Predicting tumor cell line response to drug pairs with deep learning.

BACKGROUND: The National Cancer Institute drug pair screening effort against 60 well-characterized human tumor cell lines (NCI-60) presents an unprecedented resource for modeling combinational drug activity. RESULTS: We present a computational model for predicting cell line response to a subset of drug pairs in the NCI-ALMANAC database. Based on residual neural networks for encoding features as well as predicting tumor growth, our model explains 94% of the response variance. While our best result is achieved with a combination of molecular feature types (gene expression, microRNA and proteome), we show that most of the predictive power comes from drug descriptors. To further demonstrate value in detecting anticancer therapy, we rank the drug pairs for each cell line based on model predicted combination effect and recover 80% of the top pairs with enhanced activity. CONCLUSIONS: We present promising results in applying deep learning to predicting combinational drug response. Our feature analysis indicates screening data involving more cell lines are needed for the models to make better use of molecular features.

The National Cancer Institute ALMANAC: A Comprehensive Screening Resource for the Detection of Anticancer Drug Pairs with Enhanced Therapeutic Activity.

To date, over 100 small-molecule oncology drugs have been approved by the FDA. Because of the inherent heterogeneity of tumors, these small molecules are often administered in combination to prevent emergence of resistant cell subpopulations. Therefore, new combination strategies to overcome drug resistance in patients with advanced cancer are needed. In this study, we performed a systematic evaluation of the therapeutic activity of over 5,000 pairs of FDA-approved cancer drugs against a panel of 60 well-characterized human tumor cell lines (NCI-60) to uncover combinations with greater than additive growth-inhibitory activity. Screening results were compiled into a database, termed the NCI-ALMANAC (A Large Matrix of Anti-Neoplastic Agent Combinations), publicly available at https://dtp.cancer.gov/ncialmanac Subsequent in vivo experiments in mouse xenograft models of human cancer confirmed combinations with greater than single-agent efficacy. Concomitant detection of mechanistic biomarkers for these combinations in vivo supported the initiation of two phase I clinical trials at the NCI to evaluate clofarabine with bortezomib and nilotinib with paclitaxel in patients with advanced cancer. Consequently, the hypothesis-generating NCI-ALMANAC web-based resource has demonstrated value in identifying promising combinations of approved drugs with potent anticancer activity for further mechanistic study and translation to clinical trials. Cancer Res; 77(13); 3564-76. ©2017 AACR.

Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.

Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.

Gene expression profiling of 49 human tumor xenografts from in vitro culture through multiple in vivo passages--strategies for data mining in support of therapeutic studies.

BACKGROUND: Development of cancer therapeutics partially depends upon selection of appropriate animal models. Therefore, improvements to model selection are beneficial. RESULTS: Forty-nine human tumor xenografts at in vivo passages 1, 4 and 10 were subjected to cDNA microarray analysis yielding a dataset of 823 Affymetrix HG-U133 Plus 2.0 arrays. To illustrate mining strategies supporting therapeutic studies, transcript expression was determined: 1) relative to other models, 2) with successive in vivo passage, and 3) during the in vitro to in vivo transition. Ranking models according to relative transcript expression in vivo has the potential to improve initial model selection. For example, combining p53 tumor expression data with mutational status could guide selection of tumors for therapeutic studies of agents where p53 status purportedly affects efficacy (e.g., MK-1775). The utility of monitoring changes in gene expression with extended in vivo tumor passages was illustrated by focused studies of drug resistance mediators and receptor tyrosine kinases. Noteworthy observations included a significant decline in HCT-15 colon xenograft ABCB1 transporter expression and increased expression of the kinase KIT in A549 with serial passage. These trends predict sensitivity to agents such as paclitaxel (ABCB1 substrate) and imatinib (c-KIT inhibitor) would be altered with extended passage. Given that gene expression results indicated some models undergo profound changes with in vivo passage, a general metric of stability was generated so models could be ranked accordingly. Lastly, changes occurring during transition from in vitro to in vivo growth may have important consequences for therapeutic studies since targets identified in vitro could be over- or under-represented when tumor cells adapt to in vivo growth. A comprehensive list of mouse transcripts capable of cross-hybridizing with human probe sets on the HG-U133 Plus 2.0 array was generated. Removal of the murine artifacts followed by pairwise analysis of in vitro cells with respective passage 1 xenografts and GO analysis illustrates the complex interplay that each model has with the host microenvironment. CONCLUSIONS: This study provides strategies to aid selection of xenograft models for therapeutic studies. These data highlight the dynamic nature of xenograft models and emphasize the importance of maintaining passage consistency throughout experiments.

The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology.

The NCI-60 cell lines are the most frequently studied human tumor cell lines in cancer research. This panel has generated the most extensive cancer pharmacology database worldwide. In addition, these cell lines have been intensely investigated, providing a unique platform for hypothesis-driven research focused on enhancing our understanding of tumor biology. Here, we report a comprehensive analysis of coding variants in the NCI-60 panel of cell lines identified by whole exome sequencing, providing a list of possible cancer specific variants for the community. Furthermore, we identify pharmacogenomic correlations between specific variants in genes such as TP53, BRAF, ERBBs, and ATAD5 and anticancer agents such as nutlin, vemurafenib, erlotinib, and bleomycin showing one of many ways the data could be used to validate and generate novel hypotheses for further investigation. As new cancer genes are identified through large-scale sequencing studies, the data presented here for the NCI-60 will be an invaluable resource for identifying cell lines with mutations in such genes for hypothesis-driven research. To enhance the utility of the data for the greater research community, the genomic variants are freely available in different formats and from multiple sources including the CellMiner and Ingenuity websites.

Antiproliferative mechanisms of action of the flavin dehydrogenase inhibitors diphenylene iodonium and di-2-thienyliodonium based on molecular profiling of the NCI-60 human tumor cell panel.

Flavoprotein-dependent reactive oxygen species (ROS) play a critical role in cytokine-mediated signal transduction in normal tissues and tumor cells. The flavoenzyme inhibitors diphenylene iodonium (DPI) and di-2-thienyliodonium (DTI) have been used to inhibit membrane-bound, flavoprotein-containing NADPH oxidases, including epithelial and leukocyte NADPH oxidases (Nox1-5 and Duox 1 and 2). Recent evidence suggests that DPI can decrease tumor cell proliferation; however, the molecular mechanisms involved remain poorly defined. To explore the mechanisms underlying DPI- and DTI-related tumor cell growth delay, we examined growth inhibition patterns produced by both agents in the NCI-60 tumor panel, and determined expression levels of Nox gene family members across these cell lines. Possible molecular targets were predicted using the COMPARE program. DPI was more potent than DTI (GI(50): 10nM versus 10µM); DPI and DTI exposure produced unique patterns of growth inhibition when evaluated against the small molecule anticancer database of the National Cancer Institute. Growth inhibition profiling of DPI revealed a modest positive correlation with Nox1 levels; novel mechanisms of DPI and DTI action, including alterations in Stat, Erk1/2, and Akt pathways, were inferred by correlation with NCI-60 Affymetrix(®) array data. Exposure of HT-29 colon cancer cells, which express Nox1, to DPI and DTI confirmed their inhibitory effects on steady state ROS levels, and demonstrated decreased Stat, Erk1/2, and Akt signaling mediated by IL-4, IL-6, IL-13, and IL-22, possibly due to a concomitant increase in tumor cell phosphatase activity. These findings suggest that DPI and DTI may act therapeutically by altering ROS-related signal transduction.

Downregulation of Pdcd4 by mir-21 facilitates glioblastoma proliferation in vivo.

MicroRNAs (miRNAs) are small, noncoding RNAs that play a critical role in developmental and physiological processes and are implicated in the pathogenesis of several human diseases, including cancer. They function by regulating target gene expression post-transcriptionally. In this study, we examined the role of oncogenic mir-21 in the pathogenesis of glioblastoma, the most aggressive form of primary brain tumor. We have previously reported that mir-21 is expressed at higher levels in primary glioblastoma-tissue and glioblastoma-derived cell lines than in normal brain tissue. We demonstrate that downregulation of mir-21 in glioblastoma-derived cell lines results in increased expression of its target, programmed cell death 4 (Pdcd4), a known tumor-suppressor gene. In addition, our data indicate that either downregulation of mir-21 or overexpression of its target, Pdcd4, in glioblastoma-derived cell lines leads to decreased proliferation, increased apoptosis, and decreased colony formation in soft agar. Using a glioblastoma xenograft model in immune-deficient nude mice, we observe that glioblastoma-derived cell lines in which mir-21 levels are downregulated or Pdcd4 is over-expressed exhibit decreased tumor formation and growth. Significantly, tumors grow when the glioblastoma-derived cell lines are transfected with anti-mir-21 and siRNA to Pdcd4, confirming that the tumor growth is specifically regulated by Pdcd4. These critical in vivo findings demonstrate an important functional linkage between mir-21 and Pdcd4 and further elucidate the molecular mechanisms by which the known high level of mir-21 expression in glioblastoma can attribute to tumorigenesis--namely, inhibition of Pdcd4 and its tumor-suppressive functions.

Utilizing targeted cancer therapeutic agents in combination: novel approaches and urgent requirements.

The rapid development of new therapeutic agents that target specific molecular pathways involved in tumour cell proliferation provides an unprecedented opportunity to achieve a much higher degree of biochemical specificity than previously possible with traditional chemotherapeutic anticancer agents. However, the lack of specificity of these established chemotherapeutic drugs allowed a relatively straightforward approach to their use in combination therapies. Developing a paradigm for combining new, molecularly targeted agents, on the other hand, is substantially more complex. The abundance of molecular data makes it possible, at least in theory, to predict how such agents might interact across crucial growth control networks. Initial strategies to examine molecularly targeted agent combinations have produced a small number of successes in the clinic. However, for most of these combination strategies, both in preclinical models and in patients, it is not clear whether the agents being combined actually hit their targets to induce growth inhibition. Here, we consider the initial approach of the US National Cancer Institute (NCI) to the evaluation of combinations of molecularly targeted anticancer agents in patients and provide a description of several new approaches that the NCI has initiated to improve the effectiveness of combination-targeted therapy for cancer.

Analysis of Food and Drug Administration-approved anticancer agents in the NCI60 panel of human tumor cell lines.

Since the early 1990s the Developmental Therapeutics Program of the National Cancer Institute (NCI) has utilized a panel of 60 human tumor cell lines (NCI60) representing 9 tissue types to screen for potential new anticancer agents. To date, about 100,000 compounds and 50,000 natural product extracts have been screened. Early in this program it was discovered that the pattern of growth inhibition in these cell lines was similar for compounds of similar mechanism. The development of the COMPARE algorithm provided a means by which investigators, starting with a compound of interest, could identify other compounds whose pattern of growth inhibition was similar. With extensive molecular characterization of these cell lines, COMPARE and other user-defined algorithms have been used to link patterns of molecular expression and drug sensitivity. We describe here the results of screening current Food and Drug Administration (FDA)-approved anticancer agents in the NCI60 screen, with an emphasis on those agents that target signal transduction. We analyzed results from agents with mechanisms of action presumed to be similar; we also carried out a hierarchical clustering of all of these agents. The addition of data from recently approved anticancer agents will increase the utility of the NCI60 databases to the cancer research community. These data are freely accessible to the public on the DTP website (http://dtp.cancer.gov/). The FDA-approved anticancer agents are themselves available from the NCI as a plated set of compounds for research use.

Expression profiling of nuclear receptors in the NCI60 cancer cell panel reveals receptor-drug and receptor-gene interactions.

We profiled the expression of the 48 human nuclear receptors (NRs) by quantitative RT-PCR in 51 human cancer cell lines of the NCI60 collection derived from nine different tissues. NR mRNA expression accurately classified melanoma, colon, and renal cancers, whereas lung, breast, prostate, central nervous system, and leukemia cell lines exhibited heterogeneous receptor expression. Importantly, receptor mRNA levels faithfully predicted the growth-inhibitory qualities of receptor ligands in nonendocrine tumors. Correlation analysis using NR expression profiles and drug response information across the cell line panel uncovered a number of new potential receptor-drug interactions, suggesting that in these cases, individual receptor levels may predict response to chemotherapeutic interventions. Similarly, by cross-comparing receptor levels within our expression dataset and relating these profiles to existing microarray gene expression data, we defined interactions among receptors and between receptors and other genes that can now be mechanistically queried. This work supports the strategy of using NR expression profiling to classify various types of cancer, define NR-drug interactions and receptor-gene networks, predict cancer-drug sensitivity, and identify druggable targets that may be pharmacologically manipulated for potential therapeutic intervention.

Identification of compounds that correlate with ABCG2 transporter function in the National Cancer Institute Anticancer Drug Screen.

ABCG2 is an ATP-binding cassette transporter that counts multiple anticancer compounds among its substrates and is believed to regulate oral bioavailability as well as serve a protective role in the blood-brain barrier, the maternal-fetal barrier, and hematopoietic stem cells. We sought to determine whether novel compounds that interact with the transporter could be identified through analysis of cytotoxicity profiles recorded in the NCI Anticancer Drug Screen database. A flow cytometric assay was used to measure ABCG2 function in the 60 cell lines and generate a molecular profile for COMPARE analysis. This strategy identified >70 compounds with Pearson correlation coefficients (PCCs) >0.4, where reduced drug sensitivity correlated with ABCG2 expression, as well as >120 compounds with PCCs < -0.4, indicating compounds to which ABCG2 expression conferred greater sensitivity. Despite identification of known single nucleotide polymorphisms in the ABCG2 gene in a number of the cell lines, omission of these lines from the COMPARE analysis did not affect PCCs. Available compounds were subjected to validation studies to confirm interaction with the transporter, including flow cytometry, [(125)I]IAAP binding, and cytotoxicity assays, and interaction was documented in 20 of the 27 compounds studied. Although known substrates of ABCG2 such as mitoxantrone or topotecan were not identified, we characterized three novel substrates-5-hydroxypicolinaldehyde thiosemicarbazone (NSC107392), (E)-N-(1-decylsulfanyl-3-hydroxypropan-2-yl)-3-(6-methyl-2,4-dioxo-1H-pyrimidin-5-yl)prop-2-enamide (NSC265473), and 1,2,3,4,7-pentahydroxy-1,3,4,4a,5,11b-hexahydro[1,3]dioxolo[4,5-j]phenanthridin-6(2H)-one [NSC349156 (pancratistatin)]-and four compounds that inhibited transporter function-2-[methyl(2-pyridin-2-ylethyl)-amino]fluoren-9-one hydroiodide (NSC24048), 5-amino-6-(7-amino-5,8-dihydro-6-methoxy-5,8-dioxo-2-quinolinyl)-4-(2-hydroxy-3,4-dimethoxyphenyl)-3-methyl-2-pyridinecarboxylic acid, methyl ester (NSC45384), (17beta)-2,4-dibromo-estra-1,3,5(10)-triene-3,17-diol (NSC103054), and methyl N-(pyridine-4-carbonylamino)carbamodithioate (NSC636795). In summary, COMPARE analysis of the NCI drug screen database using the ABCG2 functional profile was able to identify novel substrates and transporter-interacting compounds.

Glut-1 as a therapeutic target: increased chemoresistance and HIF-1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies.

The facilitative glucose transporter Glut-1 is overexpressed and confers poor prognosis in a wide range of solid tumours. The peri-necrotic pattern of expression often seen in human tumour samples is linked with its transcriptional control in hypoxic conditions by hypoxia-inducible factor HIF-1 or through a reduced rate of oxidative phosphorylation. Hypoxia-regulated genes offer promise as novel therapeutic targets as a means of preventing the proliferation and eventual metastatic spread of tissue originating from residual chemically and radio resistant hypoxic cells that have survived treatment. Inhibiting the expression or functionality of Glut-1 may be a way of specifically targeting hypoxic cells within the tumour that depend upon a high rate of glucose uptake for anaerobic glycolysis. We used an array of formalin-fixed, paraffin-embedded samples of the NCI-60 panel of cell lines to carry out immunohistochemical detection of Glut-1 and to select possible candidate lead compounds by COMPARE analysis with agents from the NCI diversity screen, which may work via inhibition of Glut-1 or Glut-1-dependent processes. "Positive" COMPARE hits were mostly conjugated Pseudomonas toxins binding the epidermal growth factor receptor (EGFR). However, correlations with standard anticancer agents were virtually all negative, indicating a link between Glut-1 and chemoresistance. MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls. Metabolomic analysis ((31)P-MRS and (1)H MRS) carried out using cell lysates and xenografts generated from Glut-1 overexpressing Hepa and c4 cell lines showed higher glucose levels in Glut-1 overxpressing c4 relative to parental tumour extracts occurred in the absence of an increase in lactate levels, which were in turn significantly higher in the Glut-1 overexpressing Hepa xenografts. This implies that Glut-1 over-expression without a co-ordinate increase in HIF-1-regulated glycolytic enzymes increases glucose uptake but not the rate of glycolysis. Glut-1 overexpressing xenografts also showed higher levels of phosphodiester (PDE), which relates to the metabolite turnover of phospholipids and is involved in membrane lipid degradation, indicating a mechanism by which Glut-1 may increase cell turnover.

Batracylin (NSC 320846), a dual inhibitor of DNA topoisomerases I and II induces histone gamma-H2AX as a biomarker of DNA damage.

Batracylin (8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) is an investigational clinical anticancer agent. Previous animal studies showed activity against solid tumors and Adriamycin-resistant leukemia. We initially sought to test the proposed Top2-mediated DNA cleavage activity of batracylin and identify potential biomarkers for activity. COMPARE analysis in the NCI-60 cell lines showed batracylin activity to be most closely related to the class of Top2 inhibitors. The 50% growth inhibition (GI50) value for batracylin in HT29 colon carcinoma cells was 10 micromol/L. DNA-protein cross-links, consistent with Top2 targeting, were measured by alkaline elution. DNA single-strand breaks were also detected and found to be protein associated. However, only a weak induction of DNA double-strand breaks was observed. Because batracylin induced almost exclusively DNA single-strand breaks, we tested batracylin as a Top1 inhibitor. Batracylin exhibited both Top1- and Top2alpha/beta-mediated DNA cleavage in vitro and in cells. The phosphorylation of histone (gamma-H2AX) was tested to measure the extent of DNA damage. Kinetics of gamma-H2AX "foci" showed early activation with low micromol/L concentrations, thus presenting a useful early biomarker of DNA damage. The half-life of gamma-H2AX signal reversal after drug removal was consistent with reversal of DNA-protein cross-links. The persistence of the DNA-protein complexes induced by batracylin was markedly longer than by etoposide or camptothecin. The phosphorylated DNA damage-responsive kinase, ataxia telangiectasia mutated, was also found activated at sites of gamma-H2AX. The cell cycle checkpoint kinase, Chk2, was only weakly phosphorylated. Thus, batracylin is a dual Top1 and Top2 inhibitor and gamma-H2AX could be considered a biomarker in the ongoing clinical trials.

Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop.

Adenoviral vectors are commonly used for liver-directed gene therapy following systemic administration owing to their strong propensity for hepatocyte transduction. However, many disease applications would benefit from the delivery of adenoviruses to alternate tissues via this route. Research has thus focused on stripping the virus of native hepatic tropism in conjunction with modifying virus capsid proteins to incorporate novel tropism. Recently, the KO1S* adenovirus serotype 5 fiber mutant, devoid of both coxsackie and adenovirus receptor binding in the fiber knob domain and mutated at the putative heparan sulphate proteoglycan binding site in the fiber shaft, was shown to possess strikingly poor hepatic tropism in mice, rats, and non-human primates. Thus, it is an ideal candidate for retargeting strategies. We therefore assessed the ability of peptide-modified KO1S* fibers to retarget adenovirus. Peptide insertions were well tolerated and virions produced to high titers. However, expected retargeting at the level of transduction was not observed, despite cell-binding studies showing enhanced vector targeting at the cell surface. Cy3 labeling studies showed retarded trafficking of S*-containing fibers. Taken together, our data demonstrates that KO1S* mutant fibers are ineffective for cell retargeting strategies.

Anticancer activity of BIM-46174, a new inhibitor of the heterotrimeric Galpha/Gbetagamma protein complex.

A large number of hormones and local agonists activating guanine-binding protein-coupled receptors (GPCR) play a major role in cancer progression. Here, we characterize the new imidazo-pyrazine derivative BIM-46174, which acts as a selective inhibitor of heterotrimeric G-protein complex. BIM-46174 prevents the heterotrimeric G-protein signaling linked to several GPCRs mediating (a) cyclic AMP generation (Galphas), (b) calcium release (Galphaq), and (c) cancer cell invasion by Wnt-2 frizzled receptors and high-affinity neurotensin receptors (Galphao/i and Galphaq). BIM-46174 inhibits the growth of a large panel of human cancer cell lines, including anticancer drug-resistant cells. Exposure of cancer cells to BIM-46174 leads to caspase-3-dependent apoptosis and poly(ADP-ribose) polymerase cleavage. National Cancer Institute COMPARE analysis for BIM-46174 supports its novel pharmacologic profile compared with 12,000 anticancer agents. The growth rate of human tumor xenografts in athymic mice is significantly reduced after administration of BIM-46174 combined with either cisplatin, farnesyltransferase inhibitor, or topoisomerase inhibitors. Our data validate the feasibility of targeting heterotrimeric G-protein functions downstream the GPCRs to improve anticancer chemotherapy.

Bisindenoisoquinoline bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}propane bis(trifluoroacetate) (NSC 727357), a DNA intercalator and topoisomerase inhibitor with antitumor activity.

Indenoisoquinolines are topoisomerase (Top) I inhibitors developed to overcome some of the limitations of camptothecins and expand their anticancer spectrum. Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}-propane bis(trifluoroacetate) (NSC 727357) is a novel dimeric indenoisoquinoline derivative with potent antiproliferative activity in the NCI-60 cell line panel, promising hollow fiber activity (score of 32) and activity against xenografts. Submicromolar concentrations of the bisindenoisoquinoline NSC 727357 induce Top1 cleavage complexes at specific sites in biochemical assays. At higher concentrations, inhibition of Top1 catalytic activity and DNA intercalation is observed. NSC 727357 also induces a limited number of Top2-DNA cleavage complexes. In contrast to the effect of other Top1 inhibitors, cells treated with the bisindenoisoquinoline NSC 727357 show an arrest of cell cycle progression in G(1) with no significant inhibition of DNA synthesis after a short exposure to the drug. Moreover, unlike camptothecin and the indenoisoquinoline MJ-III-65 (NSC 706744, 6-[3-(2-hydroxyethyl)aminopropyl]-5,6-dihydro-5,11-diketo-2,3-dimethoxy-(methylenedioxy)-11H-indeno[1,2-c]isoquinoline hydrochloride), the cytotoxicity of bisindenoisoquinoline NSC 727357 is only partially dependent on Top1 and p53, indicating that this drug has additional targets besides Top1 and Top2.

Spectrum of activity and molecular correlates of response to phosphatidylinositol ether lipid analogues, novel lipid-based inhibitors of Akt.

The serine/threonine kinase Akt is a promising target in cancer. We previously identified five phosphatidylinositol ether lipid analogues (PIA) that inhibited Akt activation and selectively killed lung and breast cancer cells with high levels of Akt activity. To assess the spectrum of activity in other cell types and to compare PIAs with other inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, we compared growth inhibition by PIAs against the PI3K inhibitors LY294002 and wortmannin and the mTOR inhibitor rapamycin in the NCI60 cell line panel. Although each of these compounds inhibited the growth of all the cell lines, distinct patterns were observed. The PIAs were the least potent but the most cytotoxic. The broad spectrum of activity of PIAs was confirmed in vivo in hollow fiber assays. The response to PIAs was significantly correlated with levels of active but not total Akt in the NCI60, as assessed using COMPARE analysis. However, a number of molecular targets were identified whose expression was more highly correlated with sensitivity to PIAs than active Akt. Expression of these molecular targets did not overlap with those that correlated with sensitivity to LY294002, wortmannin, or rapamycin. A COMPARE analysis of the National Cancer Institute chemical screening database revealed that the patterns of activity of PIAs correlated best with patterns of activity of other lipid-based compounds. These studies show that although PIAs are widely active in cancer cells, which correlates with the presence of its intended target, active Akt, PIAs are biologically distinct from other known inhibitors of the PI3K/Akt/mTOR pathway.

Biological evaluation of tubulysin A: a potential anticancer and antiangiogenic natural product.

Tubulysin A (tubA) is a natural product isolated from a strain of myxobacteria that has been shown to depolymerize microtubules and induce mitotic arrest. The potential of tubA as an anticancer and antiangiogenic agent is explored in the present study. tubA shows potent antiproliferative activity in a panel of human cancer cell lines irrespective of their multidrug resistance properties. It induces apoptosis in cancer cells but not in normal cells and shows significant potential antiangiogenic properties in several in vitro assays. It is efficacious in initial animal studies using a hollow fibre assay with 12 different human tumour cell lines. This study suggests that both in vitro and preclinical profiles of tubA may translate into clinically useful anticancer properties.

Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries.

A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.

Correlation of PIK3Ca mutations with gene expression and drug sensitivity in NCI-60 cell lines.

The gene that encodes the alpha-isoform of phosphatidylinositol 3-kinase (PIK3Ca) is frequently mutated in human cancers. We profiled the mutation status of the PIK3Ca gene in the National Cancer Institute (NCI)-60 panel of human cancer cell lines maintained by the Developmental Therapeutics Program of the NCI. Mutation hotspots on the gene were PCR amplified and sequenced, and the trace data were analyzed with software designed to detect mutations. Seven of the cell lines tested have PIK3Ca mutations: two lines derived from breast cancer, two from colon cancer, two from ovarian cancer, and one from lung cancer. BRAF and EGFR genes were normal in the PIK3Ca mutant lines. Two of the cell lines with mutant PIK3Ca also have a mutant version of the KRAS gene. The mutation status was correlated with array-based gene expression that is publicly available for the NCI-60 cell lines. We found increased expression levels for estrogen receptor (ER) and ERBB2 in PIK3Ca mutant lines. The PIK3Ca mutation status was also correlated with compound screening data for the cell lines. PIK3Ca-mutant cell lines were relatively more sensitive than PIK3Ca-normal cell lines to the ER inhibitor tamoxifen and the AKT inhibitor triciribine, among other compounds. The results provide insights into the role of mutant PIK3Ca in oncogenic signaling and allow preliminary identification of novel targets for therapeutic intervention in cancers harboring PIK3Ca mutations.