We validated that CUDC-101 inhibited cellular proliferation and resulted in cell death by inducing cell cycle arrest and caspase-dependent apoptosis. CUDC-101 also inhibited cellular migration in vitro. Mechanistically, CUDC-101 inhibited MAPK signaling and histone deacetylation in ATC cell lines with multiple driver mutations present in human ATC. The anticancer effect of CUDC-101 was associated with increased expression of p21 and E-cadherin, and reduced expression of survivin, XIAP, β-catenin, N-cadherin, and Vimentin. In an in vivo mouse model of metastatic ATC, CUDC-101 inhibited tumor growth and metastases, and significantly prolonged survival. Response to CUDC-101 treatment in vivo was associated with increased histone 3 acetylation and reduced survivin expression. Our findings provide a preclinical basis to evaluate CUDC-101 therapy in ATC.Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies that currently has no effective therapy. We performed quantitative high-throughput screening (qHTS) in three ATC cell lines using 3,282 clinically approved drugs and drug candidates, and identified 100 active agents. Enrichment analysis of active compounds showed that inhibitors of EGFR and histone deacetylase (HDAC) were most active. Of these, the first-in-class dual inhibitor of EGFR, HER2 and HDACs, CUDC-101, had the highest efficacy and lower IC Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. It accounts for less than 2% of all thyroid cancers, but results in nearly one-third of thyroid cancer–related deaths . The medTP53-inactivating mutations are common (42–55%), followed by mutations in genes involved in the PI3K/AKT/mTOR, EGFR/RAS/BRAF/MEK/ERK, and WNT signaling pathways [RAS and BRAF occur in approximately one-fourth of ATC cases [PIK3CA and PTEN occur in 17–23% and 12% of ATC cases, respectively [Drug discovery and development are costly and time-consuming, and rarely focused on rare malignancies. This is partly because it takes an average of 15 years and costs approximately $800 million to bring a single drug to market; this cost would not be recouped for rare cancer indications. Thus, quantitative high-throughput screening (qHTS) of existing drugs has become an emerging strategy for identifying new drugs for rare diseases/cancers such as ATC . There apathways . MutuallTC cases , 8. Mutaectively . Thus, ain vivo, resulting in prolonged survival in a mouse model of metastatic ATC. Response to CUDC-101 treatment in vivo was associated with increased histone H3 acetylation and decreased survivin nuclear staining in tumor tissues.In this study, we performed qHTS in multiple ATC cell lines and identified 100 active compounds that were enriched for inhibitors of epidermal growth factor receptor (EGFR) signaling and histone deacetylase (HDAC). One of the most potent compounds identified was CUDC-101, a first-in-class dual inhibitor of EGFR, HER2 and HDACs . We thenTP53 and the genes involved in PI3K/AKT/mTOR and MAPK pathways are frequently mutated in ATC, we first examined the mutation status of genes involved in these pathways. As summarized in Table TP53-inactivating mutations, as well as mutations in EGFR, MET, BRAF, RAS, PI3K, PTEN, and mTOR, suggesting that these cell lines are representative of human ATC. Thus, we used these three cell lines for the qHTS of the 3,282-compound library, and identified 100 compounds that were pan-active in the three ATC cell lines  at 0.15 μM for 8505c, and 1.66 μM for both C-643 and SW-1736 cells. Current and past chemotherapy agents for ATC include sorafenib, paclitaxel, carboplatin, docetaxel, and doxorubicin as monotherapy or in combination with other agents, or as radiosensitizers to external beam radiation therapy [50) than sorafenib, paclitaxel, carboplatin, docetaxel, and doxorubicin.One of the highly active agents identified was CUDC-101, a first-in-class dual EGFR, HER2 and HDAC inhibitor . CUDC-10 therapy –14. TherKRAS mutation), THJ-16T , THJ-21T , and THJ-29T (RB mutation) [As previously reported, HDAC1, HDAC2, and EGFR are over-expressed in ATC –19. Therutation) . We firsTo understand the mechanism by which CUDC-101 inhibited cellular proliferation and caused cell death at higher concentrations, we next assessed the effect of CUDC-101 on cell cycle progression and apoptosis using the three representative ATC cell lines used in the qHTS. Cell cycle analysis revealed that CUDC-101 treatment decreased the number of cells in the S phase and induced accumulation of cells in G2/M phase, which were dose-dependent Figure . To deteWe next investigated whether CUDC-101 had any effect on cellular migration because ATC is a highly invasive cancer and the EGFR/RAS/BRAF/MEK/ERK pathway has been shown to regulate cellular migration and epithelial-mesenchymal transition (EMT) –23. CompCUDC-101 is thought to be a dual HDAC, EGFR, and HER2 inhibitor; therefore, we examined its inhibitory effect on HDAC and EGFR downstream pathways in ATC cells . CUDC-10As mentioned above, CUDC-101 inhibited cell cycle progression of ATC cells by reducing the cell numbers in the S phase and inducing arrest in the G2/M phase. Therefore, we examined the expression of p21, an important cell cycle regulator. Under regular culture condition, ATC cells had very low expression of p21, matching their aggressive cellular phenotype. CUDC-101 treatment induced an increase in p21 expression Figure . FurtherTo determine if alternative activated pathways implicated in targeted therapy resistance are not activated with CUDC-101 therapy, and to further understand additional mechanism by which CUDC-101 may have an anticancer effect, we performed phospho-kinase protein arrays using 8505c and SW-1736 cells. As expected, CUDC-101 reduced the phosphorylation of ERK in both cell lines, but also decreased the expression of β-catenin Figure . We alsoin vitro observations, the effect of CUDC-101 treatment was evaluated in a metastatic mouse model of ATC (8505c-Luc2) that recapitulates the aggressive nature of human ATC [in vivo experiments. We found increased levels of acetyl-histone H3 and decreased survivin nuclear staining in tumors that responded to therapy ; C-643 and SW-1736 were purchased from Cell Lines Service ; THJ-11T, THJ-16T, THJ-21T, and THJ-29T have been described previously . Cell liThe targeted sequencing of genes involved in the PI3K/AKT/mTOR, EGFR/RAS/BRAF/MEK/ERK, and WNT signaling pathways was performed using the Ion Torrent TargetSeq platform for 409 known tumor suppressor and oncogenes at the National Cancer Institute (NCI) Frederick Laboratory of Molecular Technology .http://tripod.nih.gov/npc/. qHTS was performed in three ATC cell lines, 8505c, C-643, and SW-1736, as previously described [Two pharmaceutical collections were used: The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC) and the Mechanism Interrogation PlatE (MIPE) collection. Additional information on NPC drug libraries can be found at Cell proliferation assay was performed using the CyQuant kit . Apoptosis was determined using the Caspase-Glo 3/7 assay kit (Promega) and normalized to the cell number.Cell cycle analysis was performed at 24 hour after drug treatment on a FACScan using CellQuest software . Cell cycle data was analyzed using Modfit software .Cellular migration was determined using the Transwell chamber assay (BD Biosciences). After 24 hours of incubation with CUDC-101 or vehicle, cells were trypsinized, and an equal number of live cells were plated in each of the transwell chambers. After 22 hours of incubation at 37°C, the migrated cells were fixed, stained with Diff-Quik  and photographed.Following antibodies were used: anti-p21 (1:500), anti-vimentin , anti-E-cadherin (1:250), anti-survivin , anti-phospho-Erk1/2 , and anti-Erk1/2 were from Cell Signaling Technology ; anti-N-cadherin  was from EMD Millipore ; anti-β-catenin (1:250) and anti-XIAP  were from R&D Systems ; and anti-β-actin  was from Santa Cruz Biotechnology .Human phospho-kinase  array  were purchased from R&D Systems . Signal intensity of each spot was quantified with Image J  and averaged for each specific protein.Tissues were fixed in 10% formalin, embedded in paraffin, sections were incubated with primary antibody  at 4°C overnight, and immunostaining was performed using Vectastain ABC and DAB kits .in vivo ATC metastasis mouse model was used to assess the effect of CUDC-101 [tm1WjlPrkdcscid Il2rg/SzJ mice. Bioluminescence imaging was used to assess tumor burden using the Xenogen in vivo imaging system  [An CUDC-101 . The Aniton, MA) .CUDC-101  was reconstituted according to pharmacy manual directions. Eight days after tumor cell injection, widespread lung metastasis was established. The mice were randomized into two groups and injected with either CUDC-101 (120 mg/kg) or vehicle (10% captisol) daily, alternating between intravenous and intraperitoneal injections. Treatment continued until the first mouse reached humane euthanasia criteria endpoints. Then the mice were housed under normal care conditions with no further treatment and monitored daily.scidIl2rgtm1WjlPrkdc/SzJ mice were first injected intraperitoneally with either CUDC-101 (120 mg/kg) or 10% captisol solution. Twenty minutes later, the mice were injected with 8505C-Luc2 cells via tail vein. CUDC-101 (120 mg/kg) or 10% captisol solution was injected daily. Treatment was continued until the first mouse reached humane euthanasia criteria endpoints. Then the mice were imaged, and euthanized by CO2 inhalation.To assess the effects of CUDC-101 on metastasis and as an adjuvant therapy, a pre-treatment mouse study was also performed. In this group, six-month-old NOD.Cg-qHTS data analysis was performed as previously described . For val


None were identified by exome sequencing of 1015 cell lines, including 37 derived from hematopoietic neoplasms.1 We therefore looked for cell lines derived from patients with leukemic transformation of a preceding MPN. Given that CALR and JAK2 mutations are almost completely mutually exclusive,6 we focused on four such lines known to lack a JAK2 mutation , and also tested a further 52 other predominantly myeloid cell lines  and carries a heterozygous 61-basepair (bp) deletion in CALR exon 9 , which, like all other reported CALR mutations, results in a +1- bp shift in the reading frame and thus generates a novel C terminus  and a 5-bp insertion .8 Both the type 1 deletion and the MARIMO deletion are immediately preceded by a nucleotide sequence identical to that at the 3' end of the deletion  or CML (K562) total CALR mRNA levels were 10-fold higher in MARIMO  showed that HEL and UKE-1 (both JAK2V617F positive) had IC50 values of 217 and 430 nm, respectively , STAT5 and pSTAT5  and JAK-inhibitor-I (a pan-JAK inhibitor) . MARIMO ectively . In markd pSTAT5 . The lacd pSTAT5 . JAK2 trll lines , suggestJAK2-mutated cell lines. Our results therefore raise the possibility that mutations of CALR and JAK2 may share activation of pathways other than the STATs. Superficially our data appear to contrast with reports that JAK2-unmutated and CALR-mutated MF patients respond to ruxolitinib.15 However in JAK2V617F-positive patients studies of mutant allele burden show that Ruxolitinib has a minimal effect on the mutant clone.15 It is therefore likely that the clinical responses to Ruxolitinib  do not reflect a cytotoxic effect of the drug on the neoplastic clone, but instead are at least in part due to down-modulation of pro-inflammatory signaling cascades.16Together, our data demonstrate that the MARIMO cell line harbors a CALR mutation and yet is not dependent on JAK/STAT signaling, in marked contrast to


L, and MCL-1 have been characterized as key survival factors in multiple cancer types. Because cancer types with BCL2 and MCL1 amplification are more prone to inhibition of their respectively encoded proteins, we hypothesized that cancers with a significant frequency of BCL2L1 amplification would have greater dependency on BCL-XL for survival.Defects in programmed cell death, or apoptosis, are a hallmark of cancer. The anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins, including BCL-2, BCL-XBCL2L1 amplification, we performed data mining using The Cancer Genome Atlas (TCGA) database. We then assessed the dependency on BCL-XL in a panel of cell lines using a selective and potent BCL-XL inhibitor, A-1155463, and BCL2L1 siRNA. Mechanistic studies on the role of BCL-XL were further undertaken via a variety of genetic manipulations.To identify tumor subtypes that have significant frequency of BCL2L1 amplification across all tumor types examined. Colorectal cancer cell lines with BCL2L1 copy number >3 were more sensitive to A-1155463. Consistently, cell lines with high expression of BCL-XL and NOXA, a pro-apoptotic protein that antagonizes MCL-1 activity were sensitive to A-1155463. Silencing the expression of BCL-XL via siRNA killed the cell lines that were sensitive to A-1155463 while having little effect on lines that were resistant. Furthermore, silencing the expression of MCL-1 in resistant cell lines conferred sensitivity to A-1155463, whereas silencing NOXA abrogated sensitivity.We identified colorectal cancer as having the highest frequency of L in mediating the survival of specific tumor types, and indicate that BCL-XL inhibition could be an effective treatment for colorectal tumors with high BCL-XL and NOXA expression.This work demonstrates the utility of characterizing frequent genomic alterations to identify cancer survival genes. In addition, these studies demonstrate the utility of the highly potent and selective compound A-1155463 for investigating the role of BCL-X Cancer is a genetic disease that arises from the accumulation of somatic gene alterations. One way to identify key genes in cancer is to examine genomic regions that undergo frequent alterations. Recent technological advances in characterizing these alterations make it possible to identify genes that are essential for the initiation and/or survival of cancer, and thus encode potential therapeutic targets.L, BCL-W, BFL-1/A1, and MCL-1), 2) multi-domain pro-apoptotic , and 3) BH3-only pro-apoptotic . The BH3-only proteins contain a single BH3 domain and are bound by specific anti-apoptotic proteins [L bind and antagonize BIM but not NOXA. In contrast, MCL-1 and A1 bind and antagonize NOXA but not BAD protein. Other BH3 domain proteins such as BIM and PUMA are bound and antagonized by all anti-apoptotic proteins. BAX and BAK are known as the “effectors”. Once activated, these proteins oligomerize on the outer mitochondrial membrane and induce pore formation; this results in the release of cytochrome c and other pro-apoptotic proteins that ultimately carry out the cell death mechanism.Evasion of apoptosis is a hallmark of cancer cells. One mechanism of apoptotic pathway deregulation is via upregulation of the anti-apoptotic BCL-2 family members. Apoptotic pathway proteins belong to a family of BCL-2 Homology (BH)-domain-containing proteins comprising three classes: 1) multi-domain anti-apoptotic  subtype of DLBCL [The role of anti-apoptotic BCL-2 family proteins in various cancers has been well studied –6. In adof DLBCL . Not surof DLBCL .MCL1 was reported to be amplified in 10.9 % of tumor samples analyzed, spanning multiple cancer subtypes [MCL1 region identified lung and breast cancers as having significantly higher frequencies of focal amplification, suggesting that these tumors depend on MCL-1 for survival. This is supported by multiple studies demonstrating that cell lines with MCL1 amplification are sensitive to siRNA knockdown of MCL1 [subtypes . Fluores of MCL1 , 13.L has been implicated as a key survival factor in numerous solid tumors [BCL2 and MCL1 amplification are more prone to inhibition of their encoded proteins, we hypothesized that cancers with a significant frequency of BCL2L1 amplification are more dependent on BCL-XL for survival. In this study, we identified colorectal cancer as having a significant incidence of BCL2L1 amplification. We then dissected the role of BCL-XL in colorectal cancer cell lines using a selective small-molecule inhibitor of BCL-XL and a variety of genetic manipulations.BCL-Xd tumors . Based oL inhibitor A-1155463 and navitoclax were synthesized at AbbVie, Inc. . All the siRNAs were purchased from Dharmacon .BCL-X2.Colorectal cell lines (ATCC) were cultured in RPMI  supplemented with 10 % fetal bovine serum (FBS) (Invitrogen), 1 % sodium pyruvate (Invitrogen), and 4.5 g/L glucose , or DMEM (Invitrogen) supplemented with 10 % FBS. All the lines were maintained in a humidified chamber at 37 °C containing 5 % COL siRNA used is ACAAGGAGAUGCAGGUAUUUU (Dharmacon). The sense sequences of the MCL-1 siRNAs used is GCATCGAACCATTAGCAGATT (Dharmacon). The cells were then grown in medium without antibiotic before harvesting for western blotting analysis. LS1034 cells were transfected at 1.5–2.5 × 104 cells/100 μl in 96-well tissue culture plates with 20 nM Noxa siRNA pool (Dharmacon). The cells were grown in medium without antibiotic before harvesting.LS1034, SW1417, GEO, and RKO cells were transfected in 6-well plates with siRNAs using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). A final concentration of 20 nM siRNA was used in all cases. The sense sequences of the BCL-XCells were treated with increasing concentration of A-1155463. Cells were assayed for viability after 72 h using the CellTiter-Glo luminescent cell viability assay according to the manufacturer’s protocol . Results were normalized to cells without treatment. EC50 was calculated using the GraphPad Prism software .L , anti-MCL-1 (Epitomics), anti-BCL-2 (BD), anti-BIM (Epitomics), anti-actin and anti-NOXA . Antibody against tubulin  was used as a loading control.Cell lysates were prepared in RIPA buffer (Sigma) plus protease inhibitor cocktail (Roche). 20 μg of total protein was resolved on a 12 % SDS polyacrylamide gel and probed with anti-BCL-X6 cells/ml. The cells were stored in the dark at room temperature for at least 30 min or up to 24 h at 4 °C before the DNA content was determined by flow cytometry using a FACS Calibur .LS1034 cells were treated with DMSO or 200 nM A-1155463, with or without 50 μM Z-VAD caspase inhibitor  for 72 h. DNA content was measured by flow cytometry to determine the effect of the inhibitors on the cell cycle and cell death. Following treatment, cells were spun down, the medium was removed and the cells were resuspended in staining solution  at a cell concentration of 1 × 10L has been implicated as a key survival factor in numerous solid tumors. Since cancer types with BCL2 and MCL1 amplification are more prone to inhibition of these proteins, we reasoned that significant frequency of BCL2L1 amplification in a particular cancer type may indicate dependency on BCL-XL for survival. To identify cancer types with a significant amplification of BCL2L1, we utilized The cBio Cancer Genomics Portal , an open-access resource for interactive exploration of multidimensional cancer genomics data sets from more than 5,000 tumor samples from 20 cancer studies [BCL2, BCL2L1 and MCL1 among a majority of these tumor samples . Furthermore, there is a good correlation between BCL2L1 copy number gain/amplification and gene expression as determined by RNAseq and microarray analysis  and lung adenocarcinoma has the highest percentage of MCL1 amplification (20 %)  Fig. . These dBCL2L1. We first investigated the sensitivity of these colorectal cell lines to treatment with the selective BCL-XL inhibitor, A-1155463 .To test this hypothesis, we selected a panel of 25 colorectal cancer cell lines with known copy number of nes Fig.  as definL, and BCL-W, and ABT-199, a specific inhibitor of BCL-2 [BCL2L1 gains are sensitive to selective BCL-XL inhibition. Among the 11 lines with BCL2L1 gain (CN > 3), 9 are sensitive to A-1155463 (EC50 < 0.5 μM). In contrast, only 3 out of 11 lines with no CN gain of BCL2L1 are sensitive to A-1155463  gain, protein and mRNA expression of BCL-2 family members is a crucial determinant of sensitivity to BH3 mimetics such as ABT-737, an inhibitor of BCL-2, BCL-Xof BCL-2 , 16–18. 463 Fig. .L  between cell lines with BCL2L1 gain and BCL-XL protein expression. In addition, sensitive lines had higher protein expression of BCL-XL (p = 0.0002) and NOXA (p = 0.02), higher BCL-XL to MCL-1 ratio (p = 0.0027), and a trend towards lower expression of MCL-1, although not statistical significant (p = 0.12). The Pearson correlation between log EC50 and expression of BCL-XL, NOXA, MCL-1, and BCL-XL/MCL-1 was 0.75, 0.653, 0.363, and 0.637, respectively. The Spearman correlation between EC50 and expression of BCL-XL, NOXA, MCL-1, and BCL-XL/MCL-1 was 0.789, 0.749, 0.277, and 0.795, respectively. BCL-2 expression was detected in a single line, consistent with other studies showing that BCL-2 expression is less common in solid tumor cell lines [L. Additionally, sensitive lines had higher expression of BCL2L1 (p = 0.0078) and NOXA (p = 0.02), a higher BCL2L1 to MCL1 ratio (p = 0.0086), and a trend towards lower expression of MCL1 mRNA, although not statistical significant (p = 0.15) , an inhibitor of BCL-2, BCL-XL and BCL-W [L and the anti-apoptotic gene BIRC5. APC is part of a destruction complex that mediates the ubiquitin-targeted proteolysis of β-catenin. Mutations in APC destabilize this complex and lead to the accumulation of β-catenin, which can then translocate to the nucleus and aberrantly activate the transcription of key drivers of oncogenesis [L was described to be regulated by the β-catenin pathway and multiple components of the pathway are highly mutated in colorectal cancer, we determined whether a correlation exists between these mutations and the activity of inhibitor A-1155463 in the colorectal cancer cell lines. While the majority of these lines have mutations in APC, only two lines have β-catenin mutations according to the Cancer Cell Line Encyclopedia (CCLE) [L for survival as revealed by sensitivity to A-1155463  oligonucleotides specific to BCL2L1 in two sensitive (LS1034 and SW1417) and two resistant (RKO and GEO) colorectal cell lines. The two sensitive lines also exhibit BCL2L1 gain. BCL2L1 siRNA effectively knocked down BCL-XL protein expression completely in three cell lines, and ~80 % in SW1417 cells . In contrast, there was minimal effect on the viability of the inhibitor-resistant cell lines, thus confirming that BCL-XL is critical for survival of a subgroup of colorectal cell lines. The viability is also confirmed using another assay that quantifies the ATP present in culture as an indicator of metabolically active cells  by silencing the expression of MCL-1 with siRNA. While silencing MCL-1 alone had no effect (data not shown), MCL-1 knockdown in the presence of A-1155463 afforded complete cell killing .NOXA is a pro-apoptotic protein that antagonizes MCL-1 activity. Because NOXA expression is higher in the sensitive lines Fig. , we sougent Fig. , indicatBCL2L1 amplification using genomic mining. Our second major aim was to investigate the dependency of corresponding cell lines on BCL-XL for survival. To this end, we used not only siRNA but a recently disclosed small molecule inhibitor of BCL-XL that shows high potency and selectivity against other pro-survival proteins. Our study identified colorectal and cervical cancers as having the most frequent BCL2L1 amplification. Further studies showed that the majority of colorectal cancer cell lines with a gain in BCL2L1 are sensitive to the selective BCL-XL inhibitor A-1155463 as well as to siRNA-medited knockdown of BCL2L1. Although the gains are typically modest (3–4.4 copies), it should be noted that CN gain of other anti-apoptotic genes such as MCL1 is also modest in other tumors that nevertheless become dependent on these genes for survival [L and NOXA, and a trend towards lower protein and mRNA expression of MCL-1 than the resistant lines.In this study, we set out to first identify cancer types that show high levels of survival . In addiL dependence and the presence of β-catenin or APC mutations. Possible reasons for this discrepancy could be differences in the colorectal cell lines used in the two studies, or alternatively, the fact that the highly selective BCL-XL inhibitor was not available at the time of the earlier study by Basu et al. and the study was thus confounded by the mixed selectivity of the compound used [L as a survival factor [L may be regulated through multiple pathways in colorectal cancer, including CN gain and active β-catenin signaling. The role of other pathways cannot be ruled out.Despite a recent report that navitoclax-sensitive cell lines are enriched with β-catenin mutations , we founund used . Our datl factor . In thisL-selective inhibitor A-1155463. Mechanistically, A-1155463 and navitoclax displace pro-apoptotic protein BIM from BCL-XL, which leads to the activation of apoptosis [L inhibitor is readily sequestered by MCL-1, thereby inhibiting the activation of the apoptotic pathway. Indeed, it has been shown that MCL-1 and BCL-XL play compensatory roles in regulating apoptosis in most solid tumor cell lines [L inhibition, expanding on earlier studies with the BCL-2/BCL-XL inhibitors ABT-737 and ABT-263 [Kras or BRAF mutations are sensitized to BCL-2/BCL-XL inhibition [Similar to navitoclax, we found that MCL-1 is a resistance factor for the BCL-Xpoptosis , 25. In ll lines , 16, 26. ABT-263 –30. By thibition . Our obshibition .MET and ALK alterations [BCL2L1 gain, we identified colorectal cancers as being potentially more dependent on BCL-XL and more susceptible to BCL-XL inhibition. Indeed, by using a potent and selective BCL-XL inhibitor, A-1155643, we have shown that BCL-XL inhibition is sufficient to kill colon cancer cell lines expressing high levels of BCL-XL and the MCL-1-neutralizing protein NOXA. Of particular interest, sensitive lines are enriched in a gain of BCL2L1, suggesting BCL2L1 gain could be used as a patient stratification biomarker. This finding demonstrates the power of identifying potential new targets by examining genomic regions that undergo frequent alterations as well as the utility of highly potent and selective tool molecules. Finally, BCL-XL selective inhibitors may have utility as therapeutics for the treatment of colorectal cancer, a disease for which there is significant unmet medical need.Other gene amplifications have served as therapeutic targets for other cancers. For example, the MET and ALK tyrosine kinase inhibitor crizotinib (PF-02341066) shows differential antitumor effects in non-small cell lung and gastric cancer according to erations . By utilL inhibition could be an effective treatment for colorectal tumors with high BCL-XL and NOXA expression. Additionally, BCL2L1 gain may represent stratification biomarker for colorectal cancer patients undergoing treatment with a BCL-XL inhibitor. Future studies should investigate the utility of BCL2L1 gain as a stratification biomarker.BCL-X


Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC Anthelmintic benzimidazoles including mebendazole and flubendazole were shown to exert anti-cancer activity by mechanisms including inhibition of microtubule function123456Mebendazole affects the viability of cancer cells in experimental systems from a broad spectrum of cancer entities including lung cancer, breast cancer, ovary cancer, adrenocortical carcinoma, osteosarcoma, melanoma, glioblastoma, and colorectal carcinoma13456Here, we performed a screen of flubendazole in a panel of 321 cell lines including cell lines from 26 cancer entities. Among leukaemia and multiple myeloma, neuroblastoma was identified as a highly flubendazole-sensitive cancer entity. Flubendazole showed broad activity in primary neuroblastoma cells and a panel of 140 neuroblastoma cell lines with acquired drug resistance. The anti-neuroblastoma activity of flubendazole involved p53-mediated apoptosis and the MDM2 inhibitor and p53 activator nutlin-3 strongly enhanced the flubendazole effects. A water-soluble flubendazole-(2-hydroxypropyl)-β-cyclodextrin preparation inhibited vessel formation and tumour growth in the chick chorioallantoic membrane (CAM) model in vivo.max of 3.6 μM90 values in neuroblastoma cells to be significantly lower than those from 21 out of the 25 other investigated cancer cell types  displayed IC90s > 1 μM and <5 μM, and 173 cell lines (54%) displayed IC90s > 5 μM. There were clear differences between the individual cancer entities. In leukaemia/lymphoma 40 (82%) out of 49 cell lines, in multiple myeloma 10 (100%) out of 10 cell lines, and in neuroblastoma 28 (88%) out of 32 cell lines displayed an IC90 < 1 μM. Together, these three entities accounted for 78 (67%) of the 117 cell lines that displayed IC90s < 1 μM among the 26 cancer entities. In all other entities, except Ewing's sarcoma (4 (57%) out of 7 cell lines with an IC90 < 1 μM) and head and neck cancer (3 (60%) out of 5 cell lines with an IC90 < 1 μM), the majority of the cell lines displayed IC90s > 1 μM. None of the 9 gastric cancer, the 13 melanoma, the 6 oesophageal cancer, the 10 ovarian cancer, the 10 pancreas cancer, the 5 prostate cancer, or the 3 retinoblastoma cell lines displayed an IC90 < 1 μM (117 (36%) of the 321 cell lines displayed IC0 < 1 μM .90s < 1 μM, 11 cell lines (8%) displayed IC90s > 1 μM and <5 μM, and 10 cell lines (7%) displayed IC90s > 5 μM  of the drug-resistant cell lines the fold change was >2.0. In 14 drug-resistant cell lines it was <0.5  of the 140 drug-resistant neuroblastoma cell lines was similarly sensitive to flubendazole like the corresponding parental cell lines as indicated by fold changes ICwas <0.5 .Vinca alkaloids vinblastine, vincristine, and vinorelbine are destabilising agents that target the vinca domain. Flubendazole and 2-methoxyestradiol are destabilising agents that target the colchicine domain21290 and IC50 values for flubendazole . 2 (13%) taxane resistant cell lines showed an increased flubendazole resistance  and 1 cell line showed enhanced sensitivity . 12 (43%) of the 28 Vinca alkaloid-resistant cell lines displayed cross-resistance to flubendazole while the remaining 16 (57%) Vinca alkaloid-resistant cell lines were similarly sensitive to flubendazole like the corresponding parental cells. The 2-methoxyestradiol-resistant cell line UKF-NB-3rME1000 displayed an 8-fold increased flubendazol IC90 relative to UKF-NB-3. These data suggest that cross-resistance formation to flubendazole is more likely in cell lines resistant to destabilising agents than to stabilising agents  transporters including ABCB1 and ABCG21416CDKN1A (encodes for p21), BAX, and BBC3 (encoding for PUMA) (p53shRNA) cells showed a lack of flubendazole-induced p53 target gene expression (Cell death induced by many anti-cancer drugs involves p53 activationor PUMA)  and signor PUMA)  in UKF-Npression , and redpression . Also, fpression . Differepression . Moreovepression . Again, pression .Moreover, p53 depletion in IMR-32, UKF-NB-2, UKF-NB-3, LAN-5, NB-S-124, and SH-SY5Y cells resulted in decreased flubendazole sensitivity .rVCR10 cells and hardly influenced the effect of flubendazole in p53-depleted UKF-NB-3p53shRNA cells (p53shRNA cells treated with the combination of flubendazole 80 nM and nutlin-3 1.25 μM. This is most likely caused by some remaining p53 activity due to incomplete p53 depletion.The MDM2 inhibitor nutlin-3NA cells . Some coTo develop DMSO-free flubendazole preparations we developed flubendazole-cyclodextrin complexes. (2-hydroxypropyl)-β-CD increased the aqueous solubility of flubendazole while γ-CD did not. A 20% (m/V) (2-hydroxypropyl)-β-CD formulation containing 74 μM flubendazole and 0.1% saline was similar active against neuroblastoma cells like flubendazole in DMSO and then diluted with cell culture medium . The pre−6 relative to vehicle-treated control sponges). Representative photographs are presented in For the investigation of vessel formation, the CAM assay was performed using drug-loaded gelatin sponges as described previously90 = 102.3 ± 12.7 nM) and its cisplatin-resistant sub-line UKF-NB-3rCDDP1000 (flubendazole IC90 = 122.1 ± 16.7 nM) were selected as highly flubendazole-sensitive cell lines. The vincristine-resistant UKF-NB-3 sub-line UKF-NB-3rVCR10 (flubendazole IC90 = 552.3 ± 60.9 nM) had displayed an approximately five-fold decreased flubendazole sensitivity compared to UKF-NB-3 and UKF-NB-3rCDDP1000. Flubendazole 2 μg (applied as 100 μL of a 20 μg/mL solution) treatment resulted in all cell lines in less extensive growth and large necrotic areas. Representative photographs are presented in To analyse the effects of flubendazole on neuroblastoma xenograft growth in the CAM, three cell lines were used. UKF-NB-3 , a substantial fraction of the investigated cell lines was sensitive to flubendazole in concentrations <1 μM that appear to be pharmacologically achievableFlubendazole had been demonstrated to exert activity against leukaemia and myeloma cells90 values below 1 μM. 87 drug-resistant neuroblastoma cell lines (62%) were similar sensitive to flubendazole like the corresponding parental cells. 39 drug-resistant cell lines (28%) showed cross-resistance to flubendazole, 14 drug-resistant cell lines (10%) were more sensitive to flubendazole than their parental counterparts.Neuroblastoma is the most frequent extracranial solid childhood tumour. About half of patients suffer from high-risk disease associated with overall survival rates below 50% despite intensive therapyVinca alkaloids vinblastine, vincristine, and vinorelbine , and to 2-methoxyestradiol that is like flubendazole a destabilising agents that targets the colchicine domain21290 and IC50 values but the differences did not reach statistical significance. These findings suggest that acquired resistance to a tubulin-binding agent may not necessarily be associated with a substantially decreased sensitivity to flubendazole. In concordance, albendazole, another benzimidazole anthelminthic, had been shown to be active in cancer cell lines resistant to the stabilising tubulin-binding agents paclitaxel and epothilone B232Flubendazole has been described to interfere with microtubule function12In concordance with previous results141126112829303132The poorly water-soluble flubendazole was administered in 0.9% NaCl and 0.01% Tween-80 in a previous study8Mebendazole and albendazole are further benzimidazole anthelminthic agents that have been shown to exert anti-cancer effects13456735363738In conclusion, we show that the well-tolerated anthelminthic flubendazole4041WK-X-34 was synthesised as described beforeThe origins of the investigated parental cell lines and the cell culture media are shown in www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html)  .Cells were routinely tested for mycoplasma contamination and authenticated by short tandem repeat or variable number tandem repeat profiling.www.lentigo-vectors.de).p53-depleted cells or cells showing high expression of ABCB1  or ABCG2  were established as described previously44Fresh neuroblastoma cells (MYCN amplified) were isolated from the bone marrow aspirate of five patients with metastasised INSS stage 4 neuroblastoma following informed consent. Primary cells were cultivated in IMDM supplemented with 10% FCS, 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37°C.Cell viability was tested by the 3--2,5-diphenyltetrazolium bromide (MTT) dye reduction assay after 120 h incubation modified as described previously1650 and IC90 values were determined using the CalcuSyn software . Differences in the IC50 and IC90 values between the cancer entities were tested using the Wilcoxon rank sum testCell viability was tested in 96-well plates using 1 in 4 dilution steps. The maximum flubendazole concentration tested was 5000 nM. ICCells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Proteins were detected using specific antibodies directed against β-actin , p21, Bax, cleaved PARP, PUMA, caspase 3, caspase 9 , p53 , and phosphorylated p53 (serine 15)  and were visualised by enhanced chemiluminescence using a commercially available kit .TM Transfection System  according to the manufacturer's protocol. 2 × 106 UKF-NB-3 cells were suspended in 200 μl re-suspension buffer containing 2.5 μM siRNA. After electroporation , the cells were transferred into fibronectin (100 μg/ml)-coated 6-well-plates containing pre-warmed IMDM plus 10 % FCS. Western blot analyses were performed and cells were used for viability assays 48 h post transfection.Synthetic siRNA oligonucletides targeting BAX, PUMA, p21, or Caspase 9 and control non target siRNAs (ON-TARGETplus SMARTpool) were purchased from Dharmacon . The target sequences are presented in The activity of the caspases 3 and 7 was examined using the Caspase-Glo® 3/7 Assay  following the manufacturer's instructions. Cells were seeded in 96-well cell culture plates and allowed to adhere overnight. After drug treatment, the culture plates were adjusted to room temperature. Then, the cells were incubated for 5 min with the pre-mixed substrate and the luminescent signal was measured with a plate reader  for 30 cycles.5.0 mg flubendazole were suspended in 10.0 mL aqueous solutions of the cyclodextrin (CD) derivatives (2-hydroxypropyl)-β-CD  or γ-CD . After vortexing and ultrasonication (15 min), the samples were stirred at room temperature for 5 days. After 48 h and 120 h samples were taken, filtered through pre-saturated cellulose acetate membrane filters (0.45 μm), diluted with purified water and analysed by HPLC for the flubendazole concentration using an isocratic mixture of water and methanol (31:69) containing 0.1% trifluoracetic acid at a temperature of 30°C and a reverse phase column  in combination with a KrudKatcher™ Ultra In-line filter  in an Agilent 1200 HPLC system  with DAD detection (flow rate 1.0 mL/min). Flubendazole was detected at 234 nm (retention time 2.1 min).www.ich.org). An acceptable degree of linearity, accuracy and precision was confirmed for concentrations between 0.25 and 1 μg/mL. The limit of detection was <0.01 μg/mL; the limit of quantification was >0.2 μg/mL.Validation of the HPLC analysis was performed according to the ICH harmonised tripartite guideline “Validation of analytical procedures: Text and Methodology Q2(R1)”  assay. For the investigation of vessel formation the CAM assay was performed using drug-loaded gelatin sponges as described previously6 cells were suspended in 30 μl extracellular matrix  and implanted on the CAM of fertilised chicken eggs on day 11 of embryo development. Eggs were incubated for another 3 days to allow formation of a distinct tumour mass. On day 14, a small silicone ring was placed around the tumour mass and drugs were administered. On day 18, the tumours were sampled with the surrounding CAM, fixed using 4% paraformaldehyde, and embedded in paraffin. Sections (4 μM) were haematoxylin/eosin-stained.Tumour growth in the CAM was examined following described methodsResults are expressed as mean ± S.D. of at least three experiments. Comparisons between two groups were performed using Student's t-test. Three and more groups were compared by ANOVA followed by the Student-Newman-Keuls test. P values lower than 0.05 were considered to be significant.Ma.M. and J.C. jr. designed the study, performed experiments, analysed data, and wrote the manuscript. B.A., F.R., N.L., Y.V., Mi.M., T.S., P.N.H., K.R., B.A.A., S.F., S.S., K.L., W.G.D., R.Z. and J.C. performed experiments, analysed data, and proofread the manuscript. M.W. and F.W. provided essential materials, analysed data, and proofread the manuscript. M.N.W. performed statistical analyses, analysed data, wrote parts of the manuscript, and proofread the manuscript.Suppl Files


Aim of this study was to evaluate the relative biological effectiveness (RBE) of carbon (12C) and oxygen ion (16O)-irradiation applied in the raster-scanning technique at the Heidelberg Ion beam Therapy center (HIT) based on clonogenic survival in hepatocellular carcinoma cell lines compared to photon irradiation.Four human HCC lines Hep3B, PLC, HepG2 and HUH7 were irradiated with photons, 12C and 16O using a customized experimental setting at HIT for in-vitro trials. Cells were irradiated with increasing physical photon single doses of 0, 2, 4 and 6 Gy and heavy ionsingle doses of 0, 0.125, 0.5, 1, 2, 3 Gy (12C and 16O). SOBP-penetration depth and extension was 35 mm +/−4 mm and 36 mm +/−5 mm for carbon ions and oxygen ions respectively. Mean energy level and mean linear energy transfer (LET) were 130 MeV/u and 112 keV/um for 12C, and 154 MeV/u and 146 keV/um for 16O. Clonogenic survival was computated and realtive biological effectiveness (RBE) values were defined.For all cell lines and both particle modalities α- and β-values were determined. As expected, α-values were significantly higher for 12C and 16O than for photons, reflecting a steeper decline of the initial slope of the survival curves for high-LET beams. RBE-values were in the range of 2.1–3.3 and 1.9–3.1 for 12C and 16O, respectively.Both irradiation with 12C and 16O using the rasterscanning technique leads to an enhanced RBE in HCC cell lines. No relevant differences between achieved RBE-values for 12C and 16O were found. Results of this work will further influence biological-adapted treatment planning for HCC patients that will undergo particle therapy with 12C or 16O. Treatment options for patients with advanced hepatocellular carcinoma (HCC) are limited and prognosis still remains poor Aim of this study was to evaluate in-vitro effects of carbon and oxygen ion-irradiation applied in the raster-scanning technique on clonogenic survival in hepatocellular carcinoma cell lines compared to photon radiotherapy. Results of this work will further influence biological-adapted treatment planning for HCC patients at HIT.2 tissue plastic flasks in an incubator at 37°C in humidified air with 5% CO2 and passaged weekly. Mycoplasma screening was performed regularly by a dedicated laboratory.All cell lines were obtained from the American Type Culture Collection . The three human HCC lines Hep3B, PLC and HUH7 were grown in DMEM , HepG2 cells were maintained in RPMI 1640 medium . Both media were supplemented with 10% heat-inactivated fetal bovine serum (FBS)  and 1% penicillin-streptomycin . Cells were stored lying flat in 175 cmPhoton irradiation was performed with a biological cabinet X-ray irradiator  at single doses of 2, 4, 6 and 8 Gy. Irradiation was performed at room temperature.Carbon and oxygen ion treatment was performed at the Heidelberg Ion-Beam Therapy Center (HIT) using the raster-scanning technique. Single doses of 0.125, 0.5, 1, 2 and 3 Gy were delivered with an extended Spread-Out Bragg peak (SOBP) that was adjusted using a 3.5-cm acrylic shield and positioning cell monolayers in the middle of the SOBP. SOBP-penetration depth and extension was 35 mm +/−4 mm and 36 mm +/−5 mm for carbon ions and oxygen ions respectively. Mean energy level and mean linear energy transfer (LET) were 130 MeV/u and 112 keV/um for carbon ions, and 154 MeV/u and 146 keV/um for oxygen ions. Irradiation was performed at room temperature. Experiments were done as previously described by our group 2 flasks filled with medium and incubated for 24 h before irradiation. Secondly, after irradiation cells were incubated 5 to 14 days depending on cell line and its individual extent of forming colonies. Thirdly, cell fixation was done using 4 ml of 70% ethanol followed by 5 ml of methylene blue for 10 min. Finally, colony counting was performed under the microscope with a threshold of minimum 50 cells per colony. From the determined surviving fractions the plating efficiency and clonogenic survival were calculated. These results were used to generate survival curves, to define α- and β-parameters and to calculate RBE-values. SigmaPlots’  non-linear least-squares regression option was used to fit the linear-quadratic expression [-ln(S) = α*D+β*D2] to the resulting averaged survival fractions after normalizing plating efficiencies to the untreated samples .In order to generate reliable results every experiment was performed in triplets two or three times at independent days. Clonogenics were performed as previously described by our group In a first set of experiments HCC cell lines HepG2, HuH7, Hep3B and PLC were irradiated with increasing doses of 0, 2, 4, 6 and 8 Gy. Cell lines showed a dose-dependent suppression of clonogenic survival as can be seen by typical shoulder-shaped curves . The surIn a second set of experiments the above mentioned cell lines were treated with 12C and 16O ion beams and again clonogenic survival was assessed by a colony forming assay. Applied physical doses were 0, 0.125, 0.5, 1, 2 and 3 Gy dependent on cell line and particle modality. All cell lines showed a dose-dependent suppression of clonogenic survival .In comparison to the photon-RT derived survival data of the tested cell lines, survival fractions for 12C and 16O treated cells were even lower . Therefo10) and consistent for all 12C- and 16O-treated cell lines  were comparable for 12C and 16O and in the range of 2–3. Based on these data future adoptions and corrections in the context of biological optimized particle therapy of HCC patients at HIT will be possible with the aim of a more exact computation of RBE-tables in the treatment planning environment.Charged particle beams with carbon or oxygen ions applied using the raster-scanning technique at HIT lead to high RBE values in four tested HCC cell lines. Clonogenic cell survival was dose-dependent and detectable in all examined cell lines. Resulting RBE-values  in V79 Chinese hamster cells 60Co, in that order. The presented results are basically in accordance with our data regarding higher RBE-values for heavy ion beams. But, in contrast to our results, 12C had a higher impact on cell survival than 16O. One explanation can be the different experimental setup. The Indian group used 16O beams with a different energy and the examined cells are non-cancerous fibroblasts, so finally different biological effects may result.Previously, an Indian group examined the influence of several charged particle beams  compared to Cobalt irradiation  after photon and 12C irradiation using the premature chromosome condensation (PCC) technique and colony formation Syngo PT Planning developed by Siemens Oncology Care Systems  including biologic plan optimization, originally based on the Local Effect Model (LEM) developed by GSI (Gesellschaft für Schwerionenforschung) A further important aspect of our work is the possible integration of our data into biologically individualized dose calculation algorithms for the treatment planning procedure for HCC patients. Currently the phase-I/−II clinical trial PROMETHEUS is recruiting HCC patients for a four fraction protocol of increasing doses from 10 to 15 Gy(RBE) 3 in size. Together with the results of a recent meta-analysis on the prognostic relevance of tumor hypoxia in HCC patients, these findings have to be considered in TPS dose calculation based on in-vitro derived radiobiological parameters to optimize particle RT for future HCC patients Future improvements may also incorporate LET-painting in particle therapy treatment planning systems for tumor regions that are hypoxic and therefore more radio-resistant. For this purpose, Bassler and colleagues recently came up with strategies to overcome the relative radio-resistance of hypoxic tumor regions with LET-painting using 12C and 16O The acquired data will be valuable for biologically adapted treatment planning in clinical trials on carbon ion therapy of hepatocellular carcinoma such as our running clinical PROMETHEUS trial (hypofractionated carbon ion therapy for HCC). The presented results represent the biological fundamentals of the clinical application for charged particle treatment using the raster-scanning technique.


The structures were determined by means of extensive spectroscopic analysis. All diterpenoids isolated were evaluated for their cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7, and SW480 cell lines, and compounds 5 and 7 showed significant inhibitory effects on all cell lines. Three new Supplementary material is available for this article at 10.1007/s13659-011-0031-7 and is accessible for authorized users. Supplementary material, approximately 590 KB.


Mutant cell lines have been reported to respond to MDM2 inhibition, while many p53WT cell lines have been shown not to respond. To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies. However, upon extensive curation of this panel, these apparent exceptions could be eliminated, revealing a perfect correlation between p53 mutational status and MDM2 inhibitor responsiveness. It has been suggested that the MDM2-amplified subset of p53WT tumors might be particularly sensitive to MDM2 inhibition. To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification. Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.Previous reports have provided evidence that p53 mutation is a strong negative predictor of response to MDM2 inhibitors. However, this correlation is not absolute, as many p53 The tumor suppressor protein p53 plays a critical role in protecting cells from various stresses, such as DNA damage and hypoxia . In respMDM2 occurs at high frequency in sarcomas and at low frequency in cancers of the brain, bladder, stomach, lung, skin, and breast . Relative cell count IC50 was the inhibitor concentration that produced 50% of the cell proliferation inhibitory response or 50% cytotoxicity level, relative to DMSO control.Cell proliferation was measured by incorporation of nuclear dye to determine the relative cell count. To calculate the effect of compound treatment on cell proliferation, the data were transformed to percent of control (POC) using the following formula: POC = relative cell count (compound wells)/relative cell count (vehicle wells) × 100. ICTP53, either using 454 sequencing (Roche Diagnostics) or WAVE® Mutation Detection System and SURVEYOR® Nuclease technology  and sequenced for the presence of mutations in c, Inc.; ).http://www.sanger.ac.uk/cosmic [TP53 database (version R15), p53.iarc.fr/TP53GeneVariations.aspx [TP53 Mutation Database, http://www.p53.fr [Additional data regarding the p53 mutational status of tumor cell lines in the panel were obtained from the Wellcome Trust Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC v44–62 releases), k/cosmic , 28; theons.aspx , and thew.p53.fr .Primers to detect viral DNA sequences for human papillomavirus E6 from high-risk variants 16, 18, 31, 33, and 45, as well as simian virus 40 large T antigen and adenovirus E1B were designed using Primer Express software and synthesized by Integrated DNA Technologies . GenomicTP53 cDNA sequence from each of the samples. PCR products were electrophoresed on 1% agarose gels, and the desired PCR products were isolated, purified, cloned, and sequenced. Sequences from the cDNA clones were aligned against the TP53 cDNA reference sequence NM_000546 using Vector NTI® software (Life Technologies).Total RNA was isolated from human tumor cell lines according to the RNeasy Mini Kit protocol (QIAGEN). 3′RACE-ready cDNA was generated according to the SMARTer RACE cDNA Amplification Kit protocol (Clontech). 3′ RACE PCR, followed by additional nested PCR, was performed to amplify 2. The following day, either DMSO or AMG 232 was added to appropriate wells at a final concentration of 0.1% DMSO or 10 μM, respectively. After incubation for 24 hours at 37°C and 5% CO2, protein lysates were collected, electrophoresed on 10% Bis-Tris NuPAGE gels (Life Technologies), and transferred to Invitrolon PVDF membranes (Life Technologies). Following transfer, the membranes were blocked and incubated with primary antibodies against p53 , MDM2 (BD Pharmingen), p21 (R&D Systems), or β-actin-HRP (Sigma). For detection, the membranes were incubated with species-specific secondary antibodies conjugated to HRP. Luminescent signal was developed using ECL Plus reagent  and the membranes were exposed to film.Cells were seeded at subconfluent densities in 6-well plates and incubated overnight at 37°C and 5% COet al. [The vast majority of the gene expression data was obtained from the GlaxoSmithKline Cancer Cell Line Genomic Profiling dataset . For celet al.  and from50 for a cell line was in the lower quartile of IC50 values among all cell lines; otherwise it was set to false. An ANOVA model including a factor for tumor origin was used as a feature selection step to identify genes whose expression were highly correlated with IC50 response. The single most correlated gene from the feature selection step was used in a logistic regression model to predict response given the gene expression value and tissue source of a new cell line. If the model-based probability of response was predicted to be greater than 0.5, the new cell line was classified as a responder; otherwise it was classified as a non-responder. The leave-one-out (LOO) cross-validation procedure recommended by Simon et al. [Association analyses were performed to attempt to identify additional predictors of sensitivity. Response to MDM2 inhibition was categorized as true if the ICn et al.  was usedhttp://www.cancer.sanger.ac.uk/cancergenome/projects/cell_lines/; v61 release) [WT cell lines in the panel. There were 25 genes for which at least one cell line was called mutant. We evaluated associations between response to MDM2 inhibition and mutation status using the “aov” function in R. Test p-values were adjusted using the R “p.adjust” function with method = “BH”.Mutation data were collected from the Wellcome Trust Sanger Institute COSMIC Cell Line Project (release) . The mut


Recombinant products have reached the market in diverse areas, including pharmaceutical, veterinary food, pesticides and detergents. Noteworthy, approximately 30 recombinant products in the pharmaceutical sector account for more than 90 per cent of all recombinant product sales. Although microbial systems can be considered an attractive option for expressing certain biopharmaceutical proteins, many biopharmaceutical molecules are too large and complex and need to be expressed in mammalian cells.The most reported cell lines used for industrial processes are CHO, NS0, Sp2/O-Ag14 and HEK293. In particular, the latter has been gaining significant importance in the biopharmaceutical field during the last two decades. While initially employed for adenoviral vector production HEK293 became also one of the preferred cell lines for transient or stable protein expression. This is mainly due to its high transfection efficiency. Meanwhile genetic manipulation also includes new techniques for directing the integration of the foreign gene to highly expressed chromosomal sites e.g. by RMCE.In this work, three strategies for recombinant protein production in HEK293 cells have been compared Figure : (St.1) Viral DNA was isolated from field and the gene of the capsid (CapPCV2) was cloned into Adh5 genome using AdEasyXL kit. Virus and protein production were performed by infecting HEK293-F6 suspension cells .CapPCV2 gene sequence was codon optimized for mammalian cell expression. The obtained sequence was cloned into pIRESpuro3 bicistronicvector [Three targeting vectors encoding for different promoters were transfected by electroporation to three different HEK293 master cell lines. Positive clones were selected by neomycin and ganciclovir addition to media. Clones were isolated by single colony pick up. Upon the selection of the best promoter, RMCE was applied to 293T cell line [We generated transient Cap-PCV2 producer cells by infection of the HEK293 cell line with a recombinant adenovirus (rAdV-Cap) Figure .In contrast, a significant increase in production was achieved upon random integration of a CapPCV2 encoding plasmid Figure  which reThe main conclusion of this work is that a bioprocess based on site-integrated stable suspension cell line would be the preferred one not only because the highest qp, but also due to the easy characterization of the process (in comparison to adenovirus infection), the repeatability of batch productions (in comparison to the other approaches) and the possibility of rapid substitution of the protein of interest.


The bioproduction of therapeutic proteins such as monoclonal antibodies (mAb) continues to be a fast growing sector of advanced manufacturing. The ever increasing repertoire of therapeutic proteins coupled with the emergence of biosimilars led to increasing global demand for higher, faster, more cost-effective manufacturing process. Central to any good production platform is the capacity of the production cell line. A suitable production cell line exhibit physiological traits such as high specific productivity (qp), rapid doubling time, high peak cell density and efficient metabolism. The emergence of a suitable production cell with all the aforementioned traits is an extremely rare event, as it requires all facets of cellular transcription, translation, secretion and metabolic efficiency are individually optimized and collectively synchronized into a system capable of high level protein expression. One of the major cellular bottlenecks thought to limit protein production in mammalian cells lies in the cellular translation and secretion capacity. The over-expression of complex recombinant proteins such as mAbs driven by strong viral promoters exert considerable burden in the Endoplasmic Reticulum (ER) and Golgi apparatus. The increase in ER stress triggers the Unfolded Protein Response (UPR) which can result in cell apoptosis and possibly eliminating cells with high uptake of the Gene of Interest (GOI). We adopted host cell engineering approach using XBP1 spice ratio to expand cellular translation and secretion capacity to increase qp and improve the probability of isolating suitable high producers.We screened a panel of mAb producing clonal cell lines using qPCR to identify XBP1 mRNA splice ratio as a suitable target to augment host cell translation and secretion machinery. We then employed FACS to isolate cell population with high ratio of spliced over unspliced XBP1 in order to overcome the negative regulatory effect of unspliced XBP1. We created a host cell line with high XBP1 splice ratio, exhibiting increased expression of chaperones, secretary vesicle proteins and quality assurance proteins without increasing UPR associated apoptotic markers as identified using qPCR. This host cell line was then used for in comparative mAb transient bioproduction studies with two different mAbs. Transient studies were performed using PEI-mediated transfection in industrial relevant chemically defined medium and feeding regime.The XBP1 cell line demonstrated 7.5 fold increase in qp over the control cell line and more than 4 fold increase in final volumetric productivity (Figure In conclusion, XBP1 splice ratio engineered host cell have expanded translation and secretion capacity to alleviate the ER stress experienced from during mAb bioproduction. The augmented capacity allowed for multi-fold improvements in qp and volumetric productivity and have the potential to increase the probability for isolating high producers. The engineered host cell line showed great promise to become a commercially suitable production platform cell line to significantly reduce the time and resources associated with cell line development.


Ala 5655A → G (m.5655A → G) mutation. The destabilization of a highly conserved base pairing (A1-U72) at the aminoacyl acceptor stem by an m.5655A → G mutation altered the tRNAAla function. An in vitro processing analysis showed that the m.5655A → G mutation reduced the efficiency of tRNAAla precursor 5′ end cleavage catalyzed by RNase P. By using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA (mtDNA)-less (ρo) cells, we showed a 41% reduction in the steady-state level of tRNAAla in mutant cybrids. The mutation caused an improperly aminoacylated tRNAAla, as suggested by aberrantly aminoacylated tRNAAla and slower electrophoretic mobility of mutated tRNA. A failure in tRNAAla metabolism contributed to variable reductions in six mtDNA-encoded polypeptides in mutant cells, ranging from 21% to 37.5%, with an average of a 29.1% reduction, compared to levels of the controls. The impaired translation caused reduced activities of mitochondrial respiration chains. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These caused increases in the production of reactive oxygen species in the mutant cybrids. The data provide evidence for the association of the tRNAAla 5655A → G mutation with hypertension.In this report, we investigated the pathophysiology of a novel hypertension-associated mitochondrial tRNA Glu and tRNAA1a, reside on the cytosine-rich light (L) strand; the remaining tRNAs, including tRNALys and tRNAHis, are located on the guanine-rich heavy (H) strand  strand , 9. Mito) strand –12. Thesln genes –16. Thesn genes –, 18. Then genes –, 15, 19, tRNAMet , 21. HowAla gene in three genetically unrelated probands whose families exhibited a maternal transmission of hypertension . As shown in Ala. In particular, the mutation may affect the aminoacylation capacity and stability of this tRNA. A failure in tRNA metabolism leads to the impairment of mitochondrial translation and respiration  at the 5′ end of the tRNApiration , 18, 23.0] cells , 25. TheA cohort of 2,070 Chinese hypertensive probands were recruited from the Hypertension Clinic, Wenzhou Medical University, China, as detailed previously , 27. ThiAla gene were amplified by PCR using oligodeoxynucleotides corresponding to mtDNA at positions 5258 to 6031 (NC_012920) . Subjects' DNA fragments spanning the tRNA031 NC_0120 (29). − cell line was grown in Dulbecco's modified Eagle medium (DMEM) (Life Technologies) (containing 4.5 mg of glucose and 0.11 mg pyruvate/ml), supplemented with 100 μg of BrdU/ml and 5% FBS. Transformation by cytoplasts of mtDNA-less ρ°206 cells was performed as described elsewhere . The labeled RNA substrates (125 nucleotides [nt]) were transcribed with T7 RNA polymerase and were purified by denaturing polyacrylamide gel electrophoresis. Processing assays were carried out in parallel for wild-type and mutant substrates as detailed previously (The tRNAeviously . Enzyme 8 cells) using a ToTALLY RNA kit (Ambion), as described previously -labeled oligodeoxynucleotides specific to each RNA and 5S RNA were as detailed previously  were obtained from mitochondria isolated from mutant and control cell lines , and tRNAGly as detailed previously  (ab110258) from Abcam; ND4 (sc-20499-R) and ND6 (sc-20667) from Santa Cruz Biotechnology; and CYTB (apocytochrome b) (55090-1-AP) from Proteintech. Peroxidase Affinipure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies, and protein signals were detected using an enhanced chemiluminescence (ECL) system (CWbio). Quantification of density in each band was performed as detailed previously , as detailed previously , 37.c), III (ubiquinone cytochrome c oxidoreductase), and IV (cytochrome c oxidase) were assayed as detailed elsewhere (The enzymatic activities of complexes I (NADH ubiquinone oxidoreductase), II  was used for the measurement of cellular and mitochondrial ATP levels, according to the modified manufacturer's instructions , 39.Mitochondrial membrane potential (ΔΨm) was assessed with a JC-10 assay kit  according to the manufacturer's recommendations with some modifications, as detailed elsewhere .Measurement of total cellular ROS generation was performed as detailed elsewhere , 40, 41.t test of the Microsoft Excel program or Excel for Macintosh (version 2007). Differences were considered significant at a P value of <0.05.Statistical analysis was carried out using the unpaired, two-tailed Student Ala gene was found in three genetically unrelated probands in a cohort of 2,070 Chinese hypertensive subjects but was absent in 800 Chinese control subjects. As shown in Ala 5′ end precursors of the light strand, catalyzed by RNase P   . HoweverMembers of three Chinese families carrying the m.5655A → G mutation underwent a physical examination, laboratory assessment of cardiovascular disease risk factors, and routine electrocardiography. In the pedigree WHP20, 12 of 19 matrilineal relatives had a wide range in levels of severity of hypertension (with blood pressure greater than 140/90 mm Hg even with treatment for hypertension), while only 4 of 28 nonmaternal relatives suffered from hypertension. None of the offspring of five affected fathers exhibited hypertension, while one affected subject, IV-2, was the son of subject III-1, who married an affected wife (III-2). As shown in Table S2 in the supplemental material, the age at onset of hypertension in the maternal kindred varied from 38 years to 80 years, with an average of 56.7 years. Furthermore, 3 of 9 matrilineal relatives in the pedigree WHP21 suffered from hypertension, while 9 of 17 matrilineal relatives in the pedigree WHP22 exhibited hypertension. None of the other members in these families had hypertension. The average ages at onset of hypertension in these families were 50.3 and 56.4 years, respectively. There was no evidence that any member of these families had any other cause to account for hypertension. However, no other clinical abnormalities were observed in the maternal kindred. Further analysis showed that the m.5655A → G mutation was present in homoplasmy in all matrilineal relatives but not in other members of these families (data not shown).o206 cells, derived from the 143B.TK− cell line (The lymphoblastoid cell lines derived from one affected subject (WHP20-II-13) carrying the m.5655A → G mutation and one control individual (C1) lacking this mtDNA mutation but belonging to the same mtDNA haplogroup were enucleated and subsequently fused to a large excess of mtDNA-less human ρell line , 25. Theell line . Betweenin vitro processing system to determine if the primary defect arising from the m.5655A → G mutation is the perturbed processing of the tRNA's 5′ end by RNase P. The wild-type and mutant tRNAAla precursors corresponding to mtDNA at positions 5692 to 5567 -strand transcription unit and for tRNAGlu derived from the light (L)-strand transcription unit (Ala in mutant cells were markedly decreased compared to those in control cells (Ala in the three mutant cell lines was 56.5% (P = 0.01) of the level of the three control cell lines after normalization to 5S RNA. However, the average steady-state levels of tRNALys, tRNAHis, and tRNAGlu in the three mutant cell lines were 104.6%, 103.2%, and 91.9% of those in the three control cell lines, respectively , and tRNAGly in control and mutant cell lines were examined by the use of electrophoresis in an acid polyacrylamide-urea gel system to separate uncharged tRNA species from the corresponding charged tRNA, electroblotting, and hybridizing with the above tRNA probes. As shown in Ala, while only one band occurred in the mutated tRNAAla, which migrated more slowly than that of the wild-type tRNAAla. To further distinguish nonaminoacylated tRNA from aminoacylated tRNA, samples of tRNAs were deacylated by being heated for 10 min at 60°C at pH 8.3 and then run in parallel. As shown in Ala. However, there were no obvious differences in electrophoretic mobility and aminoacylated efficiencies of other tRNAs between the mutant cell lines carrying the m.5655A → G mutation and the control cell lines lacking this mutation.The aminoacylation capacities of tRNAP = 0.02), relative to the mean value in the control cell lines. As shown in Ser(UCN) gene or m.8344A → G mutation in the tRNALys gene  in mutant and control cells with TOM20  as a loading control. The overall levels of six mitochondrial translation products in the mutant cell lines ranged from 21% to 37.5%, with an average of 29.1% (Lys gene .P = 0.005) relative to the mean value measured in the control cell lines   of mutant and control cell lines. The basal OCR in the mutant cell lines was 64% (ll lines . The avectively) .c oxidoreductase) activity was measured as a reduction of cytochrome c (III) using d-ubiquinol 2 as the electron donor. Complex IV (cytochrome c oxidase) activity was monitored by following the oxidation of cytochrome c (II). As shown in P < 0.001, 0.847, 0.702, and 0.036, respectively).The activities of respiratory complexes were also measured by isolating mitochondria from three mutant and three control cell lines. Complex I (NADH ubiquinone oxidoreductase) activity was determined by following the oxidation of NADH with ubiquinone as the electron acceptor. Complex III (ubiquinone cytochrome d-glucose with pyruvate (P < 0.001). In contrast, as shown in d-glucose to inhibit the glycolysis , ranged from 64% to 77%, with an average of 70% relative to the mean value measured in the control cell lines (P = 0.002).The capacity of oxidative phosphorylation in mutant and wild-type cells was examined by measuring the levels of cellular and mitochondrial ATP using a luciferin/luciferase assay. Populations of cells were incubated in the medium in the presence of glucose and 2-deoxy-pyruvate . As show590/FL530) were recorded to determine the ΔΨm level of each sample. The relative ratios of the FL590/FL530 geometric mean between mutant and control cell lines were calculated to represent the level of ΔΨm. As shown in P < 0.001) of the mean value measured in the control cell lines. In contrast, the levels of ΔΨm in mutant cells in the presence of FCCP [carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone] were comparable to those measured in the control cell lines (data not shown).The mitochondrial membrane potential (ΔΨm) is the central bioenergetic parameter that controls respiratory rate, ATP synthesis, and the generation of ROS and is itself controlled by electron transport and proton leaks. The levels of ΔΨm were measured in three mutant and three control cell lines using a JC-10 fluorescent probe assay system. The ratios of fluorescence intensity at excitation/emission (Ex/Em) wavelengths of 490/590 and 490/530 nm  of the mean value measured in the control cell lines. Furthermore, the levels of mitochondrial ROS (mitoROS) among the cybrids were determined using a MitoSOX assay via flow cytometry (P = 0.001) of the mean value measured in the control cell lines.The total cellular levels of ROS generation in the vital cells derived from three mutant cell lines carrying the m.5655A → G mutation and three control cell lines lacking the mutation were measured with flow cytometry under normal conditions and under Hmulation , 40, 43.ytometry . As showAla gene. The occurrence of the m.5655A → G mutation in three genetically unrelated Chinese families affected by hypertension and differing considerably in their mtDNA haplotypes strongly indicated that this mutation is involved in the pathogenesis of this disorder. In fact, the m.5655A → G mutation was located at the 5′ end of tRNA, which is the processing site for the tRNAAla 5′ end precursors of the light strand, catalyzed by RNase P  to form a base pairing (1A-72U) on the acceptor stem of the tRNAAla  to produce a clinical phenotype   . In the mplex V) , 15. Themplex V) . The resmplex V) . In thisP demand , 52. In P demand .In fact, mitochondrial respiration is the major source of ROS, with 0.2% of oxygen consumed being normally converted into reactive oxygen species in a quiescent state . ImpairmAla 5655A → G mutation. The m.5655A → G mutation alters the secondary structure and function of tRNA. A failure in tRNA metabolism impaired mitochondrial translation and respiration. As a result, this respiratory deficiency reduced mitochondrial ATP production, increased the production of oxidative reactive species, and subsequently elevated blood pressure. Thus, our findings may provide new insights into the understanding of the pathophysiology of maternally inherited hypertension.In summary, our findings suggest the pathogenic mechanism leading to an impaired oxidative phosphorylation in cybrid cell lines carrying the hypertension-associated tRNA