HRAS is a frequently mutated oncogene in cancer. However, mutant HRAS as drug target has not been investigated so far. Here, we show that mutant HRAS hyperactivates the RAS and the mTOR pathway in various cancer cell lines including lung, bladder and esophageal cancer. HRAS mutation sensitized toward growth inhibition by the MEK inhibitors AZD6244, MEK162 and PD0325901. Further, we found that MEK inhibitors induce apoptosis in mutant HRAS cell lines but not in cell lines lacking RAS mutations. In addition, knockdown of HRAS by siRNA blocked cell growth in mutant HRAS cell lines. Inhibition of the PI3K pathway alone or in combination with MEK inhibitors did not alter signaling nor had an impact on viability. However, inhibition of mTOR or combined inhibition of MEK and mTOR reduced cell growth in a synergistic manner. Finally, Ba/F3 cells transformed with mutant HRAS isoforms Q61L, Q61R and G12V demonstrated equal sensitivity towards MEK and mTOR inhibition. Our results show that HRAS mutations in cancer activate the RAS and mTOR pathways which might serve as a therapeutic option for patients with HRAS mutant tumors. Harvey-RAS (HRAS) belongs to the RAS family of small GTPases which activate the RAS–RAF–MEK–ERK pathway. Mutations in RAS family members lead to hyperactivity of the RAS signaling pathway. HRAS is a frequently mutated oncogene especially in head and neck cancer 3.9%), bladder cancer 5.1%), vulvar squamous cell carcinoma (9.3%), cutaneous squamous cell carcinoma and lung cancer 3.8%) [%, vulvar.9%, blad% %, vul, 11 and In the present study, we investigated HRAS downstream signaling in five different cancer cell lines including lung and bladder cancer and putative drugs for targeted therapy. We observed that lung cancer cell lines harboring HRAS mutations showed significant higher sensitivity to MEK inhibitors than HRAS wild-type cell lines. Further, the effect of MEK inhibitors was enhanced synergistically by the addition of mTOR but not PI3K inhibitors. Inhibition of mTOR alone is sufficient to block cell growth in HRAS mutant but not wild-type cell lines. To our knowledge, we describe for the first time that HRAS mutations can be targeted by MEK and mTOR inhibitors.In order to study HRAS mutations we collected HRAS mutant cancer cell lines including KNS-62, NCI-H1915, T24, RL95–2 and KYSE-30 Figure . SequencSince MEK inhibitors block cell growth we wanted to analyze whether induction of apoptosis occurs in HRAS mutant cancer cell lines. We incubated cell lines with MEK inhibitors and measured apoptosis 72 hours later. Indeed, MEK inhibitor MEK162 induced apoptosis in HRAS mutant cell lines KNS-62, NCI-H1915, T24, RL95–2 and KYSE-30 but not in HRAS wild-type cell lines Figure . ApoptosPreviously, it was demonstrated that mutant NRAS melanoma cells are sensitive to the combination of MEK and PI3K inhibitors . We founEnhanced apoptosis and tumor growth suppression by combination of MEK and mTOR inhibitors was previously observed in HRAS wild-type lung cancer cell lines . TherefoWe wanted to study the functional consequences of the different HRAS mutations including Q61L, Q61R and G12V in more detail by testing their abilities to transform interleukin-3 (IL-3)-dependent murine lymphoid Ba/F3 cells to cytokine-independent growth. Removal of IL-3 lead to a clear difference in cell proliferation. Ba/F3 cells expressing ectopic HRAS Q61L, Q61R or G12V mutations showed strong cell growth whereas Ba/F3 cells with wild-type HRAS or empty vector stopped cell growth . Ba/F3 cin-vivo  and RAS pathway oncogenes such as EGFR and KRAS . FurtherTo overcome possible resistance mechanisms and to expand the list of possible drugs we investigated combinations of PI3K or mTOR inhibitors with MEK inhibitors. For NRAS mutant cancers the combination of MEK and the dual PI3K/mTOR were described to be synergistic , 35, 36.HRAS mutation status was barely tested within clinical trials. Early clinical trials investigating Everolimus in non-small cell lung cancer (NSCLC) showed partial responses (PR) for some patients, however, mutation status was not determined , 40. A vWe have shown that mutant HRAS is a potential drug target in different types of cancer including lung cancer and bladder cancer. HRAS mutations sensitize to single treatment with MEK and mTOR inhibitors which can be further enhanced by combinations of MEK and mTOR inhibitors. Thus, our data may encourage clinical studies on HRAS mutant cancer patients.Murine IL-3 was purchased from Sigma-Aldrich. AZD6244, MEK162 and PD0325901 were purchased from Selleck Chemical. Everolimus and AZD8055 were purchased from MedChemExpress. All inhibitors were solubilized in dimethyl sulfoxide (DMSO) at stock concentrations of 1 mM.T24 and KNS62 were purchased from National Institute of Biomedical lnnovation JCRB Cell Bank, Japan. NCI-H1915, Phoenix and RL952 were purchased from ATCC. KYSE-30, CAL-12T, HCC44, HCC78 and Ba/F3 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), Germany. Lung cancer cell line NCI-H1915 cells were cultured in RPMI medium supplemented with 10% fetal calf serum (FCS) and 1 mM L-glutamine. KNS62, T24 and CAL12T cells were cultured in DMEM medium supplemented with 10% fetal calf serum and 1 mM L-glutamine. RL952 cells were cultured in DMEM and F12 medium (1:1) supplemented with 10% fetal calf serum, 0.005 mg/ml insulin (1:100 Opi) and 1 mM L-glutamine. KYSE30 cells were cultured in RPMI 1640 and F12 medium (1:1) supplemented with 10% fetal calf serum and 1 mM L-glutamine. Lung cancer cell lines HCC44 and HCC78 cells were cultured in RPMI medium supplemented with 10% fetal calf serum and 1 mM L-glutamine. Lung cancer cell line HCC827 cells were cultured in RPMI medium supplemented with 20% fetal calf serum and 1 mM L-glutamine. Ba/F3 cells were maintained in RPMI-1640 supplemented with 10% FCS and 0.5 ng/ml murine IL-3 (Sigma-Aldrich). For serum reduced experiments cell lines were kept in Accell serum free media (Dharmacon) supplemented with 1% FCS. For hypoxia studies cells were kept for 96 h in a 0.2% hypoxia chamber Invivo2 400 (Ruskinn).KNS-62, NCI-H1915, T24, RL95–2, KYSE-30, CAL12T, HCC44, HCC827, and HCC78 were transfected with either control or two different siRNAs against HRAS. Accell siRNA was purchased from Dharmacon and used according to the manufacturer's protocol. In brief, siRNAs were diluted in Accell siRNA Delivery Media from Dharmacon to a final concentration of 1uM. Knock-down efficiency was observed after 96 h.6/ml, selected with puromycin at 1 μg/ml (in addition with 0.1 ng/ml IL-3) 3d after transduction. Section pressure was maintained for at least 1 week, then IL-3 was reduced under ongoing puromycin treatment. IL-3 independent clones grew out after 5–7 days and were kept under puromycin 1 μg/ml.Ba/F3 cells were transduced retrovirally with empty pMSCV-Puro  or pMSCV-Puro expression wild-type HRAS, HRAS Q61L, HRAS Q61R, HRAS G12V with the help of 293T Phoenix cells (ATCC). Synthesis of HRAS or HRAS mutants, cloning and DNA sequencing of cloned inserts was performed by LifeTechnologies. Transduced cells were seeded at concentrations below 1 × 106 cells were lysed for 30 minutes in ice-cold MPERM buffer supplemented with 25 mM NaF, 1 mM dithiothreitol, and complete protease inhibitor cocktail from Roche Diagnostics. Cell debris was removed by centrifugation at 10000 rpm for 10 minutes, loading buffer added and treated for 5 min at 95°C. Then, proteins were separated by gel electrophoresis. Separated proteins were blotted onto a nitrocellulose membrane  which was blocked with 5% bovine serum albumin in phosphate-buffered saline/Tween . The following antibodies were used: anti-phospho-ERK , anti-ERK , anti-phospho Akt , anti-panAKT , anti-phospho MEK , anti-MEK , anti-phospho mTOR ,anti-mTOR , anti-HRAS rabbit  and anti–actin (Sigma-Aldrich).A total of 0.5 × 10Isolation of cellular RNA was done by using the Qiagen RNA Purification Kit (Qiagen). RNA (1 μg) was reverse transcribed into cDNA with a reverse transcription-PCR kit (Applied Biosystems). Next, 5 μL of cDNA was taken for a PCR of 50 μL volume. We used the following primers: HRAS forward, 5_-ggggcaggagaccctgtag-3_; HRAS reverse. PCR was performed, and 30 μL of PCR product was sent for sequencing to Microsynth, Switzerland. For sequencing, we used the same primers as for the PCR. Mutations can be found in Cancer Cell Line Encyclopedia .Cell lines were treated with indicated concentrations of inhibitors and apoptosis was measured after 48 h and 72 h. Apoptosis was assessed by AnnexinV–APC (Enzo Lifescience) and propidium iodide (PI) (Sigma-Aldrich) by FACS. Both reagents were diluted to a final concentration of 1.5%. Specific cell death was calculated by the following equation: specific cell death % = /(100% - % spontaneous cell death) x 100 .Cell proliferation was determined by Cell-Titer-Glo Reagent (Promega) according to the manufacturer's instructions. Cells were plated in 96-well plates at a density of 500 - 2500 cells per well. The next day, drugs were added at indicated concentrations and cell proliferation was measured 4 days later. Measurement of proliferation was done using a 96-well plate luminometer/plate reader . Data were calculated as relative values: luminescence for a given drug concentration was compared to luminescence of untreated cells. All experimental points were set up in duplicate replicates and were conducted at least 3 independent times. IC50 were calculated with GraphPad Prims. Combination index (CI) values with CalcuSyn Software (Biosoft) according to Chou-Talalay . CI valu6 KNS-62 cells into CB17 SCID−/− mice. Engrafted tumors reached 150 mm3 after 6 days and animals were randomized into 4 groups: DMSO only (n = 5), AZD6244 (Selumetinib) at 20mg/kg (n = 5), Everolimus at 3.5mg/kg (n = 5), and combined AZD6244 and Everolimus at 20mg/kg and 3.5mg/kg respectively (n = 5). Inhibitors were kept at stock concentrations of 250 mM (AZD6244) and 10 mM (Everolimus), diluted in 1000ul DMSO each and carefully diluted in animal water. Concentrations were adjusted to 4ml drink volume per day which was the observed volume for previous experiments. Tumor size and weight was monitored three times weekly, and the mouse technician who was measuring tumor size was blinded to grouping until the trial was completed. Determination of tumor volume was done by 3 dimensional digital caliper measurements. Statistical analysis of the experiment was done by a two-tailed t test at each time point. P-values < 0.05 (*), or p-values < 0.01 (**) were considered significant between different treatment groups. The animal study was performed in accordance with the University Zurich Animal Committee and animals were euthanized as soon as tumor volume exceeded 3000 mm3 or necrosis of tumors.We tested lung cancer cell line KNS-62 and bladder cancer cell line T24 for engraftment rates by subcutaneous injection in CB17 SCID−/− mice. Engraftment and growth rate was excellent for KNS-62 already after 6 days but poor for T24 cells (tumors shrinked after 14d). Finally, we xenotransplanted 1*10


We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines . Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. Colorectal cancer is the most common gastrointestinal cancer in the world, with approximately one million new cases being diagnosed and more than 500,000 deaths occurring yearly. Approximately, one in five patients is diagnosed with metastatic disease, and an additional 30%–40% develop metastasis during the course of their disease. Unfortunately, only a minority of the patients with metastatic disease is amenable to curative resection and remains free of disease recurrence . Even thGenetic analysis of colorectal tumors over the past 30 years allowed the characterization of distinct molecular pathways altered during the development and progression of this disease . More reCell surface proteins are involved in a variety of cellular functions, including nutrient and ion transport, adhesion and signaling. These proteins also play important roles in pathological conditions such as diabetes, neurological disorders and cancer. They represent approximately 18% of all protein-coding genes in the human genome  and, dueWe have recently used a combined bioinformatics approach to generate a catalog of genes coding for transmembrane proteins located on the surface of human cells . BrieflyTo define the mutational profile of the Surfaceome in colorectal cancer, we target sequenced the coding regions of the 3,594 cell surface protein genes in a panel of 23 tumor cell lines  that altSomatic point mutations were detected using an in house computational pipeline based on SAMtools mpileup calling Figure . As matcA total of 184 (5%) putative non-synonymous substitutions were nonsense, and 529 (89%) of the InDels introduced a frame-shift alteration in the mutated protein Table . To furtTo further address the biological significance of the uncovered point mutations, we have incorporated gene expression data available for the cell lines (RNAseq and microarray) and restricted our downstream analysis to mutated and expressed genes. A list of genes coding for cell surface proteins that are mutated and expressed in >10% of the 23 cell lines analyzed is provided in Semaphorin 4C (SEMA4C) mutations were detected and validated by Sanger sequencing in 4 cell lines . Two of these mutations occur in the SEMA domain, a highly conserved sequence of approximately 500 amino acids critical for inducing targets of Semaphorin signaling. A third mutation occurs in the plexin-semaphorin-integrin (PSI) domain, another highly conserved domain, enriched in cysteine residues  alterations were detected and validated by Sanger sequencing in 4 cell lines . Three cell lines carry frameshift mutations and the remaining cell line carries a non-synonymous point mutation with a predicted damaging effect, indicating a loss of function of the FGFRL1 protein in colorectal cancer  are mutated in >10% of the colorectal cancer cell lines, literature searches did not reveal evidence of the functional role or therapeutic potential of these genes in colorectal cancer. Nevertheless, recurrent mutations (>3%) in FAM38A, SLC36A1 and WDR81 have been reported for other primary tumors in the TCGA database, and further functional studies will be necessary to address their involvement in colorectal tumorigenesis.In order to identify putative druggable mutations in cell surface proteins, we searched for mutated genes present in the Drug-Gene Interaction Database (DGIdb), which integrates drug-target information from 13 different sources, including the literature and previously established databases . We geneA significant fraction (34%) of these mutations occurred in membrane transporters. Membrane transporters, including solute carriers (SLCs) and ABC transporters, control the uptake and efflux of amino acids, sugars, lipids and vitamins, and their expression and activity are frequently altered in cancer as a consequence of the higher energy and nutritional requirements of the tumor cells . MembranWe then focused on expressed genes with putative druggable mutations that were not previously considered as potential therapeutic targets for colorectal cancer, but for which specific inhibitors have previously been developed. We particularly focused on surface proteins with kinase activity, as they represent a significant fraction of the genes mutated in cancer and are highly amenable to targeting by rationally designed small molecule inhibitors. Two druggable RTKs (AXL and EPHA2) were found to be frequently (>20%) altered in our cell lines.Five point mutations in the kinase domain and/or with predicted functional impact in the AXL Receptor Tyrosine Kinase (AXL) were detected and validated by Sanger Sequencing in 22% 5/23) of our cell lines   were detected and validated by Sanger sequencing in 3 cell lines . Three of these mutations are located in the tyrosine kinase domain and one in the Ephrin-ligand binding domain  sequences. Our local pipeline for immunogenic epitope prediction was based on two algorithms RANKPEP and NetMHC as described in Materials and Methods. Mutated epitopes were required to have a binding affinity to the HLA*0201 molecule that was at least 20% higher than the reference epitope as predicted by both algorithms. A total of 82 putative mutated epitopes were identified (73 epitopes from non-synonymous mutations and 9 epitopes from frameshift mutations). However, when we combined gene expression data with epitope prediction analysis, we found that only 30% (25/82) of the predicted epitopes are expressed, and that 92% (23/25) of these epitopes are expressed in a subset of the cell lines with the mutator phenotype. These results suggest that the use of potentially immunogenic mutations in cell surface proteins for personalized T-cell based immunotherapy in colorectal cancer is limited, as only 30% of the mutated epitopes are expressed and less than half (11/23) of the tumors cell lines express mutated epitopes.One of the major objectives of cancer genome sequencing projects is to identify therapeutically targetable mutations. This objective has been achieved with repeated success in cancer therapy, resulting in the introduction of new treatment protocols in the clinical practice. The use of Imatinib, for chronic myeloid leukemia and other solid tumors, of Trastuzumab and Lapatinib, for ERBB2 positive breast cancer, and of Vemurafenib, for BRAF mutant melanomas, are emblematic examples of how genomic alterations can be used to target cancer cells . Over thCell surface proteins constitute optimal targets for directed therapies and represent two-thirds of the protein-based drug targets , 35. Surin vitro propagation of the cell lines, our results are in agreement with a recent mutation saturation analysis of 4,742 sequenced tumors, across 21 cancer types [We found that a significant 57%) fraction of the Surfaceome is reshaped by somatic point mutations in colorectal cancer cell lines. Our analysis identified 48 genes coding for cell surface proteins that were not previously described as mutated in primary colorectal tumors in the TCGA database [7% fractier types . This ster types .in vitro and in vivo and is comparable to that elicited by other well-known angiogenic molecules, such as VEGF-A, HGF and bFGF [SEMA4C mutations were found in 17% of the cell lines and recurrent mutations in SEMA4G (17%) and SEMA4D (22%) were also observed. The effects of Semaphorins and their receptors in cancer are broad, context dependent and complex . SEMA4C and bFGF , 39. OurInactivating mutations in FGFRL1, the most recently discovered member of the FGFR family, were detected in 17% of our cell lines. FGFRL1 binds with high affinity to heparin and FGF ligands, but it does not possess an intracellular protein kinase domain and, therefore, cannot signal by trans-auto-phosphorylation . FGFRL1 in vitro, as well as in tumor angiogenesis and metastasis in vivo [Higher mutation frequencies in the RTKs AXL (22%) and EPHA2 (17%) were detected in our panel compared to those reported in the TCGA database for primary colorectal tumors (3.51% AXL and 2.63% EPHA2) . AXL has in vivo . Moreove in vivo , Nilotin in vivo  and Lapa in vivo . In lung in vivo .In colorectal cancer, AXL expression is associated to increased invasiveness of tumor cell lines with overexpression of the chemokine receptors CXCR4 and CXCR7, and AXL knock-down in these cell lines significantly hampered tumor cell invasion . Considein vitro [Somatic alterations in EPH receptors were also frequently observed in our cell lines, including frequent mutations in EPHA1 and EPHA2. Point mutations in EPHA2 and EPHA1 have not so far been described in the literature for colorectal cancer. Nevertheless, mutations in the kinase domain of EPHA3 was reported in 5% of colorectal cancer cell lines  and EPHAin vitro . On the in vitro , 31.Non-synonymous and frameshift mutations in tumor cells can generate unique T-cell mutated epitopes and induce tumor antigen-specific immune response . There iNotwithstanding, we observed that mutated expressed epitopes are predominantly found in colorectal cell lines presenting a mutator phenotype and that this specific subset of cell lines express a total of 23 mutated epitopes. In this context, it was recently demonstrated that patients with tumors showing naturally occurring immunogenic mutations presented higher cytotoxic T-cell infiltration and improved overall survival and, based on these observations, the use general immune modulators that block immune regulatory checkpoints such as anti-CTLA4 and anti-PD1 was proposed as a treatment strategy for patients with immunogenic mutations . AccordiTo the best of our knowledge, this is the first systematic and focused screen of point mutations in genes coding for cell surface proteins in colorectal cancer. By combining high-throughput sequencing, bioinformatics tools, data integration and literature searches, we have successfully discovered novel altered pathways and druggable mutations for targeted therapy in colorectal cancer. We have also uncovered the potential use of existing RTK inhibitors and immune checkpoint target drugs in specific subsets of colorectal cancer patients. Results presented here are encouraging, however our study also presents some limitations.in vitro propagation of the cell lines or to very rare germline polymorphisms not represented in public databases, nor in individuals sequenced by the 1000 Genomes Project. However, we believe that these possibilities do not significantly affect our results, since we have previously shown that the rate of mutation accumulation during in vitro propagation is not significant [in vitro growth. Second, the functional consequences of the uncovered genetic alterations were predicted primarily using computational tools, and confirmation with functional in vitro assays is further required. Similarly, additional experiments to evaluate the effects of pharmacologic inhibition of the altered pathways using pre-clinical models are compulsory to translate our findings to the bedside. Finally, although we suggest potential molecular therapeutic targets in colon cancer, it is important to recognize that a recent study matching targeted therapy with specific molecular abnormalities for patients with advanced colorectal cancer failed to confer significant clinical benefit [First, although we have described novel druggable mutations occurring in a representative panel of colorectal cancer cell lines, it will be important to confirm the prevalence of these alterations in clinical samples matched with normal tissue. At present, we cannot completely exclude the possibility that some of the alterations reported in this study correspond to mutations acquired during nificant  and stri benefit . We belioC and 5% CO2. Cell lines were authenticated and tested for Mycoplasma contamination as previously described [The panel of 23 colorectal cancer cell lines used in this study was obtained from different sources . CACO2, escribed .We target-sequenced the coding regions of 3,594 cell surface proteins in 12 cell lines . Surfaceome-capture and sequencing were performed using Sure Select Target Enrichment baits (Agilent Technologies) and the SOLiD 4.0 sequencing platform (Life Technologies), respectively. For the remaining cell lines  whole-exome capture was performed using the TruSeq Exome Enrichment Kit (Illumina) and paired-end sequencing was performed using Illumina HiSeq 2000. A local pipeline was then developed to extract the genomic sequences corresponding to the Surfaceome targeted region from whole-exome data.kinase.com/human/kinome) was used to identify genes coding for cell surface proteins with kinase activity.Exome-capture sequencing data on colorectal tumors were retrieved from TCGA and used to identify novel mutated genes and to determine mutation frequencies in colorectal cancer primary tumors. The DGIdb  was usedwww.novocraft.com). A local pipeline for point mutations was developed using Samtools mpileup and bcftools [For single nucleotide variations (SNVs) detection, SOLiD 4.0 and Illumina reads were aligned to the human reference genome sequence (GRCh37/hg19) using BioScope (Life Technologies) and BWA , respectbcftools . Duplicabcftools , PolyPhebcftools  and Mutabcftools  were useRNA-Seq data was generated for 12 cell lines  using the 5500XL sequencing platform to a depth of >100 million reads. Sequences were aligned to the human reference genome sequence (GRCh37/hg19) using TopHat  with sta50 was ≤ 500nM. To check for predicted cleavage, sequences were then analyzed using the NetChop algorithm [Peptide sequences corresponding to non-synonymous mutations and InDels, flanked by 10 aminoacids on either side, were used for epitope prediction by applying a similar approach to that described by Segal et al. 2008 . The samlgorithm , and onl


Kaempferia angustifolia has afforded a new abietene diterpene, kaempfolienol (1) along with crotepoxide (2), boesenboxide (3), 2′-hydroxy-4,4′,6′-trimethoxychalcone (4), zeylenol (5), 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3β-ol (7), sucrose, β-sitosterol, and its glycoside (8). The structures of the compounds were elucidated on the basis of spectroscopic methods . Isolation of 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3β-ol (7), and β-sitosterol-3-O-β-D-glucopyranoside (8) from this plant species has never been reported previously. The spectroscopic data of (7) is firstly described in this paper. Cytotoxic screening indicated that most of the pure compounds tested showed significant activity with (4) showing the most potent activity against HL-60 (human promyelocytic leukemia) and MCF-7 (human breast cancer) cell lines. However, all extracts and most of the pure compounds tested were found to be inactive against HT-29 (human colon cancer) and HeLa  cell lines. Similarly, none of the extracts or compounds showed activity in the antimicrobial testing.Phytochemical investigation on rhizomes of  Kaempferia is a genus of herbs of over 50 species natives of east tropical Asia belonging to the Zingiberaceae family. It is small rhizomatous herb with usually thick aromatic tuberous roots and short rhizomes. Kaempferia angustifolia is one of the Kaempferia species which is less well known in Malaysia, if compared with K. galanga and K. rotunda. Itis locally known as Kunci pepet, Kunci menir, or Kunci kunot. This plant is tuberous and stemless, has small leaves, and could be found growing wildly in the forests of west and center of Java. It has nice smell and is usually used as medicine to treat cold, stomach-ache, and dysentery, while its rhizome is used for coughs and as a masticatory D: +67.5. Rf: 0.60 . IR (KBr): 3684, 3402, 2940, 1644, 1098, 888 cm−1. UV/Vis λmax (CHCl3) nm (logε): 239.2 (3.837). 1H NMR  and 13C NMR : m/z(%) = 440 , 425 (56), 407 (31), 313 (100), 69 (72), 55(81)., Salmonella choleraesuis, and Bacillus subtilis (wild) (B29) for antibacterial screening and Candida albicans, Saccharomyces cerevisiae, and Aspergillus ochraceus (ATCC 398) for antifungal screening using disc diffusion method as described in + peak at m/z 399 which corresponds to the molecular formula mentioned. All spectral data were in accordance with the previously reported data of compound 6 which is isolated from Uvaria purpurea +, 396 [M-C6H10O5-H2O]+, 255 [M-C6H10O5-H2O-C10H21(side chain)]+, and m/z 127, 73, and 57 which were obtained from the sugar moiety suggested that compound 8 may be monoglycoside with a C29-sterol (m/z 414) aglycone moiety. Further analysis of FAB-MS spectrum demonstrated the molecular ion peak at 599 [M+Na]+ which supported the molecular formula of C35H60O6. From the evidences in spectral data and comparison with reported data [ 8 was, therefore, established as β-sitosterol-3-O-β-D-glucopyranoside which was isolated previously from Prunella vulgaris.Compoundted data , compoun Kaempferia angustifolia against HL-60 (human promyelocytic leukemia), MCF-7 (human breast cancer), and HeLa  cell lines. All crude extracts tested were inactive against all cell lines tested, with IC50 > 30 μg/mL. Among the cell lines examined, HL-60 and MCF-7 cells exhibited high sensitivity for all tested compounds (1–8), except for the weak activity shown by compound 2 against HL-60 cell and compound 5 towards MCF-7. Most of the isolated compounds were inactive against HT-29 (human colon cancer) and HeLa cell lines, with the exception of compound 7 which showed strong activity with IC50 of 1.4 μg/mL, which is comparable to standard (5-fluorouracil). Compounds 6 and 8 also showed moderate inhibition against HeLa cell line. Extracts or isolated compounds, which give IC50 < 10 μg/mL, were considered to have significant cytotoxic activity [The cytotoxic screening results  suggesteactivity . Cytotox4) appeared to be the most potent compound in cytotoxic screening against both HL-60 and MCF-7 cell lines. A recent publication [4) exhibited similar cytotoxic property against MCF-7 cell line but demonstrated better anticancer effect against HT-29 cell line as compared with our findings, with GI50 values of 17.5 μM (5.5 μg/mL) and 45.3 μM (14.2 μg/mL), respectively. The presence of methoxy substituents and α-methylation of the enone moiety and the presence of 2′ oxygenated substituents are favourable structural requirements for cytotoxic activity due to antimitotic activity while several ring-A methoxylated chalcones exhibited cytotoxic activity by inhibiting tubulin polymerization against a variety of tumor cell lines in the low micromolar range (IC50 < 50 μM) [1) showed moderate inhibition against HL-60 and MCF-7 cell lines with IC50 < 30 μg/mL while zeylenol (2) demonstrated moderate inhibition towards HL-60 only with IC50 of 11.6 μg/mL as reported previously [Interestingly, 2′-hydroxy-4,4′,6′-trimethoxychalcone  (lication  investig< 50 μM) . Kaempfoeviously . Kaempferia angustifolia were assayed against Gram-positive Bacillus subtilis, MRSA, Gram-negative Pseudomonas aeruginosa, Salmonella choleraesuis, Candida albicans (yeast), and fungi Aspergillus ochraceus and Saccharomyces cerevisiae. However, no activity was detected for all the samples against all microbes tested. Compounds (4) and (6) were not tested towards C. albicans and fungi due to insufficient amount of samples. The antifungal properties of flavokawain A (4) against Saccharomyces cerevisiae, Candida albicans, and some other fungi were reported to be inactive [ 4 did not show any inhibition towards Gram-positive and Gram-negative bacteria tested in this study.As for antimicrobial testing, crude extracts and constituents ofinactive , and the Kaempferia species is unprecedented besides reports on common plant sterols such as β-sitosterol and stigmasterol. The physical properties and spectral data of (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3β-ol (7) are firstly described in this paper. The biotaxonomic distribution of lanostanes is quite restricted [ 7) from Zingiberaceae has never been reported before and, hence, this finding has contributed to the chemotaxonomic significance.The isolation of triterpene from Kaempferia angustifolia Rosc. afforded eight compounds  which showed cytotoxicity against some human cancer cell lines. The structures of the isolates were identified and elucidated based on spectroscopic method and comparison of literature reviews.The rhizome extracts of


It has been an open question how similar human and canine lung cancers are. This has major implications in availability of human treatments for dogs and in establishing translational models to test new therapies in pet dogs. The prognosis for canine advanced lung cancer is poor and new treatments are needed. Heat shock protein 90 (HSP90) is an ATPase-dependent molecular chaperone ubiquitously expressed in eukaryotic cells. HSP90 is essential for posttranslational conformational maturation and stability of client proteins including protein kinases and transcription factors, many of which are important for the proliferation and survival of cancer cells. We investigated the activity of STA-1474, a HSP90 inhibitor, in two canine lung cancer cell lines, BACA and CLAC.50 after 72 h treatment with STA-1474 were 0.08 and 0.11 μM for BACA and CLAC, respectively. When grown as spheroids, the IC50 of STA-1474 for BACA cells was approximately two-fold higher than when grown as a monolayer (0.348 μM vs. 0.168 μM), whereas CLAC spheroids were relatively drug resistant. Treatment of tumor-stromal fibroblasts with STA-1474 resulted in a dose-dependent decrease in their relative cell viability with a low IC50 of 0.28 μM.Comparative genomic hybridization analysis of both cell lines revealed genetic relevance to human non-small cell lung cancer. STA-1474 inhibited growth and induced apoptosis of both cell lines in a dose- and time-dependent manner. The ICsHere we first established that lung adenocarcinoma in people and dogs are genetically and biochemically similar. STA1474 demonstrated biological activity in both canine lung cancer cell lines and tumor-stromal fibroblasts. As significant decreases in relative cell viability can be achieved with nanomolar concentrations of STA-1474, investigation into the clinical efficacy of this drug in canine lung cancer patients is warranted. Cancer is the leading cause of mortality in dogs –3. Lung in vitro and in vivo models of canine lung cancer Gene ID: 403612, 480438, 474919, 404019, 403438, 481033, and 403940 updated on 7-Dec-2014 and 29-Jan-2015) using the ClustalW procedure in DNASTAR Software Lasergene MegAlign® v.12.1 Madison, WI. All other primer sets used to generate PCR products were previously sequenced and aligned to verify the amplicons m where fa is the fraction of cells affected, fu is the fraction of cells not affected (1-fa), D is the dose of drug, Dm is the dose of drug to cause the median effect and m is the slope of the median-effect curve. The CI value definitions are listed in the Similarly, 4 x 10mus, NJ) . The nat® Homogeneous AMC Caspase 3/7 Assay Kit  according to the manufacturer’s instructions. Briefly, 4,000 cells per well were seeded in 100 μl of medium and incubated overnight. The next morning, cells were treated with STA-1474 (0.05–1 μM) for 24 and 48 h. Then, 50 μl of the caspase 3/7 substrate solution was added to each well and mixed in a plate shaker for 60 min at 150 rpm and wrapped in foil to protect from direct light. Fluorescence intensity was measured using a plate reader (Molecular Devices), with excitation at 245 nm and emission detection at 442 nm.Alexa Fluor 488 annexin V and 1 μl of PI solution (100 μg/ml) were used to stain cells for fluorescence-activated cell sorting (FACS). After 15 min of incubation at RT, 400 μl of annexin-binding buffer was added and the sample was gently mixed and kept on ice until analysis. Cells were analyzed within 30 min of staining. Caspase 3/7 activity was evaluated with the SensoLyte®, Danvers, MA) with 1mM phenylmethanesulfonyl fluoride and protease inhibitors  added just before use. Cells were scraped from the dishes, and the lysates were incubated in the buffer for 15 min on ice, and centrifuged for 20 min at 16,000 x g at 4°C. The supernatants were collected, and protein concentration was determined by a modified Bradford method .For the measurement of HSP90 and HSP70, client proteins and phosphorylated forms in the BACA and CLAC treated cells, cells were grown to 90% confluence in 100-mm dishes, placed on ice, rinsed with ice-cold DPBS, and lysed with 1X cell lysis buffer  at a final concentration of 33%, and the samples were heated at 100°C for 5 min. Cell protein extracts were fractioned on a precast 12% NuPAGE® Novex® Bis-Tris polyacrylamide Mini gels (10 cm x 10 cm) using NuPAGE® MES SDS running buffer for small proteins (2–200 kDa) or NuPAGE® MOPS SDS running buffer  for medium-size proteins (14–200 kDa) followed by electrophoretic transfer to nitrocellulose membranes . The membranes were incubated overnight at 4°C with 0.2% Tween-20 in TBS and 2% bovine serum albumin with rabbit monoclonal and polyclonal antibodies. The blots were incubated with secondary anti-rabbit IgG horseradish peroxidase—linked antibody in 0.2% Tween-20 in TBS and 2% nonfat dry milk for 1 h at RT. Primary antibodies were epidermal growth factor receptor (VER155008:STA-1474 CLAC) and [169:1](VER155008:STA-1474 BACA) and relative cell viability assessed. The resulting CI values are shown for effect level. For example, the doses of these two drugs needed to achieve a 97% decrease in BACA relative viability gives a CI value that would indicate moderate synergism which provides a drug reduction index score indicative of a four-fold decrease of VER155008 and two-fold decrease of STA-1474.50 for cells grown as monolayers or as three-dimensional tumor spheroids. Both BACA and CLAC cells formed tumor spheroids when grown for three days in ultra-low attachment well plates  compared to monolayer cultures. CLAC cells grown as a monolayer had a dose-dependent decrease in viability that was significant at doses of 0.5 μM or higher. CLAC spheroid cultures were resistant to all drug treatment concentrations investigated  phase IIb/III clinical trials have provided compelling rationale for investigating the feasibility of using HSP90 inhibitors for treatment of lung cancer in dogs . HSP90 iMYC, and 2-copy loss of a small part of chr11, for which the overlapping segment between the two cell lines includes only CDKN2A/B/B-AS1. These are among the most common CNAs seen in human lung adenocarcinoma [CDKN2A being involved in 43% of cases. Notably CDKN2A encodes ARF, which directly interacts with overexpressed MYC protein to block its transformation and proliferation activities [PIK3R1 and CASP3 in BACA, and NRAS and CCND1 in CLAC [TSC2, NF2, BCL6, CHEK2, CDK6, KAT2B, PKD1 and TP63. Among the mechanistic insights, the CNAs suggest relevance for HSP90 inhibition that would be expected to have therapeutic effects through the PI3K and MAPK pathways . Other pathways that are likely to be affected according to our findings are cell cycle progression  and apoptosis (CASP3). The array CGH data thus support both lung adenocarcinoma and pan cancer relevance. Additionally, the GSEA analysis of these two canine cell lines strongly implicates human lung adenocarcinoma among all other cancer data. These facts formally demonstrate that BACA and CLAC can serve as comparative oncogenomic models for development of drug treatments to mammalian lung adenocarcinomas in which a set of common driver mechanisms are present.Somatic mutations are the predominant mechanism that gives rise to cancer. The average cancer cell has approximately four sequence mutations of oncogenes (mean 1) and tumor suppressors (3), 11 very large CNAs involving whole chromosomes  or chromosome arms , and 23 focal CNAs . Here wearcinoma , with CDtivities . Other l in CLAC . Table 2in vitro models to evaluate candidate drugs that have been effective in controlling human NSCLC, it was important to establish that these canine lung cancer cell lines expressed HSP90 isoforms, HSP70, and various receptor tyrosine kinases and downstream kinases, relevant to lung cancer in humans. These included EGFR, c-Kit, HER2, VEGFR2, PDGFRα, PDGFRβ, c-Met, MAPK, c-Ret and Akt. The cell lines used for this work were from primary canine lung tumors and of pulmonary cell differentiation as the presence of cDNA transcripts for TTF-1, a nuclear protein [Once we confirmed that the cell lines were appropriate  protein . TTF-1,  protein , 11, 41.in vitro, or only had effects at drug concentrations not thought to be biologically relevant, or achievable, in vivo. For example, the IC50 of vinorelbine for BACA was 0.72 μM, which is 4- to 10-fold higher than in human NSCLC [50 dose for carboplatin is not tolerable in dogs. The tolerated carboplatin IC50 has been extrapolated from pharmacokinetic data performed in laboratory beagle dogs using Cmax at the recommended dose of 300 mg/m2 given as an IV bolus [max of 20–30 μg/mL which is equivalent to a molarity of 67 to 100 μM [50 for the lung cancer cell lines  is compared to ICs50 for other cancer cell lines of canine origin, they are higher than then those for canine mammary gland tumors or canine melanoma  [50 differed between the cell lines, with the CLAC IC50 being three-fold higher than BACA IC50. Our IC50 findings for gemcitabine are similar to the ICs50 reported for canine osteosarcoma cell lines, which ranged from 5.7 to 15.3 μM for the cell lines that had dose-dependent decreases in cell proliferation [When complete surgical resection of a primary lung tumor is not possible in canine patients, treatment with cytotoxic chemotherapy may be considered in an attempted to slow the progression of the disease. Interestingly, the cytotoxic drugs used in this study had limited to no apparent effect on cell viability an NSCLC  and the an NSCLC . The obtIV bolus . An estiIV bolus . Likewiso 100 μM . When thctively) , 47. Theferation .We evaluated the prodrug STA-1474 in lieu of ganetespib because of its greater solubility in water which facilitates its use in the dog without untoward side effects. A phase I study evaluated STA-1474 in dogs with solid tumors and reported measurable objective responses for malignant mast cell disease, osteosarcoma, melanoma and thyroid carcinoma . Based oGanetespib, a second generation HSP90 inhibitor, effectively and simultaneously destabilizes HSP90 client proteins in NSCLC cells including receptor tyrosine kinases and canonical JAK/STAT, PI3K/AKT, MAPK and mTOR signaling. Moreover, ganetespib accumulates in tumors relative to normal tissues, with a half-life in the tumor that is 10- to 19-fold longer than in normal tissues or plasma . Our expThe tyrosine kinase inhibitor toceranib is a multi-targeted kinase inhibitor that effects both tumor cell proliferation and tumor angiogenesis. We sought to determine if common tyrosine kinases which support lung tumor growth could be inhibited by torceranib. Even after 72 h of exposure to the highest dose of torceranib used (1 μM) this compound was unable to abrogate the phosphorylated and total protein expression of all the kinases evaluated in both cell lines. Further studies are needed to evaluate if torceranib demonstrates inhibitory activity against VEGFR, PDGFR and c-Kit protein expression in canine lung cancer cell lines.Exposure of tumor cells to HSP90 inhibitors induces a cellular protective and compensatory response which is to increase the expression of other heat shock proteins, notably HSP70. The increase of HSP70 expression has been shown to abrogate the extent of cell death . Indeed 50, the doublet combination effects were different not only between cell lines but also in terms of the desired effect level. Moderate synergy was seen when the BACA line was treated with the combination at concentrations higher than its’ IC50 values, whereas in the CLAC line this combination created synergism only at the IC50 value. This was an unexpected finding, as VER155008 has shown to be synergistic with other HSP90 inhibitors in human NSCLC lines [HSP70 has also been shown to be a driver of oncogenesis therefore therapy using a combination of a HSP90 inhibitor with a HSP70 inhibitor may provide a wider therapeutic window and increase the target-driven therapeutic index. We wanted to investigate if the simultaneous inhibition of HSP70 would enhance the growth inhibitory effects of HSP90 treatment. The use of VER155008, a HSP70 inhibitor, suppressed cell growth in both cell lines but only at high μM concentrations. Similar results have been described in human NSCLC cells treated with this drug . SurprisLC lines –56. MultLC lines –59. The in vivo. Tumors are three-dimensional (3D) complex tissues composed of neoplastic cells, vasculature and tumor stroma. As such, we sought to determine the response of STA-1474 in a 3D-model of canine lung cancer as well as in cells of the tumor stroma, represented by tumor-stromal fibroblasts obtained from a primary canine lung tumor cell culture. Both cell lines grew as spheres in ultra-low attachment wells, as described by Vinci et al. [50 as addition of growth factors can enhance cell proliferation and viability making direct comparison of spheroid and monolayer ICs50 impossible. Spheroid conformation was different between the cell lines. This finding was similar to previous reports that have reported different cell lines from a variety of tumors grow in different spheroid patterns [50 were higher for both spheroid cell cultures than for the monolayer adherent cultures. This was not unexpected as in general chemotherapy drugs have increased potency in 2D models compared with spheroid cultures, although exceptions do exist with a better response seen in 3D models to some pharmaceuticals [50 as lack of vascularization responsible for the delivery and distribution of the agent throughout the sphere is not present.Evaluation of drug activity using a two-dimensional monolayer of tumor cells poorly models the disease i et al. . In conti et al. . This wapatterns , 62. The50 for tumor stromal fibroblasts was higher than the cell lines it was still within a biologically achievable dose. This result was dissimilar to what has been reported with other HSP90 inhibitors, which have decreased CAFs cytokine production but not viability [The tumor stroma is composed of cancer-associated fibroblasts (CAFs), cells that are phenotypically and functionally different from their normal counterparts. CAFs can induce cancer cell stemness as well as epithelial to mesenchymal transition to promote tumor progression . CAFs caiability .50 will be increased when compared to the ICs50 obtained when cells are grown in a monolayer. This finding re-enforces the importance of evaluating cellular responses to drugs in a model that more accurately mimics the natural tumor environment. Nevertheless, the preclinical activity profile of STA-1474 demonstrated in this study provides preliminary evidence that this compound is superior to most of the currently available drugs and may offer an effective therapeutic opportunity to manage the canine lung cancer patient.According to our genetic and biochemical analyses, canine lung adenocarcinoma cell lines are relevant to the same human cancer. Likewise, the results of the canine aCGH and the variability in biological response to the chemotherpy agents found in these two cell lines suggests that the canine patient would benefit from precision medicine which has significantly improved the quality of life of the human lung cancer patient. We showed that treatment with STA-1474 decreases BACA and CLAC viability and HSP90 client protein expression at biologically relevant doses, by inducing dose- and time-dependent apoptosis. In contrast with other small molecules inhibitors, STA-1474 affects several proteins in different cellular signaling pathways and decreases viability not only in tumor cells, but also stromal fibroblasts. The efficacy of two small molecule inhibitors in canine lung cancer cell lines grown in a 3D spheroid format established that ICsS1 Tablevs. large alterations and gene annotation.Contains segmentation, classification of focal (XLSX)Click here for additional data file.


As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC.in vitro  and in vivo.Three NSCLC cell lines  were treated with PF-4708671 at five different concentrations, including 0.1μM, 0.3μM, 1μM, 3μM and 10μM, and protein levels were determined by Western-blot. Then, PF-4708671’s effects were assessed both in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo.The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis  Lung cancer, one of the most common malignancies, is the leading cause of cancer-related deaths worldwide, with the highest morbidity and mortality rates in China. More than 80% of patients with lung cancer are diagnosed as non-small cell lung cancer . DespiteIn vitro overexpression of p70S6K promotes cell proliferation, angiogenesis, and apoptosis suppression  x 100%. The study was approved by the ethics committee of West China Hospital.H460 cells, which showed the highest growth rate, were selected for P<0.05 was considered statistically significant.Data were processed by Microsoft Excel 2010. Using the SPSS 19.0 software, t-test and one way-ANOVA were applied for data analysis. Data are mean±SD, and two-sided P<0.05); p-S6 expression levels decreased with increasing drug concentrations (P<0.05). On the contrary, protein levels of downstream BAD, Caspase3 and ERK were increased after treatment with PF-4708671 with the concentration of 1μM. However, total p70S6K and S6 protein levels had no significant differences between negative control and treatment groups . In addition, H460, A549, and SK-MES-1 cell growth rates were significantly inhibited by PF-4708671 at 0.3μM, 0.1μM, and 0.1μM, respectively, 48 hour post treatment (P<0.05). All cell lines showed significantly reduced proliferation at 72 hour after treatment with 0.1μM PF-4708671 (P<0.05). Significant time- and concentration-dependent inhibition of cell proliferation was observed in all cell lines (P<0.05)  .P<0.05). Meanwhile, the proportions of cells in S and G2/M phases were significantly decreased in A549 cells (P<0.05). In addition, reduced amounts of S phase (H460) and G2/M phase (SK-MES) cells were also detected (P<0.05)  .P<0.05), with invasion inhibition rates of 75.90%, 46.23% and 82.73%, for H460, A549 and SK-MES-1 cells, respectively . Compared with negative control groups, apoptosis rates for H460, A549 and SK-MES-1 cell lines were increased by 1.537%, 2.483% and 3.475%, respectively (P<0.05)  .P<0.05). As shown in Table B in P<0.05). Furthermore, cell proliferation, as expressed by Ki-67, was significantly reduced in the H460+PF4708671 group compared with values obtained for both control groups. Finally, TUNEL staining demonstrated significantly higher apoptotic rate for tumor cells in Group 3 compared with control (Groups 1 and 2) values  values .P70S6K participates in tumorigenicity and cancer progression, but the mechanism underlying its effect is not completely understood. Studies assessing p70S6K in relation to NSCLC are scarce; we previously found that p70S6K overexpression promotes NSCLC cell proliferation, inhibits cell apoptosis and enhances invasion ability. To further understand the role of p70S6K inhibitors in non-small cell lung cancer, the present study assessed three NSCLC cell lines  treated with PF-4708671, and evaluated the changes of malignant phenotypes such as proliferation, invasion and apoptosis evasion.As shown above, the p70S6K inhibitor PF-4708671 significantly inhibited the activation of p70S6K and its key downstream effector S6, in a concentration dependent manner. Furthermore, the protein levels of downstream BAD; Caspase3 and ERK, which affecting apoptosis ability of cells, were increased after treatment with PF-4708671. This proved that PF-4708671 might affect the NSCLC cell lines’ survival by regulating relevant factors of apoptosis. In addition, NSCLC cell proliferation was time and concentration dependently inhibited by PF-4708671, corroborating previous reports –17. Thusin vivo.We hypothesized that by regulating the cell cycle, PF-4708671 may inhibit cell proliferation. Recent studies showed that S6K1 and p70S6K are critical for G1 arrest –28. Consin vitro. Interestingly, PF-4708671 could inhibit the invasion ability of all NSCLC cell lines assessed in this study. In contrast, studies in NSCLC patients showed that p-p70S6K expression is not associated with lymph node metastasis and cancer stage [in vivo.p70S6K overexpression was shown to be associated with aggressive disease and poor prognosis in breast cancer . Our preer stage –29. FurtHowever, there still had some limitations in our research. First of all, although all cells were NSCLC cell lines, the sources were different. So our results could not be completely consistent because there might be different mechanisms among different genetic backgrounds. Moreover, quantity was not big enough for experiments of nude mice in vivo. Therefore, the results need further in-depth research to confirm.In conclusion, our study demonstrated that the p70S6K inhibitor PF-4708671 could affect cell cycle distribution, inhibiting cell proliferation, apoptosis and invasion in NSCLC cells. Inhibiting the p70S6K-S6 axis resulted in potent anti-tumor activity. Therefore, combination therapy with p70S6K inhibitor and chemotherapy represents a promising new strategy for NSCLC treatment.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity.ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts  in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates.Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples.Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with  The DNA damage response (DDR), through the action of sensors, transducers and effectors, orchestrates the appropriate recognition of DNA damage and the repair and resolution of stalled DNA replication . This prImpaired cellular capacity to repair DNA damage has significant clinical implications, including the development of cancer and the response of cancer cells to cytotoxic therapies . PreviouSingle cell network profiling (SCNP) using multiparametric flow cytometry has emerged as versatile tool to quantitatively study the function of multiple specific biological pathways and signaling networks simultaneously using standardized methods and instrumentation ,6. By chBRCA1 inherited germline mutation, cell lines of known HRR status (BRCA1+/- and BRCA1+/+) were assessed. Lastly, primary AML samples were used to demonstrate the ability to quantify activation of these pathways in primary cancer cells.This manuscript describes the results of multi-parametric flow cytometry experiments measuring drug-induced DNA damage and activation of multiple DDR readouts in the HRR and NHEJ DSB repair pathways in distinct cell subsets defined based on cell cycle phase to quantify the functional integrity of the cellular DNA repair pathways. Cell lines with functional or defective DNA repair capacities were used to identify reagents capable of detecting activation of DNA damage repair pathways. To confirm that these methods are capable of identifying functional happloinsufficency in a model of ATM, BRCA2 or BRCA1 were used in the study. Following the suppliers’ instructions, cell lines obtained from ATCC  were maintained in RPMI-1640 supplemented with 10% FCS, and cell lines obtained from Coriell Cell Repositories  were maintained in complete RPMI-1640 supplemented with 15% FCS.Individual cell lines harboring mutations in ATM wild type cell lines , were compared with an ATM +/- cell line [GM03323 (Coriell)] and two ATM-/- cell lines [GM01526 and GM03189 (Coriell)] for measurement of etoposide-induced DDR readouts (details in SCNP assay section below).Cell line panel 1 was used to establish tools for measuring ATM-dependent DNA damage responses; BRCA2-/-, BRCA1+/-, and BRCA1+/+ samples in response to PARPi treatment (details in SCNP assay section below). To model BRCA2-/- status, an Epstein Barr Virus (EBV)-transformed B lymphocblast cell line [GM13023 (Coriell)] from a Fanconi’s Anemia patient with homozygous BRCA2/FANCD1 mutation was used. To model heterozygous BRCA1 mutation (BRCA1 +/-), 5 EBV-transformed B lymphoblast cell lines  from patients with BRCA1-mutated familial breast cancer were used. Similarly, to model wild type BRCA1 (BRCA1 +/+) status, 5 EBV-transformed B lymphoblast cell lines  from breast cancer patients whose tumors are negative for BRCA1 mutations or from healthy donors were evaluated.Cell line panel 2 was used to detect differences between AML samples consisted of either peripheral blood mononuclear cell (PBMC) or bone marrow mononuclear cell (BMMC) specimens obtained from pediatric or adult patients with AML. Mononuclear cells were purified by ficoll centrifugation then cryopreserved in 90% FBS, 10% DMSO. In accordance with the Declaration of Helsinki, all patients consented to the collection of biospecimens for biology studies.3), pelleted, and stained with unlabeled antibody cocktails followed by fluorochrome conjugated goat anti mouse or goat anti rabbit secondary antibodies , then blocked with normal rabbit serum and normal mouse serum (Life Technologies) and stained with cockails of fluorochrome-conjugated antibodies. Cocktails included antibodies against cell surface markers for cell gating of AML cells  and up to 3 antibodies against intracellular signaling molecules (detailed below) for 6- 8-color flow cytometry assays.SCNP assays were performed as described previously . AliquotData was acquired on an LSR II flow cytometer using the FACS DIVA software (BD Biosciences). All flow cytometry data were analyzed with FlowJo  or WinList . Daily QC of the LSRII cytometers was performed as previously described . Dead cea) For experiments measuring multiple DDR readouts after etoposide treatment, cell lines (Cell line panel 1) or primary AML samples were treated with 30 μg/mL etoposide  for 2 h or 6 h and assayed for p-BRCA1 (S1423) , pDNA-PKcs (T2609) , p-53BP1 (S1778), p-ATM (S1981), p-p53 (S15), p-Chk2 (T68), and p-H2AX (S139) .BRCA1 +/+ , BRCA1 +/- , and BRCA2 -/- cell lines (Cell line panel 2) were treated with 6 μg/mL AZD2281  +/- 2 μg/mL temozolomide (Sigma) for 48-72 h and assayed for p-H2AX (S139), p-RPA2/32 (T21) , p-DNA-PKcs (T2609), p21, p-ATM (S1981) and p-BRCA1 (S1423) in CyclinA2- and CylcinA2+ cells. In these experiments a 2 μg/mL dose of temozolomide was used due to excessive apoptosis observed with the combination of AZD2281 with higher doses of temozolomide (10 μg/mL) at later timepoints (data not shown).b) For experiments showing magnitude and reproducibility of multiple AZD2281+/- temozolomide-induced DDR readouts and the ability of these readouts to stratify for HRR status, c) For experiments measuring the cell cycle selectivity of individual genotoxic agents, cell lines in culture or AML samples pre-treated for 48 h with a panel of myeloid growth factors to induce proliferation in all AML subtypes  were challenged for 6 h with etoposide (30 μg/mL), PARP inhibitor AZD2281 (6 μg/mL), temozolomide (10 μg/mL), or the combination of AZD2281 + temozolomide (6 μg/mL + 10 μg/mL) and assessed for induced p-H2AX in distinct subsets of cells including CyclinA2+ cells, CyclinA2- cells, or “All” live, cPARP- cells regardless of CyclinA2 status.2Fold and Uu) were applied to gated cell populations containing at least 200 events and have been described previously  in all cells (regardless of CyclinA2 expression), in CyclinA2- and in CyclinA2+ cell subsets within the same cell line. This choice of conditions was based on previous experiments in cell lines demonstrating that PARPi AZD2281 +/- temozolomide selectively induced DSBs (p-H2AX) in CyclinA2+ cells while etoposide induces DSBs (p-H2AX) in both CyclinA2- and CyclinA2+ cells (data not shown).The ability of SCNP to measure DDR responses in cellular subpopulations was next exploited as a method to quantify the activity of cell cycle specific DDR pathways such as HRR. To identify robust DDR readouts which could be leveraged to recognize cells with functionally defective or impaired HRR pathway activity, the cellular response to PARPi AZD2281 was examined using an expanded panel of DDR readouts and cell lines with known mutations in HRR machinery, including BRCA2-/- cell line in CyclinA2+ cells at both 48 h and 72 h  higher in BRCA1+/- lines compared to BRCA1+/+ lines in CyclinA2+ cells but not in CyclinA2- cells or in the parent population at all timepoints and treatment conditions tested  CyclinA2+ cells only, b) CyclinA2- cells only or b) all healthy cells. While DDR readouts measured in all live cells or CyclinA2- cells demonstrated higher correlations with proliferation , DDR readouts from CyclinA2+ subsets were less correlated with proliferation (average R2: 0.194). These data confirm that analysis of DDR readouts specifically in CyclinA2+ cells helps normalize for cell proliferation rate, enabling more accurate quantification of HRR proficiency. In addition, examination of the percentages of live, healthy cells with or without PARPi treatment demonstrated no significant differences in apoptosis between BRCA1+/+ and BRCA1+/- samples (data not shown), which is consistent with published literature  are also associated with cancer predisposition and/or drug sensitivity , the futThis study demonstrates the utility of SCNP to functionally quantify multiple DNA damage-associated readouts in single cells of distinct cell cycle subsets and identify samples with defective DNA DSB repair capacity, including inherited haploinsufficiency. As multivariate analyses of SCNP data have proven useful in the prediction of clinical outcome in other oncology settings ,38, futuDBR, LYL, BL, JAC, AC, REH, and AC are employees of and/or stockholders in Nodality, Inc.DBR designed, performed, and analyzed the experiments and prepared the manuscript. LYL, JAC and AC performed experiments and analyzed data. BL analyzed and interpreted the data. REH and AC designed the study, interpreted data and prepared the manuscript. IS and SZF helped interpret the data and prepare the manuscript. All authors read and approved the final manuscript.


Primary culture of respiratory epithelial cells is useful to study the pathophysiology of respiratory diseases. However, such primary culture has been very limited because of its high dependence on the availability of biopsies and the long time required to reach confluence. Therefore, cell lines are an alternative to primary cultures because they reach confluence faster and some can maintain their differentiation abilities. However, unlike primary cultures and native tissues just some cell lines are able to polarize, with normal channel functionality and transepithelial ionic flux. Therefore, the authors concluded that the BCi NS1.1 cell line accurately mimics the airway epithelium, and can be considered as a model to study the interactions with environmental stimuli, cytokines, cigarette smoke or as a target for assessing of pharmacologic agents. However, studies of such interactions with the environment or drugs require not only a 100 % confluent culture but also polarization. The authors examined the Rte but this parameter is not sufficient to make conclusions about the polarization status of the cell line. Additionally, the Rte was 57 ± 4 Ω × cm2 (day 10) and 165 ± 67 Ω × cm2 (day 14) [2). Similar to native epithelium, passage 2 and 3 tracheobronchial epithelial cell cultures show Rte (500–800 Ω × cm2), but the equivalent Isc is much lower than that in native tissue (3 μA/cm2) [2 and 2 μA/cm, respectively, at passage 4. In another study, human airway epithelial cells were successfully passaged up to six times with an Rte of 1000–4000 Ω × cm2 and Isc of 5 μA/cm [I read with great enthusiasm the recent article written by Matthew Walters and colleagues in BMC Respiratory Research . The autday 14) . Each of(day 14) . However . Each o 5 μA/cm . Therefo


Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL. Follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) compose 60% of non-Hodgkin lymphomas (NHLs), both are derived of germinal center or post germinal center B cells. FL is uTo study the transforming mechanisms for germinal B cell derived lymphomas at the protein level several proteomics based studies have been conducted. The follicular lymphoma derived cell line SUDHL-4 was used to identify secreted proteins. In thisIn this study we compared the 2-D proteome profiles of NHL cell lines to Epstein Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) to identify differentially expressed proteins. Expression of a selection of the differentially expressed proteins  was validated in the cell lines and in primary patient material. Inhibition of PPIA with cyclosporine A (CsA) showed a clear effect on cell growth in all NHL cell lines with a correlation between PPIA expression and sensitivity to CsA induced cell death.2 in RPMI 1640 supplemented with 10% fetal calf serum (FCS), ultra-glutamine, penicillin and streptomycin (100U/ml). SUDHL5, SUDHL6 and SUDHL10 cells were cultured with 20% FCS. Five LCLs were generated from peripheral blood mononuclear cells by infection with B95.8 virus. One LCL was used to compare in the 2-D experiments, the other four were used in the validation and functional studies. LCLs were routinely grown in RPMI 1640 with 10% FCS. For the production of LCLs from peripheral blood permission was granted by the Institutional Review board  and written informed consent was obtained.DOHH2, SUDHL4 (FL), SUDHL6, SUDHL10, OCILY3, Karpas 422 and SUDHL5 (DLBCL) were obtained from DSMZ . DOHH2, SUDHL4, OCILY3, Karpas 422 cells were routinely grown at 37°C at 5% COTissue samples of 46 patients were collected from the pathology biobank for validation by immunohistochemistry. These 46 cases, consisted of 13 low grade FL, 8 FL transformed to DLBCL with evidence of FL in the sample or earlier diagnosis of FL (TFL), and 25 nodal DLBCL. The 25 DLBCL cases were stained for CD10, BCL6 and MUM1 and classified according to the Hans algorithm in GCB (8) were homogenized in 1 ml of the Homogenize Buffer Mix  in an ice-cold Dounce homogenizer. The homogenate was centrifuged at 700g for 10 minutes at 4°C. The supernatants were transferred to a new tube and centrifuged at 10,000g for 30 minutes at 4°C. The total cellular membrane protein pellet was lyzed in lysis buffer  and kept on ice for 30 minutes. The supernatants were harvested by centrifuging at 16,000g for 5 minutes at 4°C. The protein concentrations of the lysates were determined by Bradford assay.Cells . Immobilized pH gradient strips  were rehydrated for 12–16 hours after the protein was loaded. Isoelectric focusing  was performed at 20°C by the following program: a linear increase from 0–500V over 30 minutes, 500–1000V over 1 hour, 1000–5000V over 4 hours, 5000–8000V 4 hour and then held at 8000V for a total of 64,000Vh. This was followed by a two-step equilibration; first, strips were put into 10 ml equilibration buffer , which contained 1% dithiothreitol for 15 minutes; next, strips were put into 10 ml equilibration buffer with 2.5% iodoacetamide for 25 minutes, and transferred to 12% SDS polyacrylamide gels. All proteins were visualized by silver staining of the gel, according to standard protocols.In each experiment, two gels were run in parallel, one with the LCL sample and the second gel with one of the lymphoma cell lines. In order to assure reproducibility, all samples were run at least twice.All gels were scanned with a GS-710 calibrated imaging densitometer imager. The comparative analysis of gels was performed with PD Quest software (BioRad). The density of each spot was evaluated by normalizing volumes of all spots. Spots which were consistently up or downregulated (≥ 2-fold) or spots that appeared or disappeared and were showing consistent differences between LCL and NHL were carefully cut out. For LC-MS/MS spots with the highest density were selected, destained and digested overnight with 5ng/μl trypsin (freshly made in 20mM ammonium bicarbonate pH 8–8.5). After incubation, formic acid was added and gels were incubated 5 minutes on a shaker. They were centrifuged at 5,000rpm for 1 minute and the supernatant was collected for LC-MS/MS analysis with the LTQ-Orbitrap XL .The peaks and sequences of peptides from selected protein spots were identified by ProteinPilot 3.0 (Applied Biosystems). Proteins were identified by using the UniprotKB/Swiss-Prot database. ProteinCells were collected by centrifuging at 1200rpm for 5 minutes at 4°C and incubated with an anti-B2M antibody  for 30 minutes on ice. Cells were washed with 1ml 1% PBS/BSA and FITC labeled goat anti rabbit antibody  was added as the secondary antibody. Acquisition was performed on a Calibur flow cytometer  and data were analyzed with Winlist software.™ 1000 Spectrophotometer  and RNA integrity was evaluated by 1% agarose electrophoresis. cDNA was synthesized using 500ng input RNA, Superscript II and random primer according to the manufacturer’s protocol . Primers used were for PRDX1 forward 5’-AGCCTGTCTGACTACAAAGGAAAATAT-3’ and reverse 5’- GGCACACAAAGGTGAAGTCAAG-3’ and for PPIA forward 5’- AGCTGTTTGCAGACAAGGTCC-3’ and reverse 5’-GCAGGAACCCTTATAACCAAATCC-3’. The qPCR reaction was performed in triplicate in a final volume of 10μl consisting of 5μl SYBR Green mix , 2μl of forward and reverse primer (300mM) and 2,5μl 1ng of cDNA. Amplification was performed on a Roche LightCycler® 480 Instrument . TBP was used as a housekeeping gene and 2-ΔCp values were calculated.Total RNA was isolated using QIAzol  and samples were DNAse treated  according to the manufacturer’s protocol for cell lines. RNA concentration was quantified using the NanodropImmunohistochemistry was performed according to standard protocols with appropriate positive and negative controls. Antibodies used were: anti-B2M , anti-PRDX1  and anti-PPIA .5 cells/ml with different concentrations (0–10μg/ml) of Cyclosporine A and Alamar Blue . Cultures were measured every 24 hours for 3 days at an emission of 560nm and extinction of 590nm. Experiments were performed 3 times.Cell lines were cultured in triplicate at 10Statistical analysis was performed with IBM SPSS Statistics 22. The Mann-Whitney U-test was used to compare B2M, PRDX1, and PPIA expression levels in NHL groups and LCLs for MFI and mRNA levels. Differences of B2M, PRDX1, and PPIA staining were defined by Chi-square test for immunohistochemistry. A paired T-test was performed to define the difference in cell viability before and after cyclosporine A treatment. The correlation of PPIA expression level and cell viability was defined by Spearman-test. All analyses were two-tailed. P<0.05 was considered as significant.An average of 1,133 (±355) and 1,119 (±330) spots were detected in the 2-D gels of the LCL and NHL cell lines, respectively , PPIA (modified), MDH2, PRDX1 (modified), MDH1, ENO1, PRDX2 and PCBP1 were upregulated . The remWe selected B2M, PRDX1 and PPIA for further validation based on the differential expression patterns in the NHL cell lines and availability of suitable antibodies for immunohistochemistry. B2M plays a role in the immune response, PRDX1 has an anti-oxidant function, and PPIA is involved in signal transduction.Expression of B2M was absent or reduced in all NHL cell lines compared to the LCL cell line in the 2-D analysis . These 2PRDX1 (modified) was upregulated in 3 NHL cell lines  compared to the LCL cell line. Expression of PRDX1 was upregulated in 2 NHL cell lines (SUDHL4 and SUDHL10) and downregulated in 1 NHL cell line (SUDHL5). PRDX1 mImmunohistochemistry of the 46 primary NHL cases showed positive staining in 33 cases (72%). In FL, 5 of the 13 cases were positive with 4 cases showing weak positive and 1 case showing strong positive staining. Of the TFL cases, 3 showed weak positive staining and 3 strong staining. In GCB DLBCL 10 cases were weak positive and 3 cases were strong positive. Of the ABC DLBCL cases 5 showed weak positive staining and 4 showed strong staining . When coThe expression of PPIA (modified) was upregulated in 4 cell lines  compared to LCL in the 2-D gels. The unmodified PPIA was upregulated in 2 cell lines (DOHH2 and SUDHL4) and downregulated in 1 cell line (SUDHL6) .PPIA mRNA expression levels were upregulated in all NHL cell lines compared to LCLs . After treating the cells for 72 hours with different concentrations  of CsA the viability of LCL and NHL cell lines was assessed . The NHLOur 2-D gel electrophoresis approach revealed 28 differentially expressed proteins in lymphoma cell lines compared to LCLs. LCLs were chosen since they are cell lines and have similar proliferation patterns, while normal B cells are in a resting stage. For LCLs the transformation mechanism is by EBV and its proteins, and should be different from the NHL cell lines. The identified proteins are involved in various processes relevant to the pathogenesis of B cell lymphoma including cell motility and metabolism.The proteins included in the cell motility gene ontology are ARPC5, TUBB, CFL1, MYL6, CAPZA1 and PFN1. With the exception of the modified CFL1 and PFN1, these proteins are all downregulated in NHL. ARPC5, CFL1, CAPZA1 and PFN1 have been shown to play roles in metastasis and invasion of solid tumors. Downregulation of ARPC5 blocks metastasis, whereasVarious proteins identified by us as differentially expressed in NHL compared to LCL have been identified in B cell lymphoma previously. In the Eμ-driven mouse model Romesser et al found differential expression of ENO1, CALR, CFL1 and PPIA by comparing the proteome of activated B cells to B cell lymphoma ConsisteB2M, expression is downregulated or absent in all NHL cell lines compared to LCL cells. This observation is partially supported by flow cytometry and IHC staining. B2M is one of the polypeptide chains of human leukocyte antigen class I (HLA class I), and both chains are essential for membrane expression of HLA class I. Loss of HLA class I expression provides an immune escape mechanism for tumor cells. A number of studies have shown loss of HLA class I in DLBCL patients, especially in DLBCL presenting at immune privileged sites. Loss ofPRDX1 expression is elevated at protein and mRNA level in part of NHL cell lines compared to LCL cells. In patient samples, we observed a higher proportion of strong positive and positive PRDX1 expression in the more aggressive TFL and DLBCL as compared to the indolent FL, this difference is most pronounced in the comparison between GCB-DLBCL and FL. Peroxiredoxins are thiol peroxidases, that play a role in maintaining the redox balance and have anti-apoptotic ability. PRDX1 is expressed in germinal center B cells and plasma cells and in germinal center derived B cell lymphomas and multiple myeloma. The expPPIA, expression was elevated in the 2-D in 3 cell lines and mRNA levels are consistently higher in NHL cell lines compared to LCL cells. In addition, PPIA protein expression is higher in aggressive TFL and DLBCL as compared to the indolent FL. PPIA belongs to the immunophilin family, which catalyzes cis-trans isomerization during protein folding. Elevation of PPIA expression has been observed in some solid cancers, such as non-small cell lung carcinoma and gastIn summary, we identified 28 differentially expressed proteins and validated the expression in primary tissue samples for three selected proteins. The finding of B2M and PRDX1 confirmed validity of our approach as it has been shown previously in NHL. Elevated PPIA expression in lymphoma compared with LCL cells is novel and its oncogenic potential is supported by the inhibition of cell growth upon CsA treatment.S1 File(DOCX)Click here for additional data file.


Selective phosphoinositide 3-kinase (PI3K)/AKT/mTOR inhibitors are currently under evaluation in clinical studies. To identify tumor types that are sensitive to PI3K pathway inhibitors we screened compounds targeting PI3Kα/δ (AZD8835), PI3Kβ/δ (AZD8186), AKT (AZD5363) and mTORC1/2 (AZD2014) against a cancer cell line panel (971 cell lines). There was an enrichment of hematological malignancies that were sensitive to AKT and mTOR inhibition, with the greatest degree of sensitivity observed in T-cell acute lymphoblastic leukemia (T-ALL). We found that all NOTCH mutant T-ALL cell lines were sensitive to AKT and mTORC1/2 inhibitors, with only partial sensitivity to agents that target the PI3K α, β or δ isoforms. Induction of apoptosis only occurred following AKTi treatment in cell lines with PTEN protein loss and high levels of active AKT. In summary, we have demonstrated that T-ALL cell lines show differential sensitivity to inhibition at different nodes in the PI3K/AKT/mTOR pathway and inhibiting AKT or mTOR may have a therapeutic benefit in this disease setting. NOTCH1 are present in 55–60% of T-ALL and mutations in PTEN and the phosphoinositide 3-kinase (PI3K) pathway are present in 47% of pediatric cases [T-cell acute lymphoblastic leukemia (T-ALL) is the most common form of malignancy in children and is classified by the abnormal accumulation of immature lymphoblasts of the T-cell lineage . T-ALL aic cases –5. The dPIK3CA, AKT, and PTEN), amplifications  and deletions  [PTEN, a PIP3 phosphatase, display increased sensitivity to PI3Kβ and AKT inhibitors [PIK3CA mutations generally do not respond to PI3Kβ inhibitors but encouraging results have been observed with PI3Kα and AKT inhibitors [The PI3K/AKT/mTOR signaling pathway is the most frequently activated signaling network in cancer and recurring mutations in this network have been identified, including mutations ( INPP4B) –12. The  INPP4B) . Studieshibitors –15. In chibitors , 17.The development of selective PI3K pathway inhibitors allows us to better understand intrinsic target dependencies in the PI3K pathway to determine the optimal strategy for pathway inhibition. In addition, advances in screening technologies have enabled investigation of inhibitor sensitivity across large panels of cell lines spanning multiple cancer types . To this50 values were generated from 72 hour proliferation assays using a nine-point concentration range for each compound. The complete screening results can be found in To further understand the contribution of the different nodes in the PI3K pathway, we used four kinase inhibitors that are currently in clinical development, AZD8835 a PI3Kα and PI3Kδ inhibitor (PI3Kα/δi); AZD8186 a PI3Kβ and PI3Kδ inhibitor (PI3Kβ/δi); AZD5363 an pan-AKT inhibitor (AKTi) and AZD2014 an mTORC1/2 inhibitor (mTORC1/2i) , 19, 20.PTEN gene (loss of function/deletion mutations) or PIK3CA mutations, which is in keeping with previously published data , chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin lymphoma (NHL)   or similar to the ICI3Kα/δi) . FollowiNOTCH is frequently mutated in T-ALL and genetics data for the seven cell lines suggest that NOTCH is mutated in all of these cell lines [PTEN, which is observed at the protein level. Ser473 and Thr308 AKT phosphorylation was elevated in four of the T-ALL cell lines. In the same four cell lines, there was also strong phosphorylation of NDRG1 compared with the other three cell lines. p-PRAS40 was also elevated in the same four cell lines but not to the same extent as for p-NDRG1 and p-AKT. MYC protein levels were variable across the cell lines, and no correlation could be made between PI3K isoform expression and PI3K inhibitor sensitivity. These results demonstrate that a subset of NOTCH mutant T-ALL cell lines can be grouped through their protein expression profile as being PTEN null and p-AKT/p-NDRG1 high.In addition to examining the regulation of pathway biomarkers, we also determined the basal levels of protein expression and activation across the T-ALL cell lines. ll lines , 23. In PTEN null/p-AKT/p-NDRG1 high. These results suggest that at relevant concentrations of the inhibitors, only AKTi induces apoptosis and this is in a defined subset of the T-ALL cell lines.Given the strong phenotype following AKT or mTOR inhibition, we wanted to determine whether these inhibitors induced apoptosis in the T-ALL cell lines. Cells were dosed with increasing concentrations of each compound and Annexin V flow cytometry was performed 72 hours post drug treatment. At the concentrations defined in the Western blot experiments, AKTi induced apoptosis in four out of seven cell lines, whereas we did not observe an increase in apoptosis following PI3K or mTOR inhibitor treatment Figure . At highHere we screened four PI3K pathway inhibitors, targeting PI3Kα/δ, PI3Kβ/δ, AKT and mTORC1/2, against a panel of 971 cancer cell lines. Using elastic net regularization, we confirmed the robustness of the cell panel screen and observed a number of sensitivity correlations that have been previously described. Whilst we focussed on hematological malignancies and T-ALL, this PI3K pharmacology dataset can be used as a resource for further interrogation and validation of PI3K pathway biology and sensitivity. In addition to T-ALL, there were a number of other hematological subtypes that displayed enhanced sensitivity to the PI3K pathway inhibitors, which are currently being investigated.PTEN null, we did not observe strong sensitivity to the PI3K inhibitors across the T-ALL cell line panel. Previous reports have described that the PI3Kδ and PI3Kγ isoforms are required for the development of PTEN null T-ALL [NOTCH mutations negate the PI3Kδ/γ dependency in the T-ALL cell lines. PI3K isoform selectivity of the PI3K-selective inhibitors was determined in enzyme and cell based assays and demonstrate that GDC0941 and BYL719 inhibit the PI3Kδ and PI3Kγ isoforms at sub-micromolar concentrations : Cleaved Notch1 (#4147), PTEN (#9559), pAKT T308 (#2965), pAKT S473 (#9271), pNDRG1 T346 (#5482), pS6RP S235/236 (#2211), PIK3CA (#4249), PIK3CB (#3011), PIK3CG (#5405), TUBULIN (#5346), GAPDH (#3683). PIK3CD (sc-7176), pPRAS40 T246 (44-1100G) and MYC (ab32072) were purchased from Santa Cruz , Life Technologies  and Abcam , respectively.50 values were determined using live cell counts. Formulas used to calculate growth curves and to analyse combination data have been previously described [The Sanger cancer cell line pharmacology screen was performed as previously described . For theescribed . BrieflyCells were lysed in RIPA buffer (Life Technologies) supplemented with 1× Protease Inhibitor Cocktail , 1× phosphatase inhibitor (Life Technologies) and 1:5,000 benzonase  and equal amounts of protein were loaded and separated by SDS-PAGE. Horseradish peroxidase-linked secondary antibodies  and ECL or supersignal (Life Technologies) were used to detect immune complexes.The PE Annexin V apoptosis detection kit  was used for flow cytometry, performed using a Guava easyCyte flow cytometer .50 responses for each of the 971 cell lines together with a matrix of genomic features (gene mutations and lineage) represented as binary values for each of 1758 cancer-relevant genes and 39 cancer lineages. Strongly damaging loss-of-function mutations comprising nonsense, frameshift and splice site mutations in 189 well-characterised tumor suppressor genes were manually curated and classified into binary values. Prediction performance was determined using tenfold cross-validation and the elastic net features were validated to retain only those that were consistent across runs. The glmnet 1.9-5 software package and R 3.0.1 were used to perform the elastic net regression analysis [For the elastic net regression analysis, we used as input variables to the elastic net algorithm the natural log ICanalysis , 43.