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Supplementary Table 2 from Analysis of Food and Drug Administration–Approved Anticancer Agents in the NCI60 Panel of Human Tumor Cell Lines

Supplementary Table 1 from Analysis of Food and Drug Administration–Approved Anticancer Agents in the NCI60 Panel of Human Tumor Cell Lines

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.

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. Mol Cancer Ther; 9(5); 1451–60. ©2010 AACR.

This file contains: Supplementary Methods for pharmacodynamic biomarker analyses, clinicaltrials.gov searches, and molecular correlation analyses; Supplementary Figures S1 (overview of the NCI-ALMANAC web resource), S2 (apoptotic biomarker response to bortezomib-clofarabine), and S3 (associations between ABC transporter activity and nilotinib-paclitaxel combination activity); and Supplementary Tables S1 (FDA-approved agents used in the NCI-ALMANAC screen), S2 (tumor doubling time analysis of xenograft experiments with clinically tested NCI-ALMANAC-derived combinations), S3 (in vitro, in vivo, and clinical activity of NCI-ALMANAC-derived combinations), and S4 (tumor doubling time analysis of xenograft experiments with novel NCI-ALMANAC-derived combinations).

PDF file - 880K, Volcano plots for 28 FDA approved cancer drugs of interest.

XLSX file - 713K, Excel sheet containing additional data for main Figure 4.

XLSX file - 271K, Excel sheet containing additional data for the main text.

Supplementary Figure S1 from 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. (Cancer Res 2006; 66(1): 34-40)

Supplementary Table S1 from Ligand-Directed Surface Profiling of Human Cancer Cells with Combinatorial Peptide Libraries

XLSX file - 337K, Excel sheet containing the list of compounds from Supplemental Figure 9.

PDF file - 3388K, Supplementary Figure 1: Variant filtering decision tree. Supplementary Figure 2: Distribution of variants by annotation. Supplementary Figure 3: The NCI-60 cell lines. Supplementary Figure 4: Distribution of variants by type. Supplementary Figure 5: Phylogenic conservation of POL epsilon-P286. Supplementary Figure 6: A snapshot of the WES dataset in Variant Analysis by Ingenuity Systems (http://www.ingenuity.com/NCI60_WES). Supplementary Figure 7: Pattern comparison for cell with wild-type TP53 status. Supplementary Figure 8: Receiver operating curves (ROC) for cross-validated drug predictors.

PDF file - 4628K, Volcano plots for 46 cancer genes of interest.

PDF file - 125K

Supplementary Legend and Methods from Ligand-Directed Surface Profiling of Human Cancer Cells with Combinatorial Peptide Libraries

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.

For more than 10 years the National Cancer Institute (NCI) has tested compounds for their ability to inhibit the growth of human tumor cell lines in culture (NCI screen). Work of Ken Paull [J. Natl. Cancer Inst. 81 (1989) 1088] demonstrated that compounds with similar mechanism of cell growth inhibition show similar patterns of activity in the NCI screen. This observation was developed into an algorithm called COMPARE and has been successfully used to predict mechanisms for a wide variety of compounds. More recently, this method has been extended to associate patterns of cell growth inhibition by compounds with measurements of molecular entities (such as gene expression) in the cell lines in the NCI screen. The COMPARE method and associated data are freely available on the Developmental Therapeutics Program (DTP) web site (http://dtp.nci.nih.gov/). Examples of the use of COMPARE on these web pages will be explained and demonstrated. Published by Elsevier Science Inc.

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.

Anticancer drug discovery has historically been done empirically, screening for compounds that inhibit the growth of tumor cells in culture, or that are effective against implanted tumors in mice. This approach is analogous to the highly effective screens carried out in the 1950s and 1960s for antimicrobial agents and has had its successes – indeed, many of the anticancer drugs currently in clinical use were developed in this fashion. However, as our understanding of the molecular mechanisms underlying the development and growth of cancer cells has improved, new approaches to drug discovery have begun to build on this knowledge. The recognition that not all tumors arising in the same tissue are due to the same underlying defects calls for treatments regimes that will be tailored to these molecular alterations. The drug screen described in this chapter was initiated with the hypothesis that single gene changes that are often associated with particular hereditary and sporadic forms of cancer may serve as determinants of drug sensitivity [27]. In principle, there are two possible mechanisms by which a drug can be more toxic to a cell containing a particular genetic alteration. First, by a mechanism in which damage caused by the drug is normally repaired in a wild-type cell by a protein that has been deleted or altered in the mutant. An example of this is the sensitivity of mutants defective in recombinational repair of DNA double-strand breaks (DSBs) (e.g., yeast rad50 mutants) to agents that cause DSBs (e.g., the topoisomerase poison

Sixty cancer cell lines have been extensively characterized and used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as screening tools for anti-cancer drug development. An extensive database has been accumulated that could be used to select individual cells lines for specific experimental designs based on their global genetic and biological profile. However, information on the human leukocyte antigen (HLA) genotype of these cell lines is scant and mostly antiquated since it was derived from serological typing. We, therefore, re-typed the NCI-60 panel of cell lines by high-resolution sequence-based typing. This information may be used to: 1) identify and verify the identity of the same cell lines at various institutions; 2) check for possible contaminant cell lines in culture; 3) adopt individual cell lines for experiments in which knowledge of HLA molecule expression is relevant. Since genome-based typing does not guarantee actual surface protein expression, further characterization of relevant cell lines should be entertained to verify surface expression in experiments requiring correct antigen presentation.

We identified five structurally related dimethane sulfonates with putative selective cytotoxicity in renal cancer cell lines. These compounds have a hydrophobic moiety linked to a predicted alkylating group. A COMPARE analysis with the National Cancer Institute Anticancer Drug Screen standard agent database found significant correlations between the IC50 of the test compounds and the IC50 of alkylating agents (e.g., r = 0.68, P0.00001 for chlorambucil). In this report, we examined whether these compounds had activities similar to those of conventional alkylating agents. In cytotoxicity studies, chlorambucil-resistant Walker rat carcinoma cells were 4- to 11-fold cross-resistant to the test compounds compared with 14-fold resistant to chlorambucil. To determine effects on cell cycle progression, renal cell carcinoma (RCC) line 109 was labeled with bromodeoxyuridine prior to drug treatment. Complete cell cycle arrest occurred in cells treated with an IC90 dose of NSC 268965. p53 protein levels increased as much as 5.7-fold in RCC line 109 and as much as 20.4-fold in breast cancer line MCF-7 following an 18-hour drug exposure. Finally, DNA-protein cross-links were found following a 6-hour pretreatment with all compounds. Thus, the dimethane sulfonate analogues have properties expected of some alkylating agents but, unlike conventional alkylating agents, appear to possess activity against RCC.

Thymidylate synthase (TS) is a critical cellular target for cancer chemotherapeutics, particularly the fluoropyrimidine and antifolate classes of antineoplastic agents. One of the primary mechanisms of clinical insensitivity to these agents is through the overexpression of the target enzyme, TS. Thus, there is a need for the development of agents which selectively target TS-overexpressing malignant cells. To this end, we conducted a search for agents which potentially selectively target TS-overexpressing cells using two separate algorithms for identifying such compounds in the NCI Drug Repository by comparing cytotoxicity profiles of 30000 compounds with the TS expression levels measured by Western blot analysis in 53 cell lines. Using the traditional COMPARE analysis we were unable to identify compounds which maintain a selective ability to kill high TS-expressing cells in a subsequent four cell line validation assay. A new algorithm, termed COMPARE Effect Clusters analysis, enabled the identification of a particular drug cluster which contained compounds that maintained a selective ability to kill TS-overexpressing cell lines in the validation assay. While the identified compounds were selectively cytotoxic to TS-overexpressing cells, we found that they were not specifically targeting TS as a mechanism of action. Apparently, the overexpression of TS was providing a marker for sensitivity. This identified class of compounds which appears to be selectively cytotoxic against cells which overexpress TS may be useful for the development of therapeutics for those whose cancers overexpress TS de novo.

Recombinational repair of double-strand breaks (DSBs), traditionally believed to be an error-free DNA repair pathway, was recently shown to increase the frequency of mutations in a nearby interval. The reversion rate of trp1 alleles (either nonsense or frameshift mutations) near an HO-endonuclease cleavage site is increased at least 100-fold among cells that have experienced an HO-mediated DSB. We report here that in strains deleted for rev3 this DSB-associated reversion of a nonsense mutation was greatly decreased. Thus REV3, which encodes a subunit of the translesion DNA polymerase ζ, was responsible for the majority of these base substitution errors near a DSB. However, rev3 strains showed no decrease in HO-stimulated recombination, implying that another DNA polymerase also functioned in recombinational repair of a DSB. Reversion of trp1 frameshift alleles near a DSB was not reduced in rev3 strains, indicating that another polymerase could act during DSB repair to make these frameshift errors. Analysis of spontaneous reversion in haploid strains suggested that Rev3p had a greater role in making point mutations than in frameshift mutations.

Restriction endonuclease digestion of genomic DNA from 24 lymphoblastoid cell lines homozygous for the HLA class II specificity DQw3, followed by hybridization with a DQ beta-chain cDNA probe, identified a genomic polymorphism with variable BamHI and HindIII recognition sites. This restriction fragment pattern was found for several haplotypes associated with the DQw3 specificity, including some haplotypes positive for the HLA-DR specificities DR4, DR5, DRw8, and DRw12. The variant fragment pattern corresponds precisely with the reactivity of a monoclonal antibody, A-10-83, previously shown to define a serologic split of DQw3. Serologic detection of the specific DQw3.1 genomic polymorphism indicated that the corresponding DQ beta-chain variants are expressed. This polymorphic restriction fragment pattern, then, represents a selective marker for DQ beta-chain genes that appear to define a DQ beta-chain-associated specificity, here called DQw3.1.

A modification of the ELISA assay is described which significantly enhances the chromogenic signal, thereby increasing the sensitivity of the assay. This enhanced ELISA employs a protein A-HRP conjugate to detect antibody bound to antigen coated polystyrene plates, followed by incubation with an anti-protein A-HRP conjugate as the amplification step. This enhanced ELISA is rapid, inexpensive, uses commercially available reagents and maintains the low background and high specificity of standard ELISA.

HLA class II genes have been implicated in susceptibility to a number of diseases. We have previously identified two allelic variants of DQw3 and have shown that DR4-DQβ3.2 haplotypes are associated with increased risk of IDDM whereas DR4-DQβ3.1 haplotypes are not. DR5 and DR8 DQw3 + individuals are exclusively DQβ3.1 and share numerous restriction sites within the DQβ genes with DR4-DQβ3.1 individuals. In order to compare the DQβ3.1 genes associated with different haplotypes, we have sequenced coding and noncoding regions of the DQβ genes from a DR4-DQβ3.1 HTC (ER) and a DR8-DQβ3.1 HTC (LUY). LUY and ER DQβ genes share nucleotide substitutions in both the β1 and β2 exons, yielding six amino acid replacements distinguishing them from DQβ3.2. In the noncoding regions as well, LUY and ER share nucleotide substitutions distinguishing their DQβ3.1 genes from DQβ3.2. These data support the concept that the DQβ3.1 allele was introduced onto different backgrounds via homologous recombination.

The HLA class II-related susceptibility to type I insulin-dependent diabetes mellitus (IDDM) is examined in 94 multiplex families sorted by the presence or absence of a DR4+ haplotype in at least one diabetic family member. The families with DR4+ haplotypes are then sorted by the presence or absence of a DR4-linked DQ beta 3.2 allele. Further analysis assumes each multiplex family to represent a single diabetic genetic event and identifies the HLA class II haplotype(s) present in all affected members. The DQ beta 3.2 allele is present in over 95% of the multiplex families where DR4+ haplotypes segregate with IDDM, implying a major permissive role in determining susceptibility to IDDM.

A DQ beta locus-specific oligonucleotide probe is used to identify DQ beta alleles present in six insulin-dependent diabetes mellitus (IDDM) multiplex families. Results of genotyping using this technique agree with those from cDNA probes and are often clearer. Allele-specific oligonucleotide probes for the DR beta locus are used to identify subtypes (Dw4 and Dw14) of the DR4 specificity in five IDDM multiplex families. This distinction cannot be made with available cDNA probes.

Locus-specific oligonucleotides have been used as probes to detect polymorphic alleles for the HLA genes DQ beta, DQ alpha, DX alpha, and DO beta. These genes lie between the HLA DR and DP genes on chromosome 6, a region frequently implicated in intra-HLA recombination. In order to distinguish among these highly homologous HLA class II genes, we have identified sequences in the coding regions that are locus specific and have synthesized short oligonucleotide probes corresponding to these regions. We have used these probes in a gel hybridization procedure to analyze restriction-enzyme-digested genomic DNA and to assign polymorphic bands to a particular locus. Taq I-digested DNA hybridized with a DQ-beta-specific probe detects a single band per haplotype. The size of this band corresponds precisely to the expressed DQ beta gene and distinguishes among the serologically defined DQ alleles, providing a rapid method for genomic DQ typing. Similar analysis with an oligonucleotide probe specific for DX alpha, the nonexpressed alpha gene in the DQ subregion, detects 2 DX alleles, and hybridization with an oligonucleotide specific for the newly described DO beta gene detects 2 alleles at DO beta. Heterogeneity in linkage patterns among DQ, DX, and DO genes suggests that frequent recombinational events at multiple intergenic sites contributed to the generation of present-day haplotypes. One such recombinational event was identified directly in a family with serologically HLA-identical siblings, in which genomic analysis indicated a parental recombination event mapping between DR and DX, which correlated with unexpected alloreactivity.

The HLA genetic region consists of a large multigene complex which includes a number of highly homologous α and β genes encoding class II polypeptides, clustered in three major loci, DP, DQ, and DR. Analysis of genomic polymorphisms at each of these loci is of considerable interest due to the role of particular structural polymorphisms in immune function, but this analysis has been hampered by difficulty in distinguishing between such highly homologous loci. We have identified locus-specific and exon-specific class II gene sequences in order to produce synthetic oligonucleotide probes which hybridize specifically to DQ β genes. Two such oligonucleotide probes are described which are specific for the β 1 and β 2exons of DQ (DC) β which identify DQ β genes in digests of cellular DNA and which can be used to characterize restriction sites flanking the two oligonucleotide-specific regions. By sequentially hybridizing these probes in modified Southern analyses, we have been able to generate a tentative “restriction map” of a newly identified DQ β allele from digests of total genomic DNA. This oligonucleotide mapping technique discriminates between two HLA-DQw3 + alleles, DQ3.1 and DQ3.2, permitting the recognition of structural polymorphisms with DQ β which are highly associated with type I diabetes mellitus.

Anti-idiotypic antibodies are described that were raised against murine monoclonal antibody 8.2, an antibody specific for a human melanoma-associated cell surface marker called p97. The 8.2 idiotopes recognized by this anti-idiotypic antiserum are binding site-associated and are shared by other monoclonal anti-p97 antibodies with the same specificity as antibody 8.2. Mice immunized with the anti-idiotype demonstrate delayed-type hypersensitivity reactions when challenged with melanoma (p97-positive) tumor cells.

HLA class II genes (termed DR, DQ, DO and DP) encode proteins which are important in generation of the immune response. These proteins are heterodimers, composed of an alpha and a beta chain. Each individual has at least 7 beta genes and 6 alpha genes (some of which are non-functional) clustered together on chromosome 6 (Fig. 1). These genes are highly related to one another. Most of these HLA class II genes are polymorphic, having multiple allelic forms. Thus the number of genes and the large number of alleles make this a complex system to study and provides a good example of the power of molecular genetic techniques.