Bala S. Balakumaran

Pharmacogenomic Strategies Provide a Rational Approach to the Treatment of Cisplatin-Resistant Patients With Advanced Cancer

PURPOSE Standard treatment for advanced non-small-cell lung cancer (NSCLC) includes the use of a platinum-based chemotherapy regimen. However, response rates are highly variable. Newer agents, such as pemetrexed, have shown significant activity as second-line therapy and are currently being evaluated in the front-line setting. We utilized a genomic strategy to develop signatures predictive of chemotherapeutic response to both cisplatin and pemetrexed to provide a rational approach to effective individualized medicine. METHODS Using in vitro drug sensitivity data, coupled with microarray data, we developed gene expression signatures predicting sensitivity to cisplatin and pemetrexed. Signatures were validated with response data from 32 independent ovarian and lung cancer cell lines as well as 59 samples from patients previously treated with cisplatin. RESULTS Genomic-derived signatures of cisplatin and pemetrexed sensitivity were shown to accurately predict sensitivity in vitro and, in the case of cisplatin, to predict treatment response in patients treated with cisplatin. The accuracy of the cisplatin predictor, based on available clinical data, was 83.1% (sensitivity, 100%; specificity 57%; positive predictive value, 78%; negative predictive value, 100%). Interestingly, an inverse correlation was seen between in vitro cisplatin and pemetrexed sensitivity, and importantly, between the likelihood of cisplatin and pemetrexed response in patients. CONCLUSION The use of genomic predictors of response to cisplatin and pemetrexed can be incorporated into strategies to optimize therapy for advanced solid tumors.

Analysis of sample set enrichment scores

MOTIVATION Gene expression profiling experiments in cell lines and animal models characterized by specific genetic or molecular perturbations have yielded sets of genes annotated by the perturbation. These gene sets can serve as a reference base for interrogating other expression datasets. For example, a new dataset in which a specific pathway gene set appears to be enriched, in terms of multiple genes in that set evidencing expression changes, can then be annotated by that reference pathway. We introduce in this paper a formal statistical method to measure the enrichment of each sample in an expression dataset. This allows us to assay the natural variation of pathway activity in observed gene expression data sets from clinical cancer and other studies. RESULTS Validation of the method and illustrations of biological insights gleaned are demonstrated on cell line data, mouse models, and cancer-related datasets. Using oncogenic pathway signatures, we show that gene sets built from a model system are indeed enriched in the model system. We employ ASSESS for the use of molecular classification by pathways. This provides an accurate classifier that can be interpreted at the level of pathways instead of individual genes. Finally, ASSESS can be used for cross-platform expression models where data on the same type of cancer are integrated over different platforms into a space of enrichment scores. AVAILABILITY Versions are available in Octave and Java (with a graphical user interface). Software can be downloaded at http://people.genome.duke.edu/assess.

A genomic approach to identify molecular pathways associated with chemotherapy resistance

Resistance to chemotherapy in cancer is common. As gene expression profiling has been shown to anticipate chemotherapeutic resistance, we sought to identify cellular pathways associated with resistance to facilitate effective combination therapy. Gene set enrichment analysis was used to associate pathways with resistance in two data sets: the NCI-60 cancer cell lines deemed sensitive and resistant to specific chemotherapeutic agents (Adriamycin, cyclophosphamide, docetaxel, etoposide, 5-fluorouracil, paclitaxel, and topotecan) and a series of 40 lung cancer cell lines for which sensitivity to cisplatin and docetaxel was determined. Candidate pathways were further screened in silico using the Connectivity Map. The lead candidate pathway was functionally validated in vitro. Gene set enrichment analysis associated the matrix metalloproteinase, p53, methionine metabolism, and free pathways with cytotoxic resistance in the NCI-60 cell lines across multiple agents, but no gene set was common to all drugs. Analysis of the lung cancer cell lines identified the bcl-2 pathway to be associated with cisplatin resistance and the AKT pathway enriched in cisplatin- and docetaxel-resistant cell lines. Results from Connectivity Map supported an association between phosphatidylinositol 3-kinase/AKT and docetaxel resistance but did not support the association with cisplatin. Targeted inhibition of the phosphatidylinositol 3-kinase/AKT pathway with LY294002, in combination with docetaxel, resulted in a synergistic effect in previously docetaxel-resistant cell lines but not with cisplatin. These results support the use of a genomic approach to identify drug-specific targets associated with the development of chemotherapy resistance and underscore the importance of disease context in identifying these pathways. [Mol Cancer Ther 2008;7(10):3141–9]

MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E–Binding Protein 1–Mediated Inhibition of Autophagy

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

Characterizing the developmental pathways TTF-1, NKX2–8, and PAX9 in lung cancer

We investigated the clinical implications of lung developmental transcription factors (TTF-1, NKX2–8, and PAX9) that we recently discovered as cooperating oncogenes activated by way of gene amplification at chromosome 14q13 in lung cancer. Using stable transfectants of human bronchial epithelial cells, RNA expression profiles (signatures) representing activation of the biological pathways defined by each of the 3 genes were determined and used to risk stratify a non-small-cell lung cancer (NSCLC) clinical data set consisting of 91 early stage tumors. Coactivation of the TTF-1 and NKX2–8 pathways identified a cluster of patients with poor survival, representing ≈20% of patients with early stage NSCLC, whereas activation of individual pathways did not reveal significant prognostic power. Importantly, the poor prognosis associated with coactivation of TTF-1 and NKX2–8 was validated in 2 other independent clinical data sets. Furthermore, lung cancer cell lines showing coactivation of the TTF-1 and NKX2–8 pathways were shown to exhibit resistance to cisplatin, the standard of care for the treatment of NSCLC. This suggests that the cohort of patients with coactivation of TTF-1 and NKX2–8 pathways appears to be resistant to standard cisplatin therapy, suggesting the need for alternative therapies in this cohort of high-risk patients.

Immune Signatures Predict Prognosis in Localized Cancer

ABSTRACT The host immune response can impact cancer growth, prognosis, and response to therapy. In colorectal cancer, the presence of cells involved with T-cell-mediated adaptive immunity predicts survival better than the current staging method. We used the expression of genes recently associated with host immune responses (TH1-mediated adaptive immunity, inflammation, and immune suppression) to perform hierarchical clustering of multiple large cohorts of cancer specimens to determine if immune-related gene expression resulted in clinical significant groupings of tumors. Microarray data from prostate cancer (n = 79), breast cancer (n = 132), lung cancer (n = 84), glioblastoma multiforme (n = 120), and lymphoma (n = 127) were analyzed. Among adenocarcinomas, the TH1-mediated adaptive immunity genes were consistently associated with better prognosis, while genes associated with inflammation and immune suppression were variably associated with outcome. Specifically, increased expression of the TH1-mediated adaptive immunity genes was associated with good prognosis in breast cancer patients under 45 years of age (p = .04, hazard ratio [HR] = 0.42) and in prostate cancer patients (p = .03, HR = 0.36) but not in lung cancer patients (p = 0.45, HR = 1.37). In lymphoma, patients with increased expression of inflammation and immune suppression genes had better prognosis than those expressing the TH1-mediated adaptive immunity genes (p = .01, HR = 0.43) and in glioblastoma multiforme, the expression of inflammation genes conferred improved prognosis than those expressing immune suppression genes (p = 0.05, HR = 0.62). In aggregate, the gene expression signatures implicating specific components of the immune response hold prognostic import across solid tumors.

MYC activity mitigates response to rapamycin in prostate cancer through 4EBP1-mediated inhibition of autophagy

Cancer cells have evolved exquisitely to ignore both intrinsic and extrinsic cell death signals, and resistance to cell death is a critical challenge facing clinical oncology. Autophagy, the catabolic recycling process that involves the fusion of autophagosomes containing sequestered cargo with lysosomes, has an enigmatic role in tumorigenesis. In times of metabolic stress due to deprived nutrition or hypoxia, tumor cells use autophagy as a scavenging mechanism for maintenance of critical processes and survival. However, modulation of the extent of autophagy plays a critical role, as excessive autophagy can result in a non-apoptotic and non-necrotic cell death (sometimes referred to as Type II programmed cell death). It is likely that the genetic context of specific cancers will have an impact upon whether autophagy is primarily a mechanism for survival or cell death.

Retraction in part: A genomic approach to identify molecular pathways associated with chemotherapy resistance.

Using previously published annotations for chemotherapy sensitivity in the NCI-60 series of cancer cell lines (2), we performed gene set enrichment analysis on predefined groups of sensitive and resistantNCI-60 cell lines for a range of chemotherapies to identify biological pathways associated with resistance. We purposefully used the annotations for sensitivity and resistance published in the Nature Medicine article and applied a complementary computational approach in order to glean biological insight from the differential gene expression. The article upon which our annotations were based has now been retracted (3). After re-examination, the annotations for the cell lines with respect to chemotherapy sensitivity were erroneous. Thus, our manuscript propagates this error and the results in Table 1 and Supplemental Table 1 from our manuscript are invalid.

Pharmacogenomic strategies provide a rational approach to the treatment of cisplatin-resistant patients with advanced cancer (Journal of Clinical Oncology (2007) 25, (4350-4357))

“Pharmacogenomic Strategies Provide a Rational Approach to the Treatment of Cisplatin-Resistant Patients With Advanced Cancer” David S. Hsu, Bala S. Balakumaran, Chaitanya R. Acharya, Vanja Vlahovic, Kelli S. Walters, Katherine Garman, Carey Anders, Richard F. Riedel, Johnathan Lancaster, David Harpole, Holly K. Dressman, Joseph R. Nevins, Phillip G. Febbo, and Anil Potti J Clin Oncol 25:4350-4357, 2007 The authors wish to retract this article because they have been unable to reproduce the experiments demonstrating a capacity of a cisplatin response signature to validate in either a collection of ovarian cancer cell lines or ovarian tumor samples. Because these results are fundamental to the conclusions of the paper, the authors formally retract the paper. We deeply regret the impact of this action on the work of other investigators. This article was retracted on November 16, 2010.

Abstract C61: The PI3K pathway inhibition in prostate cancer cells is enhanced by dual targeting with the mTOR inhibitor RAD001 and a mTOR/PI3K inhibitor leading to decreased cell proliferation and increased autophagy

Introduction: Metastatic prostate cancer patients often become resistant to standard treatments such as androgen ablation therapy and docetaxel treatment and novel treatments are needed. Loss of PTEN and subsequent activation of the PI3K pathway is common in patients with advanced prostate cancer. However, use of mTOR inhibitors such as rapamycin analogues as single agents has had limited clinical impact and the identification of a potent therapeutic strategy for PI3K inhibition remains a critical question. Experimental procedures: We have used gene expression data generated from metastatic tumors collected from patients with advanced prostate cancer both before and after treatment with RAD001 along with in vitro experiments in established prostate cancer cell lines to determine the impact of TORC1 inhibition, PI3K kinase inhibition, and dual inhibition on PI3K pathway signaling and cancer cell kinetics. Summary: Treatment with RAD001 decreased tumor levels of pS6 but failed to result in a single clinical response. Gene set enrichment analysis of metastatic castration‐resistant prostate cancer biopsies prior to and following treatment with RAD001 was associated with increased PI3K activity following treatment. Analysis of the activity of the downstream components of the PI3K/AKT pathway following RAD001 treatment showed reduced pS6 levels, had minimal impact on p4EBP1 levels, and increased phosphorylation of AKT1. Cytotoxic synergy is observed when RAD001 is combined with the PI3K inhibitor, LY294002 and dual PI3K/mTOR kinase inhibitors. These combinations result in more profound reduction of TORC1 activity resulting in decreased cell proliferation and increased autophagy but a continued persistence of pAKT. Reverse phase proteomics supports increased AKT activity in an androgen‐receptor positive prostate cancer cell line treated with the combination as multiple AKT targets experience increased phosphorylation. Conclusion: TORC1 inhibition results in an upregulation of PI3K activity and therapeutic combinations that inhibit both TORC1 and PI3K demonstrate cytotoxic synergy. The signaling manifestations of these combinations are a profound inhibition of TORC1 activity together with persistent phosphorylation and activity of AKT. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C61.