughput models for drug discovery and environmental chemical safety assessment.12–17 Thus far, several studies utilizing iPSC-based models reported neuronal dysfunction reminiscent to mutations in PD-linked genes LRRK2 (leucine-rich repeat kinase 2), PINK (PTEN-induced putative kinase 1), and PARK2 (encodes PARKIN),18–21 or in the acid beta-glucocerebrosidase gene (GBA1), which encodes a lysosomal enzyme that is deficient in Gaucher’s disease, and which renders a risk of developing PD.23 Therefore, iPSCs appear to be robust models for understanding early pathogenic events occurring in familial forms of PD. Importantly, iPSCs may provide an ideal platform for studying diseases where no genetic cause has thus far been identified, such as MSA and idiopathic PD.12–17The reprogramming of human somatic cells using “stemness” transcription factors into induced pluripotent stem cells (iPSC)25 to date, none have been described for PD. Here, we report the generation of iPSCs carrying various mutations in PD-associated genes, as well as iPSCs generated from patients diagnosed with MSA and healthy controls  or human OCT3/4, KLF4, SOX-2, and c-MYC (for UEF-3A and UEF-5G lines). The lines UEF-1A , UEF-3A and UEF-5G were generated with a polycistronic lentivirus carrying all the reprogramming factors in the same viral vector (STEMCCA) while the lines UEF-2A, B and C, -3B, -4A and B, and -5B, E, F and G, were transduced with four separate Sendai viruses, encoding the same pluripotency genes  lines: number=patient and letter=clone; e.g., UEF-2A→line generated at the UEF lab, patient 2, clone A. The skin biopsies were collected from individuals with confirmed diagnosis and mutation, and the fibroblast populations were transduced with viruses carrying genes encoding mouse cy genes . Followicy genes . An averSC lines , and conSC lines . All iPSSC lines , and theSC lines . ChromosSC lines  and 3 exSC lines . In addiSC lines .All lines formed embryoid bodies when plated in suspension dishes . Immunoc32 to generate iPSC-derived mesencephalic dopaminergic neurons. The differentiation of iPSCs was started on human feeder layer. On day 14, small pieces of colonies were plated in an adherent culture. On day 20, the neuroepithelium differentiated into cells resembling neural cells. The neuroprogenitors were positive for LMX1A and FOXA2, confirming they acquired a midbrain floor-plate identity  lab, patient 2, clone A. The lines CSC-1, -2, -3, -4, -6, -7, 8-, and -9 were generated by retroviral transduction of 3  or 4 (the 3 plus c-MYC) factors; while the lines CSC-10, -11, -13, -14, -16, -18, -19, -20, -21, and -22 were generated by Sendai virus transduction of the four factors  as positive control. In some assays, e.g., determination of telomerase activity, both embryonic stem cells and parent fibroblasts can be used as positive or negative controls, as we previously reported,4 as well as heat-inactivated samples.We commonly thaw 3 putative iPSC clones for each patient lines generated, grow all clonal cell lines in 6-well plates for up to 10 (some times more) passages (with one simultaneous passage per week), and perform within the latest passages, in addition to finger print analysis, a battery of tests allowing us to determine the efficiency of reprogramming from putative to well-established/mature iPSC lines. The tests comprise: (1) expression of nuclear and cell surface markers reminiscent to pluripotent stem cell stage , (2) alkaline phosphatase activity, (3) telomerase activity, (4) embryoid body formation, (5) loss of viral agents or downregulation of viral transgenes, and (6) differentiation towards the three germ layers. The lines can further be tested for their ability to generate midbrain neural cells types: LMX1A/FOXA2 co-expressing neural floor-plate progenitors, FOXA2/tyrosine hydroxylase-expressing neurons, glial fibrillary acidic protein-expressing astrocytes, or O4-positive oligodendrocytes, which can be used for studies of development and disease phenotypes. All these tests are performed 35 Interestingly, two clonal cell lines (CSC-11C at passage 13 and CSC-13A at passage 8) showed resistance to Sendai virus elimination over passages , regardless whether they were generated using three or four factors and if they carried karyotype abnormalities , strong expression of reprogramming factor due to transgene insertion (line CSC-7B), and persistence of Sendai virus (lines CSC-11C and CSC-13A). In summary, we obtained 8 “good” control iPSC lines and 33 “good” disease iPSC lines .P-value of <0.05 was considered significant.All quantitative data was analyzed using Prism 6.0 . Sample groups were subjected to one-way analysis of variance and unpaired t-test. A Details methods are described in


TP53 gene accounts for 50% of all human cancers. In the remaining malignancies, non-genotoxic activation of wild-type p53 by small molecule inhibition of the MDM2-p53 binding interaction is a promising therapeutic strategy. Proof of concept was established with the cis-imidazoline Nutlin-3, leading to the development of RG7388 and other compounds currently in early phase clinical trials. This preclinical study evaluated the effect of Nutlin-3 and RG7388 as single agents and in combination with cisplatin in a panel of ovarian cancer cell lines. Median-drug-effect analysis showed Nutlin-3 or RG7388 combination with cisplatin was additive to, or synergistic in a p53-dependent manner, resulting in increased p53 activation, cell cycle arrest and apoptosis, associated with increased p21WAF1 protein and/or caspase-3/7 activity compared to cisplatin alone. Although MDM2 inhibition activated the expression of p53-dependent DNA repair genes, the growth inhibitory and pro-apoptotic effects of p53 dominated the response. These data indicate that combination treatment with MDM2 inhibitors and cisplatin has synergistic potential for the treatment of ovarian cancer, dependent on cell genotype.Ovarian cancer is the fifth leading cause of cancer-related female deaths. Due to serious side effects, relapse and resistance to standard chemotherapy, better and more targeted approaches are required. Mutation of the  Ovarian cancer is the most lethal of all gynecological malignancies and was reported to be responsible for approximately 152,000 deaths worldwide in 2012 ).TP53 tumor suppressor gene is referred to as the most frequently altered gene in human cancers. It has been substantially established that p53 protects cells against environmental and intra-cellular stress stimuli by playing a central role in regulating cell cycle control, differentiation, proliferation, DNA repair and apoptosis . TP53 mWAF1, BAX and BBC3 (PUMA) [de novo mutations [MDM2 is the main negative regulator of p53, regulating p53 through ubiquitin dependent degradation. The imidazoline Nutlin-3, was the first non-genotoxic specific small-molecule antagonist of the MDM2-p53 binding interaction to be developed  and has 3 (PUMA) , 10. Usiutations  and (revutations ). It is The aim of the present study was to test a panel of established ovarian cancer cell lines for their response to MDM2-p53 antagonists, Nutlin-3 and RG7388, alone and in combination with cisplatin and examine the mechanistic basis of these responses in relation to the genotype and induced gene expression of the cells.TP53 ovarian cancer cell lines derived from tumours of different histological subtypes [50) showed that wild-type TP53 ovarian cancer cell lines were significantly more sensitive to Nutlin-3/RG7388 compared to mutant, which is consistent with their mechanism of action (p<0.0001 Mann-Whitney test). Also, RG7388 was more potent compared to Nutlin-3 (p<0.0001 Mann-Whitney test). The GI50 values for wild-type TP53 cell lines for RG7388 and Nutlin-3 were in the nanomolar range (253.3 ± 73.1 (SEM) nM) and micromolar range (1.76 ± 0.51 (SEM) μM) respectively. In contrast, TP53 mutant cell lines had GI50 values greater than 10 μM (17.8 ± 2.9 (SEM) μM) for RG7388 and range 21.2->30 μM for Nutlin-3 (Table Growth inhibition by Nutlin-3/RG7388 was investigated using the sulforhodamine-B (SRB) assay for a panel of wild-type and mutant subtypes –16  their respective GI50 concentration for 24, 48 and 72 hours. They were then analyzed by flow cytometry for cell cycle phase distribution changes and evidence of apoptosis in response to treatment. The 48 and 72 hour treatment data is presented in the Wild-type TP53 cell lines, Nutlin-3 only showed a modest increase in the proportion of cells in G0/G1 phase in a dose and time dependent manner. Nutlin-3 also increased the percentage of SubG1 events, a surrogate marker of apoptosis, in A2780 and IGROV-1 cell lines in a treatment time and dose-dependent manner  μM); however, IGROV-1 (LC50= 11 ± 2.1(SEM) μM) and OAW42 (LC50= 6.25 ± 0.50 (SEM) μM) were substantially less sensitive to Nutlin-3 . Changes in the expression of cell cycle/apoptosis-related genes as well as those involved in nucleotide excision repair (NER) and mismatch repair (MMR) for the three wild-type CDKN1A, SESN1 and GADD45A gene expression in all three cell lines, with CDKN1A consistently showing the highest level of induction (p<0.05)  genes in all three cell lines, with increases of BBC3 mRNA being highest in the IGROV-1 cell line (p<0.05)  Figure . Both tr-1 genes .TP53BP1, DDB2, ERCC1, XPC, MLH1, MSH2, RAD51 and RRM2B genes in response to Nutlin-3 and RG7388 was investigated. A significant increase was measured in the expression of DDB2 in response to Nutlin-3 and XPC in response to both Nutlin-3 and RG7388 for A2780 cells (p<0.05). In the case of IGROV-1, XPC and MSH2 gene expression levels were significantly induced and reduced respectively in response to both Nutlin-3 and RG7388 (p<0.05). For OAW42 cells there was a significant increase in the expression of DDB2 and XPC genes and a significant decrease in the MLH1 and MSH2 expression levels in response to Nutlin-3 and RG7388 treatment (p<0.05). With Nutlin-3 treatment, the TP53BP1 gene expression decreased in all three cell lines, although statistically this trend was not significant (p>0.05).To study the effect of Nutlin-3 and RG7388 on the expression of genes implicated in the response to DNA repair induced by cisplatin, the expression of CDKN1A and BAX genes was consistent with the western blot analysis for p21WAF1 and BAX proteins  and RG7388 was kindly provided by Professor Herbie Newell and made available by the Newcastle Anticancer Drug Development Initiative. Both were dissolved in dimethyl sulfoxide (DMSO). Cisplatin  was dissolved in distilled water.TP53 status and histological subtype are listed in Table TP53 mutation  [MLH1-corrected CP70+ cell line was grown in RPMI-1640 supplemented with 10% (v/v) FBS and Hygromycin B  [TP53 mutation . SKOV-3 and MDAH-2774 cell lines were cultured in DMEM supplemented with 10% and 5% (v/v) FBS and penicillin/streptomycin respectively. As information on the TP53 status of SKOV-3 in the literature was contradictory, sequencing was performed and a frame shift deletion  was confirmed  [The ovarian cancer cell lines used in this study, their l172Phe) . The MLHs, Inc.) . This ceonfirmed . The MDAg273His) .50 values, the required concentrations of each compound leading to 50% growth inhibition, were determined by Sulforhodamine B (SRB) growth inhibition assays for drug exposure over 72 hours and the absorbance of the re-dissolved SRB protein stain was measured at 570 nm using a 96-well plate spectrophotometer (Spectramax 250 Molecular Devices) [TP53 cell lines were treated for 72 hours with each agent alone and in combination simultaneously at constant 1:1 ratios of 0.25x, 0.5x, 1x, 2x, and 4x their respective GI50 concentrations. Median-effect analysis was used to calculate Combination Index (CI) and Dose Reduction Index (DRI) values [The GIDevices) . Growth ) values  using CaTM C membrane . Monoclonal Mouse Anti-Human primary antibodies Actin 1:1000 , MDM2 1:300 , p21WAF1 1:100 , p53 1:500  and Polyclonal Rabbit Anti-Human primary antibody BAX 1:1000  were used. Secondary goat anti-mouse/rabbit HRP-conjugated antibodies  were used at 1:1000. All antibodies were diluted in 5% milk/1XTBS-Tween (w/v). Enhanced chemiluminescence (GE Life Sciences) and X-ray film (Fujifilm) were used to visualize the proteins.Lysis buffer  was used to harvest whole-cell lysates, followed by sonication. The concentration of protein in the cell lysates was estimated by using a bicinchoninic acid (BCA) assay. Novex® 4-20% Tris-Glycine 12-well polyacrylamide gradient gels  were used to separate proteins. The separated proteins were transferred by perpendicular electrophoresis to a nitrocellulose Hybond50 concentrations for 24, 48 and 72 hours. Cells harvested by trypsinisation were washed with PBS and resuspended in 500 μL PBS with 1mg/mL sodium citrate , 100 μg/mL propidium iodide (Sigma), 200 μg/mL RNAse A (Sigma) and 0.3% Triton-X (Sigma). Samples were analyzed on a FACSCaliburTM flow cytometer using CellQuest Pro software . Cell cycle distribution was determined using Cyflogic .Cells were treated with Nutlin-3, RG7388 and cisplatin alone and with combinations of Nutlin-3 or RG7388 with cisplatin simultaneously at constant 1:1 ratios of 1x and 2x (A2780 and IGROV-1) or 0.5x and 1x (OAW42) their respective GI50 concentrations, depending on the cell line and its single agent LC50 values, for 48 hours. Then, the medium including drug was removed and drug-free medium was added. The cells were incubated for 2 to 3 weeks to form colonies for counting (a colony was defined as a focus of ≥50 cells). After that, the plates were washed with PBS, fixed with methanol/acetic acid (3:1) and stained with 0.5% (w/v) crystal violet. Plates were again washed and then left at room temperature to dry. The LC50 values were calculated using GraphPad Prism statistical analysis software version 5.04. DRI and CI values were calculated by median-effect analysis [Based on the drug concentration and sensitivity of cell lines, 100 to 100,000 cells were seeded into triplicate six-well plates. Cells were treated with Nutlin-3, RG7388 and cisplatin alone and in combination at constant 1:1 ratios of 0.25x, 0.5x, 1x, 2x and 4x or 0.25x, 0.5x and 1x their respective LCanalysis  using CaCaspase 3/7 activity was measured using a Caspase- Glo 3/7 assay following the manufacturer's instructions .GAPDH was used as endogenous control and the DMSO solvent control sample used as the calibrator for each independent repeat. Data analysis using the ΔΔCt Method was carried out using SDS 2.2 software (Applied Biosystems).Total RNA was extracted using an RNeasy Mini Kit . RNA purity and concentration were estimated with an ND-1000 spectrophotometer . Total messenger RNA was converted to cDNA using the Promega Reverse Transcription System  as described by the manufacturer. Validated primers used (Sigma-Aldrich UK) are listed in All statistical tests presented were carried out using GraphPad Prism version 5.04 software. A p-value of <0.05 was considered to be statistically significant based on at least n=3 experimental repeats.


Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification.The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibody-dependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model.T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinib-resistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance.Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC. In patients with advanced Non-Small Cell Lung Cancer (NSCLC) harbouring Epidermal Growth Factor Receptor (EGFR) activating mutations, disease progression occurs after a median of 10–12 months of treatment with EGFR-Tyrosine Kinase Inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib. The acquisition of the EGFR-T790M secondary mutation is the main mechanism of acquired resistance, occurring in 50–60% of the cases  with ImageJ software and the Fold Increase (FI) index was calculated as the ratio between the spheroid volume after 6 days and the volume at T0.Spheroids were generated using LIPIDURE®-COAT PLATE A-U96  as previously described . BrieflyOne million of NSCLC cells were incubated, for one hour at room temperature, with Isotype control Monoclonal Mouse IgG1/R-PE , mouse anti-Human HER-2/PE or mouse anti-Human EGFR/PE  to determine EGFR or HER-2 protein membrane levels as previously described . The anaPC9-T790M and H1975 cells were plated in 96-well plates at a density of 350 cells/well and treated with osimertinib and/or T-DM1 following the indicated schedules in the experiment. Medium with drugs was changed every 7 days. Wells were followed daily over time. Taking into account the different sensitivity to osimertinib, cells were scored as resistant when colonies with at least 20 cells were detected in PC9-T790M cell cultures or when >80% confluence was reached in H1975 cell cultures .Highly purified CD56+ natural killer (NK) cells were obtained by magnetic separation and ADCC was measured with the CytoTox 96 non-radioactive cytotoxicity assay  as previously described .Tyr1068, HER-2 (29D8), p-HER2 Tyr1221/1222 (6B12), AKT, pAKTser473, p-p44/42 MAPK (D13.14.4E), MAPK (137F5), Bim (34C5) and Mcl-1 (RC-13) were from Cell Signaling Technology ; antibody against Actin was from Sigma. HRP-conjugated secondary antibodies were from Pierce  and chemoluminescence system (Immobilion™ Western Chemiluminescent HRP Substrate) was from Millipore . Reagents for electrophoresis and blotting analysis were from BIO-RAD . The chemiluminescent signal was acquired by C-DiGit® Blot Scanner and the spots were quantified by Image Studio™ Software, LI-COR Biotechnology .Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere . Antibod6 PC9/HER2c1 cells were subcutaneously injected on the flank of Balb/c-Nude female mice . The animals were housed in a protected unit for immunodeficient animals with 12-h light/dark cycles and provided with sterilized food and water ad libitum. When tumor volume reached an average size of 200 mm3, animals were randomly allocated into four groups (n = 8 tumors per treatment group): control, osimertinib, T-DM1 and osimertinib combined with T-DM1. Osimertinib (10 mg/kg in 1% Tween 80) was given once per day, five times per week, by oral gavage. T-DM1 (15 mg/kg in 0.9% NaCl) was given intraperitoneally twice per week. Tumors were measured as previously described . However, also for this agent, resistance is expected and new therapeutic approaches are needed to prolong its benefit [Osimertinib is an effective treatment in NSCLC patients with T790M EGFR mutation, progressed after first-line therapy with first- or second-generation EGFR-TKIs . MoreoveHER-2 was shown to be overexpressed (IHC 3+) in 2–6% of NSCLC, with gene amplification found in 2–4%, mainly in the adenocarcinoma histotype . In partWe tested T-DM1 in preclinical experiments suggesting its activity against HER-2-driven lung cancer cell lines in vitro . Only reAs previously reported by Scaltriti et al. for lapatinib in breast cancer cells  and by oHere we demonstrated that, differently from trastuzumab, T-DM1 showed anti-proliferative effects in PC9, PC9-T790M and H1975 cells and exerted an additive effect when combined with osimertinib in terms of inhibition of proliferation, cell death and ADCC induction. Because osimertinib is highly effective in mutated NSCLC cell lines, the addition of T-DM1 caused a modest benefit when evaluated in short-term growth assay, by contrast the combination significantly delayed the acquired resistance to osimertinib in PC9-T790M and in the less osimertinib-sensitive model H1975. The combination was effective either when the cells were treated continuously with osimertinib and a simultaneous treatment with TDM1 was cyclically performed  anticipated the appearance of resistance to osimertinib compared to cells continuously treated with osimertinib alone, by contrast in the protocols in which cells were simultaneously exposed to both drugs (osimertinib→osimertinib + T-DM1 or osimertinib + T-DM1→osimertinib), no resistant colonies were detected up to 50 days of treatment. In H1975 cells, all schedules delayed the acquisition of resistance; however, also in this model the less effective was the intermittent schedule (osimertinib→T-DM1) both in terms of percentage of resistant wells and of median time to resistance.Overall, these data may support the use of a combination of osimertinib with T-DM1 using a specific schedule to delay or prevent the acquisition of resistance to the EGFR-TKI, similarly to other combinations of third-generation EGFR-TKIs (with MEK or MET inhibitors) tested in clinical trials currently recruiting patients .In the last part of this study we investigated the role of HER-2 amplification in the responsiveness to osimertinib and the efficacy of T-DM1 in vitro and in vivo to overcome osimertinib resistance. HER-2 amplification, indeed, has been reported as a mechanism of acquired resistance to EGFR-TKIs with a frequency of 12–13% .PC9/HER2c1 cells were exposed to osimertinib in vitro and in vivo in xenotransplanted mice. These cells were significantly more resistant to osimertinib than the parental cells in vitro and tumors grew as the controls indicating intrinsic resistance to osimertinib. T-DM1 significantly reduced tumor growth but the combination of osimertinib with T-DM1 showed a complete suppression of tumor growth, suggesting an important role of T-DM1 in overcoming osimertinib resistance. These data confirm the role of HER-2 amplification as a potential cause of intrinsic resistance to osimertinib, as demonstrated by Ortiz-Cuaran S et al. , and indIn conclusion, the combination of osimertinib with T-DM1 exerted an additive effect on cell growth inhibition when given as a concomitant administration. However, when cells exposed to osimertinib were subsequently treated with T-DM1, an antagonist interaction was observed. The combination of T-DM1 with osimertinib retarded the appearance of resistance in PC9-T790M cells and overcome osimertinib-resistance in cells with HER-2 amplification. Our results may contribute to define new potential future drug-combinations in the treatment of EGFR mutated NSCLC patients.


KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration. A clinical score based on the activity of genes that regulate cell signaling can predict drug sensitivity and patient outcomes across a range of cancer types. Marie-Claire Wagle, Daniel Kirouac and their colleagues at Genentech in South San Francisco, California, USA developed an index that aggregates expression levels of 10 genes involved in modulating the mitogen-activated protein kinase (MAPK) pathway. This “MAPK Pathway Activity Score”, or MPAS, performed as well or better than other more complicated, genome-based tools at predicting whether drugs that inhibit MAPK-related enzymes were active against tumor cell lines. Retrospective analyses of clinical datasets also showed that MPAS correlated with survival outcomes in patients with melanoma, colon cancer, and breast cancer. The authors suggest that MPAS should be evaluated more broadly and perhaps implemented as a clinically informative biomarker. MAP2K1/2), and extracellular signal-regulated kinase 1/2 (ERK1/2) (MAPK3/1). Nuclear translocation of activated ERK1/2 then triggers the transcriptional activation of multiple target genes involved in modulating the cellular processes of differentiation, proliferation, survival, migration, and angiogenesis.1 Enhanced MAPK pathway activity can be driven by genetic alterations (including amplification and genetic mutations) in RTKs and RAS/RAF family members, as well as aberrant growth factor signaling.2 The high prevalence of aberrant MAPK signaling in cancer has motivated the clinical development of inhibitors targeting critical pathway nodes, including BRAFV600-mutant selective inhibitors, pan-RAF inhibitors, MEK1/2 inhibitors, and ERK1/2 inhibitors.6 Currently, there are at least 20 different MEK, RAF, and ERK inhibitors being tested alone or in combination with other drugs in active clinical trials according to www.clinicaltrials.gov. While single genetic events may not always correspond to pathway hyperactivation in tumors,7 in some instances, these drugs have demonstrated therapeutic benefit alone or in combination in pre-selected patient populations defined by genetic mutations. For example, treatment of BRAFV600E or BRAFV600K mutant melanoma patients with BRAF inhibitors (vemurafenib or dabrafenib) increases overall survival,8 and the combination with MEK1/2 inhibitors (cobimetinib or trametinib) achieves deeper MAPK pathway inhibition, resulting in improved clinical efficacy.11 However, clinical responses to MAPK pathway inhibition in mutant BRAF populations outside of melanoma are more variable,13 and the therapeutic benefit of MEK1/2 inhibitors trametinib, selumetinib, and refametinib does not appear to be associated with KRAS mutation status in lung cancer or pancreatic cancer.16 ERK phosphorylation also varies extensively across cell lines and does not predict MEK inhibitor sensitivity.17 Together, these observations suggest that neither BRAF/ KRAS mutation status nor ERK phosphorylation status alone are sufficiently reliable biomarkers of MAPK pathway activity in multiple cancer indications and that additional, clinically translatable biomarkers are needed.The mitogen-activated protein kinase (MAPK) pathway is a conserved developmental pathway that regulates organ development and tissue homeostasis by transmitting signals through a series of phosphorylation events emanating from receptor tyrosine kinases (RTKs), RAS family members, RAF family members, mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2)  pathway activity and GLI1 transcript levels have been measured in SHH inhibitor clinical trials as a pharmacodynamic biomarker to assess on-target activity.21 For the MAPK pathway, while multiple predictive transcriptional gene signatures have been identified from in vitro data, clinically translating these large gene sets to multivariate biomarkers is challenging due to many technical and practical issues. However, genes that are common between these signatures include direct targets of the MAPK pathway.24 We hypothesized that a simple aggregated gene signature consisting of direct, biologically conserved transcriptional targets of MAPK/ERK signaling may sufficiently capture the activation status of the pathway across different tumor types.Developmental pathways such as MAPK often regulate the activity of a conserved set of transcriptional targets across multiple tissues.BRAFV600E melanoma.9 Furthermore, we demonstrate that MPAS is associated with poor prognosis in adjuvant and metastatic colorectal cancer (CRC), adrenal cancers, and HER2-positive breast cancer. These findings illustrate that MPAS could potentially serve as a predictive and/or prognostic biomarker, thus paving the way for further exploration of the clinical utility of MPAS in the near future.Here we show that a MAPK Pathway Activity Score (MPAS), derived from the transcript levels of 10 MAPK target genes, correlates exclusively with MAPK inhibitor sensitivity in multiple cell lines and primary tumor samples. Retrospective analysis of CoBRIM clinical trial data shows that high MPAS correlates with improved progression-free survival (PFS) of vemurafenib-treated patients in 24 Drugs used in these studies include the MEK inhibitors selumetinib, PD0325901, CI-1040, and RDEA-119 and the BRAF inhibitors, vemurafenib, dabrafenib, and AZ628. These genes also correlate with the extent of tumor response to cobimetinib and the ERK inhibitor, GDC-0994, observed in both pancreatic ductal adenocarcinoma and non-small cell lung cancer (NSCLC) genetically engineered mouse models.25 Furthermore, these genes were the most consistently and dose-dependently inhibited by cobimetinib in three cobimetinib-sensitive cell lines: MEL-JUSO (melanoma), NCI-H2347 (NSCLC), and HUP-T4 (pancreatic cancer), compared to other MAPK pathway targets  that have also been identified as responsive genes in the literature  that have been reported in multiple gene signatures predictive of sensitivity to MAPK inhibition.50 values of cell growth inhibition and MPAS inhibition of three cell lines were similar , MPAS was calculated at each dose of cobimetinib in the three aforementioned cell lines treated with increasing doses of cobimetinib. As shown in Fig. lar Fig. .Fig. 1Ag26 Across all cell lines combined , as well as in breast, lung, colorectal, and melanoma subgroups, cell line sensitivity correlated with MPAS for both MEK and BRAF inhibitors but not inhibitors targeting phosphoinositide-3 kinase (PI3K) (GDC-0941), AKT (GDC-0068), or other pathways (data not shown). Interestingly, there was a modest positive correlation between resistance to AKT inhibition (GDC-0068) and MPAS in breast cancer, supporting reports that, in certain subsets of this disease, the MAPK signaling pathway may mediate resistance to AKT inhibition.27 Of note, hierarchical clustering separated the cancer indications into two classes: those in which MPAS predicted MEK1/2 inhibitor sensitivity , and those in which it did not (pancreatic and melanoma).Given the difference in baseline MPAS values between these three cell lines, we next queried whether such differences in baseline values are predictive of MAPK inhibitor sensitivity. To do so, we examined sensitivity to various signaling pathway inhibitors as measured by mean viability (MV) across a large cancer cell line collection Fig. .26 AcrosBRAFV600-positive melanoma, there was greater variance in sensitivity to this drug across the melanoma cell lines leading to a significant correlation of sensitivity with MPAS.As the pancreatic and melanoma cell lines were broadly sensitive to MAPK pathway inhibition they showed minimal variance in both MPAS and in sensitivity to MEK pathway inhibitors , leading to the absence of significant correlations  model to predict the sensitivity of a large panel of cell lines to the clinically approved MEK1/2 inhibitors cobimetinib and trametinib.26 Cross-validation of the E-Net model for cobimetinib and trametinib gave R values of 0.65 and 0.7, respectively , comparable to results of other such models.29 Predictive features that were identified by the model included a cluster represented by high PHLDA1 . However, the correlation between MPAS and measured MV was more significant .To validate model predictions and compare accuracy of the E-Net model to MPAS, 40 NSCLC cell lines that had not been used for model training were tested for cobimetinib sensitivity .30 Predicted sensitivities using either MPAS or E-Net gene predictors were likewise categorized as “sensitive” or “resistant” using a median cut-off. True positive and false positive rates were then calculated for MPAS, the E-Net model, and KRAS mutation status. A negative control score (CTRL score) derived from four housekeeping genes  was established as a reference  was only slightly better than using a CTRL score, which was equivalent to random chance (50%). In the 40 cell lines that were tested, all the KRAS-mutant cell lines were sensitive to cobimetinib . However, there was a high false negative rate given that many KRAS wild-type cell lines were equally sensitive to cobimetinib . MPAS and the E-Net model had identical accuracies (72%), therefore predictions derived from either applying advanced statistical models to whole transcriptome expression data (E-Net model) or from compiling known biological knowledge into a simple score (MPAS) converge on a surprisingly similar outcome.To further assess the accuracy of the different approaches in classifying MEK1/2 inhibitor sensitive vs. resistant cell lines, we categorized cell lines with ICnce Fig. . The acc28 While the set of cell lines and MEK1/2 inhibitor used were different, the results from DREAM still serve as a reasonable benchmark to compare MPAS against a variety of world-class methodologies. The cumulative distribution of Spearman correlation coefficients for all 45 teams’ predictions of MEK1/2 sensitivity are shown as a solid line in Fig. ρ = 0.55) mapping to the 36th percentile and MPAS (ρ = 0.65) to the 28th percentile. Accordingly, MPAS appeared to perform better than two-thirds of the predictions submitted to this competition. Overall, these results show that MPAS, despite being derived from the expression of only 10 genes, and without the use of regression coefficients or more advanced statistical techniques, predicted sensitivity to MEK inhibition with better or comparable accuracy to using KRAS mutational status or to other genome-based, multivariate predictive models, respectively.To further benchmark the predictive accuracy of MPAS against other gene expression-based predictors of drug sensitivity, we used data generated in the DREAM Drug Sensitivity Prediction Challenge, which engaged 45 bioinformatics teams using gene expression data from 32 breast cancer cell lines to predict sensitivity of 18 blinded cell lines to drugs (including the MEK1/2 inhibitor PD184352).BRAF (blue) or RAS  (red) mutation status for any indication analyzed . Median MPAS of each tissue type was indeed strongly correlated with the median cobimetinib sensitivity of cell lines from the same tissue type . Skin cancer (melanoma) had the highest median MPAS of all the clinical samples, and this was associated with the greatest sensitivity to cobimetinib in cell line studies , the strength of the correlations appeared to diminish, though still significant, with skin cancer remaining the most sensitive indication to cobimetinib treatment  tissues that were analyzed from TCGA varied widely (−1.5 to 2) and independently of mutational status ) than those patients with tumors who had an “immune” signature, characterized by high expression of immune-regulatory genes and CD8+ T-cell infiltration (median PFS: 9.05 (6.21–15.01)).37Approximately 50% of tus Fig. , suggestBRAF-mutant patients, we used the gene expression data to develop a multivariate Cox proportional hazard model with MPAS and the published cell cycle vs. immune categorization as two features and PFS as the measure of outcome. The “cell cycle” vs. “immune” tumor subtype was indeed a significant determinant of responsiveness to vemurafenib as published (hazard ratio (HR) = 2.15; P = 0.0085; Fig. 37 However, BRAF-mutant tumors with higher MPAS  did significantly better than those with low MPAS across all patients on vemurafenib  and resected tumor specimens from CRC patients enrolled in the AVANT phase III adjuvant trial (bevacizumab as a single agent or in combination with either FOLFOX4 or XELOX standard adjuvant therapies40). Of note, the AVANT trial demonstrated no significant difference in overall survival between bevacizumab as a single agent or in combination with adjuvant therapies. As such, data were combined for subsequent analysis. For both mCRC and AVANT datasets and across all patients, high MPAS was associated with significantly worse overall survival, whereas KRAS/BRAF mutational status, microsatellite instability status, and treatment regimens were not ; however, this prognostic effect was not significant in the KRAS-mutant or KRAS/BRAF-wild-type patients . Notably, HRs based on individual MAPK genes rather than the composite MPAS were lower, supporting the use of the aggregate signature as a more robust clinical biomarker  than any single constituent gene.MAPK signaling is known to play an important role in the development of CRC and in responsiveness to EGFR inhibitor treatments.not Fig. . FocusinP = 0.003 at an 85th-percentile cutoff)  patient populations, we performed a Kaplan–Meier scan by computing HRs for MPAS-high vs. -low patients and using a threshold varying from the 10 to 90 percentile. In mCRC patients, the relationship evidently increased monotonically, whereby patients with increasingly higher MPAS did progressively worse  Fig. . Such a ff) Fig. .41 poor overall survival in adrenocortical carcinoma patients has been correlated with MAPK pathway activity based on a recently developed 100-gene MAPK transcriptional signature.24 Likewise, despite the low prevalence of RAS/RAF family mutations in breast cancer, MAPK signaling is thought to be active in some breast cancer subtypes, e.g., triple-negative breast cancer (TNBC), due to KRAS or BRAF amplification.42 In addition, a recent study showed that a significant fraction of HER2+ cell lines are dependent on MAPK signaling for proliferation and survival.43 We therefore assessed whether MPAS is prognostic in these indications in which the activation state of the MAPK pathway seems to be independent of RAS/RAF mutational status.Although adrenal cancers have a low prevalence of RAS/RAF family mutations,P = 0.02) ; P = 0.008), suggesting that variable MAPK signaling dependence in this subgroup has an impact on prognosis  Fig. . Next, wsis Fig. .45 Whilesis Fig. .Fig. 5MP43 We therefore assessed associations between MPAS and EGFR, ERBB3, or CDKN1B transcript levels . High EGFR expression in the HER2+ breast cancers was significantly associated with high MPAS , while HER3 and p27 expression were negatively associated with MPAS  in this subgroup, implying that higher relative expression of EGFR to ERBB3 may be responsible for the higher MAPK signaling and poorer survival outcomes.A previous report demonstrated that cellular dependency on PI3K or MAPK signaling in HER2+ breast cancer could be predicted by RNA and protein expression levels of EGFR, ERBB3/HER3, and CDKN1B/p27.46 In addition, the functional activity of RAS/RAF mutations can be modified by the cellular context, including compensatory feedback through the PI3K pathway, which could alter cellular dependence on MAPK signaling. Therefore, a downstream readout of pathway signal flux based on direct transcriptional targets might better represent the extent of pathway activity. As such, we assessed whether the expression of a core set of MAPK genes would be sufficient to capture the extent of MAPK pathway activation and therefore be of use in the clinic. The 10 genes that constituted MPAS have already been identified in other published gene expression signatures that correlate with MAPK inhibitor sensitivity.24 Some were also identified by the E-Net prediction model of cobimetinib sensitivity described in this report.The MAPK pathway may be activated by mutations in RTK and RAS/RAF oncogene family across many human cancer types. While these mutations are often used as predictive biomarkers for MAPK targeted therapies, other genetic alterations  and epigenetic mechanisms may also contribute to hyperactivation of this oncogenic pathway.BRAF mutational status, supporting the idea of a potential disconnection between mutation status and the activity of the MAPK pathway  population, there existed a wide range of, and perhaps dependency on, MAPK signaling that may partially explain some instances of unresponsiveness to vemurafenib in melanoma.Melanoma had the highest levels of MAPK activity relative to other tissue types (measured by median MPAS) in our study, consistent with the robust clinical activity of BRAF and MEK1/2 inhibitors observed in this indication Fig. . However40 high MPAS was significantly correlated with poorer outcomes, with a trend for wider separation in patients within the BRAF-mutant subgroup, albeit less statistically significant due to smaller patient number ,cer Fig. , which mIn adrenal and breast cancers, RAS and RAF mutations are rare. However, high MPAS in adrenal tumors Fig.  and in a47 Note that the absolute value of the score depends on not only the cell biology but also the assay characteristics and normalization. Thus it is likely not possible currently to directly translate absolute MPAS thresholds from preclinical experiments or between different clinical studies. This is exemplified by examining the variation in the median and distribution of MPAS scores from the five clinical studies reported . The PD0325901 MEK inhibitor was purchased from Selleck Chemicals .26 and cultured according to the supplier’s specifications. The NCI-H2347 cell line  and the MEL-JUSO cell line  were routinely cultured in a growth medium of RPMI-1640 with 10% fetal bovine serum (FBS). The HUP-T4 cell line ACC 223  was routinely cultured in a growth medium of Minimum Essential Medium with 10% FBS.Cell lines were obtained from the Genentech cell line repository (gCSI)48 With this method, cell growth after treatment can be compared to that before treatment . Three replicate wells per treatment were used, such that, at 5% standard deviation, viability differences of >9% should be detectable with a power of 80%. Data shown represent three independent experiments.Cells were seeded in 96-well plates and treated in triplicate for 72 h with 0–10 μM concentrations of the relevant drug diluted in the appropriate growth medium (please see above). Cell viability was measured using CellTiter-Glo® reagent per the manufacturer’s instructions  and ATP measurements were read using an Envision reader . Cell viability measurements were corrected to the day 0 viability (day of treatment) as measured by CellTiter-Glo® and plotted relative to the dimethyl sulfoxide control (100% viability).n = 50), CRC (n = 50), and lung patients (n = 100)  tissue blocks were procured from melanoma . For the Nanostring data, gene expression levels were normalized by sample, using a set of housekeeping genes, and across samples for the RNA-Seq data. The score is thus a relative metric of MAPK gene expression, the absolute values of which will depend on the assay characteristics and choice of normalization, in addition to the underlying biology. As such, MPAS values should be interpreted as relative to other samples within an experimental or clinical context.MPAS for each cell line, tumor, or patient sample was derived from expression data for 10 MAPK-specific genes . Gene expression was measured using either RNA-Seq  sequencing (reads per counts per million)49 This model was trained on gene expression features with alpha = 0.5 and optimal lambda chosen by 5-fold cross validation. After applying a variance stabilizing transformation, gene expression was normalized to a normal distribution  to remove absolute expression bias.49 To cross-validate the model, predicted MVs of the cell lines used to train the model were correlated with experimentally derived MVs to both trametinib and cobimetinib , cell lines in the validation set (40 NSCLC cell lines) were categorized as either cobimetinib sensitive (IC50 < 1 µM) or resistant (IC50 > 1 µM) based on the average clinical serum concentration of cobimetinib (0.1–1 µM).30 Based on numbers of True Positive (TP) True Negative (TN), False Positive (FP), and False Negative (FN) calls, the true positive rate (TPR = TP/(TP + FN)), false positive rate (FPR = FP/(FP + TN)), and accuracy (ACC = (TP + TN)/N) were then computed using a median-based classifier of the E-Net predictions and MPAS.To assess the overall accuracy of each predictor  approval and informed consent were analyzed using a custom melanoma-specific Nanostring expression panel consisting of 800 genes including the 10 MAPK-specific genes constituting MPAS   and from 1262 curatively resected stage III CRC patients enrolled in the AVANT phIII adjuvant trial44mRNA expression (RPKM) data for the 10 MAPK genes, as well as patient survival and subtype classifications (for Breast Cancer) were obtained from the BROAD Firehose TCGA portal. Breast cancer data were from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study.Rank-sum tests were used to compute all correlation coefficients. Cox proportional hazard regression models were developed using MPAS as a continuous univariate predictor or as part of a set of multivariate predictors, i.e., mutational status or treatment. Log-rank tests were performed for all Cox hazard regression models. For the CRC data, a Kaplan–Meier scan was performed using MPAS as a discrete predictor, varying the threshold for calling positive/negative over a range from the 10th to 90th percentiles, and HRs were computed along this range. For the melanoma data, a sliding 30th-percentile window was used, and HRs were computed in comparison to the unselected population  vs. the entire population, and increasing the window by 2 points successively across the range to the 70–100th percentile (median 85%)). The E-Net regression model was developed in R, and all computational analyses were performed in the MATLAB v2015b software .Expression data for Figs. Supplementary figures and tables


Neuroblastoma (NB) is a devastating disease. Despite recent advances in the treatment of NB, about 60% of high-risk NB will have relapse and therefore long-term event free survival is very minimal. We have reported that targeting glycogen synthase kinase-3 (GSK-3) may be a potential strategy to treat NB. Consequently, investigating LY2090314, a clinically relevant GSK-3 inhibitor, on NB cellular proliferation and may be beneficial for NB treatment.The effect of LY2090314 was compared with a previously studied GSK-3 inhibitor, Tideglusib. Colorimetric, clonogenic, and live-cell image confluency assays were used to study the proliferative effect of LY2090314 on NB cell lines . Western blotting and caspase glo assay were performed to determine the mechanistic function of LY2090314 in NB cell lines.LY2090314 treatment exhibited significant growth reduction starting at a 20 nM concentration in NGP, SK-N-AS, and SH-SY-5Y cells. Western blot analysis indicated that growth suppression was due to apoptosis as evidenced by an increase in pro-apoptotic markers cleaved PARP and cleaved caspase-3 and a reduction in the anti-apoptotic protein, survivin. Further, treatment significantly reduced the level of cyclin D1, a key regulatory protein of the cell cycle and apoptosis. Functionally, this was confirmed by an increase in caspase activity. LY2090314 treatment reduced the expression levels of phosphorylated GSK-3 proteins and increased the stability of β-catenin in these cells.LY2090314 effectively reduces growth of both human MYCN amplified and non-amplified NB cell lines in vitro. To our knowledge, this is the first study to look at the effect of LY2090314 in NB cell lines. These results indicate that GSK-3 may be a therapeutic target for NB and provide rationale for further preclinical analysis using LY2090314. Neuroblastoma (NB) is the most common extracranial solid tumor found in children with over 650 cases diagnosed each year in the United States and it is responsible for 15% of pediatric cancer deaths . Timely Several intracellular signaling pathways have been demonstrated to play a key role in embryonal tumor biology, including growth factors controlling tumor proliferation, survival, differentiation and metastasis –14. Furt2 in air at 37 °C. LY2090314 and Tideglusib  were dissolved in dimethyl sulfoxide . An equivalent amount of DMSO alone in the treatment group served as a control.Human NB cell lines SK-N-AS (CRL-2137) and SH-SY-5Y (CRL-2266) were purchased from American Type Culture Collection  and NGP cells were a kind gift from Dr. Thiele at the National Cancer Institute  and were authenticated before receipt and use. NGP, SH-SY-5Y, and SK-N-AS cell lines were maintained in RPMI1640 medium , supplemented with 10% fetal bovine serum , and a combination of 100 IU/mL penicillin and 100 μg/mL streptomycin  in a humidified atmosphere of 5% COCell viability of each cell line was measured using a 3--2, 5-diphenyltetrazolium bromide colorimetric (MTT) assay. A 96-well plate was seeded with each cell line and incubated overnight for adhesion. Tideglusib treatment in increasing micromolar concentrations (0 – 30 μM) was added to each well, and for drug comparison, LY2090314, was added separately in increasing nanomolar concentrations, (0 – 1000 nM). Control wells were treated with DMSO. All wells were made in quadruplicate, and all plates were incubated for 48, 72, or 96 h. Cell viability was measured as described , 26. TheClonogenic (CFU) assays were performed to determine the ability of NB cells to form colonies after treatment with LY2090314 using the standard protocol as described previously , 26. BriIncuCyte Live-cell Imaging system (Essen Bioscience) was used to measure cellular proliferation/confluence of NB cell lines as described previously , 26. AppS9 and S21 , and active GSK-3Y279 and Y216 . Next day, the membranes were washed three times in a 1× phosphate buffered solution with .05% Tween-20 buffer and incubated with horse-radish peroxidase linked anti-mouse or anti-rabbit secondary antibody  depending on the source of the primary antibody. The membranes were developed using Supersignal West Dura or West Femto (Thermo Fisher Scientific) and imaged using the Molecular Images Chemi-Doc XRS+ imager (Bio-Rad).Cell lysates were prepared and quantified as described previously , 26. Bri+ imager (Bio-Rad). The relative number for the band intensities were calculated by normalized against GAPDH.After visualizing proteins bands on the membrane, the band intensities were quantified using Image Lab software version 5.2.1, Molecular Images Chemi-Doc XRSA Caspase-Glo 3/7 Assay  kit was used to measure cleaved caspase-3 and -7 activities from cell lysates after LY2090314 treatment as described previously , 26. Brip value of < 0.05 was considered significant. Data were represented as ± SE.One-way ANOVA analysis was performed using a statistical analysis software package (IBM SPSS Statistics version 22). A Several assays and imaging techniques were utilized to determine cellular growth patterns of 3 NB cell lines  treated with LY2090314 or Tideglusib. Cells were plated and treated with LY2090314 in increasing nanomolar concentrations , and proliferation was recorded using a colorimetric, MTT assay at 48 h, 72 h, and 96 h Fig. . Lastly,To determine the pathway mechanism behind the reduction in cell growth, western blotting was performed, proteins of interest were sorted, and the band intensities were measured compared to GAPDH control Fig. . LY20903Neuroblastoma is a rare developmental cancer of the sympathetic nervous system found primarily in infants and young children under 5. Its unique presentation and tumor biology present challenges for effective treatment regimens, notably in patients diagnosed as high risk (>MYCN expression) , 27, 28.Our results confirm that inhibition of GSK-3 by LY2090314 results in NB growth suppression. Importantly, low concentrations of LY2090314 significantly reduced NB cellular growth compared to Tideglusib, another GSK-3 inhibitor. This is the first study in NB, and LY2090314 is a potential agent for the treatment of NB in future.