Theresa Zhang

Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters

ABSTRACT We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles.

Farnesoid X Receptor Activates Transcription of the Phospholipid Pump MDR3

The human multidrug resistance gene MDR3 encodes a P-glycoprotein that belongs to the ATP-binding cassette transporter family (ABCB4). MDR3 is a critical trans-locator for phospholipids across canalicular membranes of hepatocytes, evidenced by the fact that human MDR3 deficiencies result in progressive familial intrahepatic cholestasis type III. It has been reported previously that MDR3 expression is modulated by hormones, cellular stress, and xenobiotics. Here we show that the MDR3 gene is trans-activated by the farnesoid X receptor (FXR) via a direct binding of FXR/retinoid X receptor α heterodimers to a highly conserved inverted repeat element (a FXR response element) at the distal promoter (-1970 to -1958). In FXR trans-activation assays, both the endogenous FXR agonist chenodeoxycholate and the synthetic agonist GW4064 activated the MDR3 promoter. Deletion or mutation of this inverted repeat element abolished FXR-mediated MDR3 promoter activation. Consistent with these data, MDR3 mRNA was significantly induced by both chenodeoxycholate and GW4064 in primary human hepatocytes in time- and dose-dependent fashions. In conclusion, we demonstrate that MDR3 expression is directly up-regulated by FXR. These results, together with the previous report that the bile salt export pump is a direct FXR target, suggest that FXR coordinately controls secretion of bile salts and phospholipids. Results of this study further support the notion that FXR is a master regulator of lipid metabolism.

Pathway-Based Identification of Biomarkers for Targeted Therapeutics: Personalized Oncology with PI3K Pathway Inhibitors

Phosphorylation sites on proteins in the phosphatidylinositol 3-kinase pathway that are regulated by candidate drugs can serve as useful biomarkers to predict tumor sensitivity to AKT inhibitors. Toward Customizing Tumor Treatment Just as our view of Earth has become increasingly global, cells are now seen as complex networks of interacting and intersecting signaling pathways rather than a collection of regulated genes. This new view applies to cancer cells as well, which we now know have entire dysregulated pathways and not just dysregulated genes. Andersen and colleagues have identified phosphoprotein biomarkers for a pathway often altered in cancer—the phosphatidylinositol 3-kinase (PI3K) pathway—and have shown that one of these predicts the sensitivity of cancer cells to a promising class of cancer drugs: inhibitors of AKT, a kinase that promotes growth and inhibits cell death. To find useful markers of the PI3K pathway, the authors focused on a vital biochemical event—the addition of phosphate groups to serines and threonines in cellular proteins. Cells use this simple covalent modification over and over again to regulate protein-protein binding and activity of key enzymes. Measurement of this modification in specific proteins reveals their activation. The authors monitored 375 phosphorylation sites in the PI3K pathway after treating prostate cancer cells with three different PI3K pathway inhibitors, potentially useful drugs. They found that each drug modulated a specific array of phosphoproteins, with some overlap, many of them within proteins that participate in cytoskeletal remodeling, vesicle transport, and protein translation. In theory, each phosphopeptide that decreased in abundance after drug treatment could, if elevated in cancer cells, serve as a biomarker of sensitivity to that drug. To show that this was the case, the authors chose one of the phosphorylated sites (the threonine at position 246 of the cytoplasmic protein PRAS40) and generated a high-quality antibody to it. The amount of phosphorylation at Thr246 correlated with activation of the PI3K pathway in human cancer cell lines, in a mouse prostate tumor, and in triple-negative breast tumors. Of potentially even more utility, Thr246 phosphorylation predicted the sensitivity of these cells to AKT inhibitors. Cancers are extremely heterogeneous, even within tissues, and for optimal effectiveness, treatments need to be customized accordingly. As this work shows, phosphorylated amino acids can serve as biomarkers for activated pathways in cancer and, because specific antibodies can easily be made to these phosphorylated peptides, can be readily measured. These results point to a way, after further development of more biomarkers, to routinely characterize the activated pathways in patients’ cancers. A tumor characterized in this way can then be treated with the appropriate pathway-specific drugs, optimizing the chances of eradicating the tumor. Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K–mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway–relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40Thr246. Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)–deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40Thr246 positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKTThr308, the phospho-PRAS40Thr246 epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

BackgroundHyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence.MethodsWe used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets.ResultsThe RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer.ConclusionsThese data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

DWE: Discriminating Word Enumerator

MOTIVATION Tissue-specific transcription factor binding sites give insight into tissue-specific transcription regulation. RESULTS We describe a word-counting-based tool for de novo tissue-specific transcription factor binding site discovery using expression information in addition to sequence information. We incorporate tissue-specific gene expression through gene classification to positive expression and repressed expression. We present a direct statistical approach to find overrepresented transcription factor binding sites in a foreground promoter sequence set against a background promoter sequence set. Our approach naturally extends to synergistic transcription factor binding site search. We find putative transcription factor binding sites that are overrepresented in the proximal promoters of liver-specific genes relative to proximal promoters of liver-independent genes. Our results indicate that binding sites for hepatocyte nuclear factors (especially HNF-1 and HNF-4) and CCAAT/enhancer-binding protein (C/EBPbeta) are the most overrepresented in proximal promoters of liver-specific genes. Our results suggest that HNF-4 has strong synergistic relationships with HNF-1, HNF-4 and HNF-3beta and with C/EBPbeta. AVAILABILITY Programs are available for use over the Web at http://rulai.cshl.edu/tools/dwe.

Combination of the mTOR Inhibitor Ridaforolimus and the Anti-IGF1R Monoclonal Antibody Dalotuzumab: Preclinical Characterization and Phase I Clinical Trial

Purpose: Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin–like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination. Experimental Design: In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10–40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored. Results: Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER+/high-proliferative breast cancer showed antitumor activity. Conclusions: Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER+/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379). Clin Cancer Res; 21(1); 49–59. ©2014 AACR.

Dual role of Nr2e3 in photoreceptor development and maintenance

Nr2e3 is a photoreceptor-specific nuclear receptor believed to play a role in photoreceptor development, differentiation, and survival. Much research has focused on the interaction of Nr2e3 with other transcription factors in determining the milieu of target gene expression in photoreceptors of the neonatal and adult retina. To investigate the downstream targets of Nr2e3 and thereby shed light on the functional pathways relevant to photoreceptor development and maintenance, expression profiling was performed on retinas from two different mouse knockout lines, one containing a targeted disruption of the Nr2e3 gene (Nr2e3 -/-), the other containing a spontaneous null allele of the Nr2e3 locus (rd7). Using whole genome microarrays, mRNA expression profiles of retinas from the two mutant strains were compared to those of wildtype C57BL/6 mice over a time course that ranged from postnatal day (p) 2 to 6months of age (p180). Additionally, expression profiling was performed on retinal explants treated with a putative NR2E3 agonist. The molecular profiling of Nr2e3 -/- and rd7/rd7 retinas identified 281 putative Nr2e3-dependent genes that were differentially expressed between wildtype and mutant retinas during at least one time point. Consistent with previous reports that Nr2e3 is necessary for the repression of cone-specific genes, increased expression of cone-specific genes was observed in the mutant samples, thereby providing proof-of-concept for the microarray screen. Further annotation of these data sets revealed ten predominant functional classes involved in the Nr2e3-mediated development and/or maintenance of photoreceptors. Interestingly, differences in the expression of Nr2e3-dependent genes exhibited two distinct temporal patterns. One group of genes showed a sustained difference in expression as compared to wildtype over the entire time course of the study, whereas a second group showed only transient differences which were largest around p10. Comparison of gene expression changes in Nr2e3 -/- and rd7/rd7 retinas with those uncovered by treating retinal explants with a putative NR2E3 agonist revealed four genes that were down-regulated in mutant retinas that lack Nr2e3 function but were up-regulated in agonist-treated explants. These results strongly suggest that the four genes may be direct targets of Nr2e3. Our identification of two sets of Nr2e3-regulated genes provides further evidence of a dual role for Nr2e3 in specification of photoreceptor fate during development as well as photoreceptor maintenance in the adult.

MCL1 and BCL-xL Levels in Solid Tumors Are Predictive of Dinaciclib-Induced Apoptosis

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclibs antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

Promoter Extraction from GenBank (PEG): automatic extraction of eukaryotic promoter sequences in large sets of genes

UNLABELLED Promoter Extraction from GenBank (PEG) extracts promoter sequences for large sets of genes using information present in GenBank. For a gene whose promoter sequence is not found, PEG will attempt to extract promoter sequences of the orthologous genes instead. AVAILABILITY It is freely available to academic users at ftp://cshl.org/pub/science/mzhanglab/theresa/. CONTACT zhangt@cshl.org; mzhang@cshl.org

An Unbiased Oncology Compound Screen to Identify Novel Combination Strategies

Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo. This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155–62. ©2016 AACR.