Use of yeast chemigenomics and COXEN informatics in preclinical evaluation of anticancer agents

Abstract

Bladder cancer metastasis is virtually incurable with current platinum-based chemotherapy. We used the novel COXEN informatic approach for in silico drug discovery and identified NSC-637993 and NSC-645809 (C1311), both imidazoacridinones, as agents with high-predicted activity in human bladder cancer. Because even highly effective monotherapy is unlikely to cure most patients with metastasis and NSC-645809 is undergoing clinical trials in other tumor types, we sought to develop the basis for use of C1311 in rational combination with other agents in bladder cancer. Here, we demonstrate in 40 human bladder cancer cells that the in vitro cytotoxicity profile for C1311 correlates with that of NSC-637993 and compares favorably to that of standard of care chemotherapeutics. Using genome-wide patterns of synthetic lethality of C1311 with open reading frame knockouts in budding yeast, we determined that combining C1311 with a taxane could provide mechanistically rational combinations. To determine the preclinical relevance of these yeast findings, we evaluated C1311 singly and in doublet combination with paclitaxel in human bladder cancer in the in vivo hollow fiber assay and observed efficacy. By applying COXEN to gene expression data from 40 bladder cancer cell lines and 30 human tumors with associated clinical response data to platinum-based chemotherapy, we provide evidence that signatures of C1311 sensitivity exist within nonresponders to this regimen. Coupling COXEN and yeast chemigenomics provides rational combinations with C1311 and tumor genomic signatures that can be used to select bladder cancer patients for clinical trials with this agent.

Figure 1

C1311 and NSC exhibit similar, favorable in vitro activities, comparable to standard-of-care agents. (A) IC₅₀ values for C1311 were determined by Spline regression for the BLA-40 cell line panel, plotted ranked ordered from left to right, then each corresponding cell line's expression of TOP2A and FLT3 expression (Affymetrix probes 201291_s_at and 206674_at, both log10 values for visualization in scale). (B) Scatter plot of NSC compound (ordinate) and C1311 (abscissa) IC₅₀ values across the BLA-40 panel, nonparametric Spearman correlation, and P value. (C) Comparison of C1311 and standard-ofcare drugs by IC₅₀ across the BLA-40 panel. IC₅₀ values C1311, cisplatin, and gemcitabine were rank ordered for the 40 cell lines for each drug and plotted in ascending order on the log-scale y axis. The green, pink, and blue arrows indicate the percentage of the BLA-40 cell lines that exhibit IC₅₀ values below the 1-µM range, demonstrating that C1311 exhibits similar activity to agents currently in clinical use.

Figure 2

Chemigenomic analysis of C1311 and paclitaxel between yeast and human cells. (A) Two-way hierarchical cluster of a 1521 x 2804 binary matrix where a black pixel represents either a synthetic lethal interaction between two yeast ORFs or reduced fitness between a drug and a yeast deletion mutant, or a white pixel represents all other two-ORF or drug-ORF pairs. We highlight the C1311 (red) and benomyl (green) clusters. (B) We see that C1311 is in a relatively sparse area of the matrix, and the four treatments with C1311 (two concentrations after 10 or 20 generations of competitive growth) cluster immediately next to each other and among several deletion strains involved with membrane lipid biogenesis, implicating this pathway in the function of C1311. (C) Enlarged inset view of the benomyl cluster from (A) showing benomyl treatments for 10 and 20 generations and neighboring deletion strains, enriched for microtubule and spindle components, as would be expected for this microtubule poison. (D) IC₅₀ patterns across 60 cell lines for ∼4400 drugs from the 60 cell lines of NCI-60 screen [20] were correlated to that of C1311 and the probability distribution function of the coefficients was plotted. Paclitaxel, cisplatin, and gemcitabine exhibited the indicated correlation coefficients.

Figure 3

Prediction of C1311 sensitivity between cell line panels and in human tumors. (A) COXEN analysis was used to develop a set of probe sets associated with sensitivity to C1311 in the NCI-60 cell line panel and concordantly expressed between the NCI-60 panel, the BLA-40 panel, and Sanchez-Carbayo et al. tumor gene expression data sets. Then, a nearest neighbor-based classification approach used to classify the BLA-40 cell line panel based on the NCI-60 panel, and the ROC curve was plotted for the classes assigned (sensitive or resistant) to test its ability to discriminate (area under the curve = 0.74, 95% CI = 0.59-0.90, P = .01). (B) Clustering of multiple data sets by C1311 sensitivity genes. A two-dimensional hierarchical cluster of NCI-60, BLA-40, Sanchez-Carbayo et al., and Als et al. data sets across 28 three-way concordant probe sets. Individual NCI-60 (actual) and BLA-40 (predicted) cells are indicated by boxes, showing resistance in yellow and sensitivity in green. Individual Sanchez-Carbayo et al. and Als et al. (both also predicted) tumor data sets are also indicated as for the BLA-40 cells. The clustergram illustrates how the COXEN methodology may select concordant biomarkers between platforms such that such gene expression patterns allow visualization or computational prediction of interpretable relationships between diverse biologic systems. (C) C1311 prediction values were dot-scatter plotted for response classes from the Als et al. data set, including CR (complete responder), PR (partial responder), NC (no change), and PD (progressive disease), finding no significant difference by nonparametric analysis of variance. (D) Kaplan-Meier analysis of survival by C1311 prediction indicates no systematic association between C1311 prediction class and survival in either study.