Cell Circuitry Research Articles

Abstract B197: Proteomic based analysis of ovarian cancer pathways.

Abstract Recent genomic-based studies have suggested ovarian cancer is a disease of copy instability, with 63 regions of focal amplification, rather than a disease driven by mutation. However, this analysis has been unable to identify changes in protein expression, modification, and activity. In this study we developed a highly quantitative MS/MS approach to examine the ratio of protein levels, combining TMT labeling with a number of motif and site-specific antibodies to identify deregulated signaling pathways between tumor and normal specimens. We identified over 1400 uniquely upregulated proteins, and over 1600 upregulated post-translational modification sites across 79 tumors and 19 normal tissue samples. We were able to survey activity of over 400 kinases and over 100 other enzymes as well as the substrates associated with them. Our study was able to map these upregulated proteins based on abundance and activity level to their regions of focal amplifications. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels. This approach may reveal new connections among pathways and candidates for therapeutics and diagnostics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B197. Citation Format: Klarisa Rikova, Benjamin Hall, Tyler Levy, Anthony Possemato, Yi Wang, Kimberly Lee, Joan MacNeil, Ailan Guo, Daniel Mulhern, Caroline Keroack, Jian M. Ren, Sean A. Beausoleil, Scott Lonning, Michael J. Comb. Proteomic based analysis of ovarian cancer pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B197.

An Interactive Resource to Identify Cancer Genetic and Lineage Dependencies Targeted by Small Molecules

The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene β-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.

Downregulation of miR-140 promotes cancer stem cell formation in basal-like early stage breast cancer

The major goal of breast cancer prevention is to reduce the incidence of ductal carcinoma in situ (DCIS), an early stage of breast cancer. However, the biology behind DCIS formation is not well understood. It is suspected that cancer stem cells (CSCs) are already programmed in pre-malignant DCIS lesions and that these tumor-initiating cells may determine the phenotype of DCIS. MicroRNA (miRNA) profiling of paired DCIS tumors revealed that loss of miR-140 is a hallmark of DCIS lesions. Previously, we have found that miR-140 regulates CSCs in luminal subtype invasive ductal carcinoma. Here, we find that miR-140 has a critical role in regulating stem cell signaling in normal breast epithelium and in DCIS. miRNA profiling of normal mammary stem cells and cancer stem-like cells from DCIS tumors revealed that miR-140 is significantly downregulated in cancer stem-like cells compared with normal stem cells, linking miR-140 and dysregulated stem cell circuitry. Furthermore, we found that SOX9 and ALDH1, the most significantly activated stem-cell factors in DCIS stem-like cells, are direct targets of miR-140. Currently, targeted therapies (tamoxifen) are only able to reduce DCIS risk in patients with estrogen receptor α (ERα)-positive disease. We examined a model of ERα-negative/basal-like DCIS and found that restoration of miR-140 via a genetic approach or with the dietary compound sulforaphane decreased SOX9 and ALDH1, and reduced tumor growth in vivo. These results support that a miR-140/ALDH1/SOX9 axis is critical to basal CSC self-renewal and tumor formation in vivo, suggesting that the miR-140 pathway may be a promising target for preventative strategies in patients with basal-like DCIS.

Abstract 4366: ShRNA-based cellular proliferation signaling analysis revealed DRD2 as a novel therapeutic target for glioblastoma.

Abstract Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within one year of diagnosis. Our best current understanding of its etiology is that glioblastoma involves genetic and epigenetic changes that subvert the inherent molecular circuitry of glial cells to drive uncontrolled cellular proliferation. Unfortunately, this molecular circuitry in glioblastoma appears sufficiently complex and redundant that disruption at any particular point tends to redirects signaling through the remaining circuit. In this context, meaningful therapeutic gains will require the identification of therapeutic agents that target critical nodes within this circuit. To identify novel therapeutic agents that target critical nodes which regulate cellular proliferation, a genome-wide retroviral shRNA library screen was conducted in two glioblastoma cell lines (U87MG and A172) and two lung carcinoma lines (A549 and NCI-H460). Pathway analysis of the pro-proliferative genes using both PANTHER and Ingenuity software revealed an over-representation of G-protein coupled neurotransmitter receptors (GPCR), including dopamine receptor subtype 2 (DRD2), which was also highly expressed in glioblastoma specimens relative to the matched normal parentchymal. Further investigation demonstrated that DRD2 signal regulates cell growth by GNAI2-induced ERK activation. Moreover, Haloperidol, one of DRD2 antagonists, enhances significantly anti-tumor efficacy of EGFR inhibitor AG1478 using U87 and mesenchymal neuroshpere xenografy mouse models. Given that both AG1478 and DRD2 antagonists have been approved for the treatment of glioblastoma and psychotics, separtately, Haloperidol combined with chemotherapy represents a novel and likely conducted strategy for the treatment of glioblastoma. Citation Format: Jie Li, Shan Zhu, David Kozono, Diahnn Futulan, David Gonda, Deepa Kushwaha, Kristopher Kahle, Stephen Elledge, Clark C. Chen. ShRNA-based cellular proliferation signaling analysis revealed DRD2 as a novel therapeutic target for glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4366. doi:10.1158/1538-7445.AM2013-4366

A central role for TFIID in the pluripotent transcription circuitry

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

IL4 and CD40L Prevent Apoptosis of Chronic Lymphocytic Leukemia Cells Via Intracellular pSTAT6 and NFkB Signaling and Not Via Receptor Kinetics

Abstract 3893Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental input for their extended survival in vivo, but the underlying molecular mechanism is still unclear. Compared to non-malignant B-cells, CLL cells are more responsive to contact dependent complex stimuli like coculture on bone marrow derived stromal cell lines of both human (p<0.0001) and murine origin (p<0.01), but also to soluble factors (human conditioned medium p<0.0001, murine conditioned medium p<0.001, all student′s t-test). In order to understand the intrinsic difference of the anti-apoptotic phenotype of CLL cells, the signalling circuitry of the malignant cells was modelled. Compared to candidate ligands like SDF-1 (at concentrations between 10–1000ng/ml), BAFF (250–1000ng/ml), APRIL (250–1000ng/ml) and soluble anti-IgM (1–25μg/ml), the factors CD40L (10–2000ng/ml) and IL4 (0.1–10ng/ml) were the most efficient ligands in rescuing CLL cells from spontaneous death in vitro. The dose response of IL4 and CD40L displayed different saturation and cooperativity between CLL cells and non-malignant B-cells. Using IL4, saturation was reached both for CLL cells and B-cells at 0.2pM, but at 52% survival (+/− 8%) for CLL cells and 28% (+/−7%) for B-cells, and the estimated dissociation constant Kd was 0.01pM for both ligands. For CD40L, CLL cell survival reached saturation at 40nM, while no saturation was reached for B-cells. Intriguingly, B-cells showed cooperativity in their response to CD40L, with a cooperativity coefficient of 2.0 and a Kd of 70pM, while cooperativity for CD40L was lost in CLL cells (Kd of only 2.6pM). This pointed towards distinct differences in ligand-receptor interactions or in downstream signaling between CLL cells and non-malignant B-cells. However, high-throughput spatial analysis with a microscope-coupled cytometer did not show differences of receptor quantity or receptor distribution between malignant and non-malignant cells. In contrast, quantity and phosphorylation levels of downstream signalling nodes like STAT6 (measured by flow cytometry and validated by Western-blot) and the activity of NF-kB (p65 binding to DNA measured by oligonucleotide-coupled ELISA) were higher in CLL cells compared to B-cells from healthy donors. Therefore, the defect in IL4 and CD40L signalling that leads to an enhanced survival in CLL cells is likely caused by changes in the intracellular circuitry. Disclosures:No relevant conflicts of interest to declare.

Efficient entry to diversely functionalized spirooxindoles from isatin and their biological activity

A collection of structurally complex and chemically diverse small molecules is a useful tool to explore cell circuitry. In this article, we have reported the two step synthesis of diverse spirooxindoles. The key reaction to assemble the spirooxindole core is a Lewis acid catalyzed three component coupling. The final library of compounds was then analyzed for their cytotoxic activity against U87 human glioma cells. It is noteworthy to mention that this is the first report on the pharmaceutical evaluation of such compounds. Although the activity is moderate, it opens the door for new chemical modifications of spirooxindoles.

Mathematical analysis of maximum power generated by photovoltaic systems and fitting curves for standard test conditions

The rural electrification is characterized by geographical dispersion of the population, low consumption, high investment by consumers and high cost. Moreover, solar radiation constitutes an inexhaustible source of energy and in its conversion into electricity photovoltaic panels are used. In this study, equations were adjusted to field conditions presented by the manufacturer for current and power of small photovoltaic systems. The mathematical analysis was performed on the photovoltaic rural system I-100 from ISOFOTON, with power 300 Wp, located at the Experimental Farm Lageado of FCA/UNESP. For the development of such equations, the circuitry of photovoltaic cells has been studied to apply iterative numerical methods for the determination of electrical parameters and possible errors in the appropriate equations in the literature to reality. Therefore, a simulation of a photovoltaic panel was proposed through mathematical equations that were adjusted according to the data of local radiation. The results have presented equations that provide real answers to the user and may assist in the design of these systems, once calculated that the maximum power limit ensures a supply of energy generated. This real sizing helps establishing the possible applications of solar energy to the rural producer and informing the real possibilities of generating electricity from the sun.

Abstract IA1: Cancer dependencies defined by genomic alterations and targeted by small molecules

Abstract The ability to sequence cancer genomes coupled with advances in small-molecule science provides a new foundation for creating safe and effective cancer therapeutics – ones delivered to patients based on the specific genomic alterations present in their cancer. Several examples now exist of small-molecule cancer drugs targeting protein kinases encoded by both wild-type and oncogenic alleles, and yielding high clinical response rates in patients with specific genetic features. However, these drugs target only a narrow range of proteins, benefit &amp;lt;1% of patients suffering today from cancer, and provide therapeutic benefits that are not always durable. All current genetically matched drugs target the driver oncogene or its wild-type allele directly (“oncogene dependencies”). It is unknown whether similar clinical responses will result from drugs targeting non-oncogenes working in cooperation with oncogenes in patients with cancers having the relevant genetic features (“non-oncogene co-dependencies”). To accelerate the discovery of genetically matched therapies, systematic approaches are needed to identify: 1) the direct and indirect dependencies cancers acquire as a consequence of their specific mutations, translocations, and copy number alterations, and 2) small-molecule drugs that target the dependencies. Genomic cancer cell line profiling has been used to reveal patterns of small-molecule sensitivities across diverse cancer cell lines (CCLs). These efforts initially focused on relating sensitivity to the lineage of CCLs (ref NCI-60 and others), but now increasingly focus on relating sensitivity to genetic and epigenetic features of the cell lines. But these are still early days of genomic CCL profiling – there are many issues to iron out. Nevertheless, there are also signs that this approach will eventually be fruitful and impacting. For example, there are no examples of targeted cancer therapeutics today that were not predicted by genomic CCL profiling. In my lecture, I will describe an Interactive Resource to analyze and visualize a rich body of quantitative CCL sensitivity measurements with the aim of identifying novel candidate cancer dependencies targeted by small molecules. To enable the Resource, we measured the sensitivity of 243 genetically characterized CCLs to 355 small-molecule probes and drugs (the ‘Informer Set’) at eight concentrations in duplicate. The CCLs are part of a 949-member Cancer Cell Line Encyclopedia (CCLE) whose extensive genomic characterization includes the mutational status of &amp;gt;1,600 genes, global assessment of DNA copy number, and global gene expression analysis. These characterizations are also available freely via an interactive web portal (broadinstitute.org/ccle). The compounds included in the Informer Set are known to have selective interactions with their target, and collectively target many distinct nodes in cancer cell circuitry, including but not limited to pathways modulating apoptosis, oxidative stress, chromatin signaling, mitotic stress, hypoxic stress, proteotoxic stress, and metabolism. Using robust analytical methods including cell-line and compound filtering, we correlated the sensitivity measurements with genomic alterations in cancer cell lines to identify dependencies conferred by specific genotypes. I will describe the current content of the Resource and provide multiple illustrations of its use, including the use of additional analytical tools not included in the Resource but readily and freely accessible elsewhere. Citation Format: Stuart L. Schreiber. Cancer dependencies defined by genomic alterations and targeted by small molecules [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr IA1.

The Forkhead Transcription Factor FOXK2 Promotes AP-1-Mediated Transcriptional Regulation

The transcriptional control circuitry in eukaryotic cells is complex and is orchestrated by combinatorially acting transcription factors. Forkhead transcription factors often function in concert with heterotypic transcription factors to specify distinct transcriptional programs. Here, we demonstrate that FOXK2 participates in combinatorial transcriptional control with the AP-1 transcription factor. FOXK2 binding regions are widespread throughout the genome and are often coassociated with AP-1 binding motifs. FOXK2 acts to promote AP-1-dependent gene expression changes in response to activation of the AP-1 pathway. In this context, FOXK2 is required for the efficient recruitment of AP-1 to chromatin. Thus, we have uncovered an important new molecular mechanism that controls AP-1-dependent gene expression.

Phosphorylation Provides a Negative Mode of Regulation for the Yeast Rab GTPase Sec4p

The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p.

Abnormalities in the Climbing Fiber-Purkinje Cell Circuitry Contribute to Neuronal Dysfunction inATXN1[82Q] Mice

One fundamental unanswered question in the field of polyglutamine diseases concerns the pathophysiology of neuronal dysfunction. Is there dysfunction in a specific neuronal population or circuit initially that contributes the onset of behavioral abnormalities? This study used a systems-level approach to investigate the functional integrity of the excitatory cerebellar cortical circuitry in vivo from several transgenic ATXN1 mouse lines. We tested the hypotheses that there are functional climbing fiber (CF)-Purkinje cell (PC) and parallel fiber (PF)-PC circuit abnormalities using flavoprotein autofluorescence optical imaging and extracellular field potential recordings. In early-symptomatic and symptomatic animals expressing ATXN1[82Q], there is a marked reduction in PC responsiveness to CF activation. Immunostaining of vesicular glutamate transporter type 2 demonstrated a decrement in CF extension on PC dendrites in symptomatic ATXN1[82Q] mice. In contrast, responses to PF stimulation were relatively normal. Importantly, the deficits in CF-PC synaptic transmission required expression of pathogenic ataxin-1 (ATXN1[82Q]) and for its entrance into the nucleus of PCs. Loss of endogenous mouse Atxn1 had no discernible effects. Furthermore, the abnormalities in CF-PC synaptic transmission were ameliorated when mutant transgene expression was prevented during postnatal cerebellar development. The results demonstrate the preferential susceptibility of the CF-PC circuit to the effects of ATXN1[82Q]. Further, this deficit likely contributes to the abnormal motor phenotype of ATXN1[82Q] mice. For polyglutamine diseases generally, the findings support a model whereby specific neuronal circuits suffer insults that alter function before cell death.

LincRNAs act in the circuitry controlling pluripotency and differentiation

While thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem cells (ESC) and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ESC regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ESCs and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ESC state.

Shedding light on class-specific wiring: development of intrinsically photosensitive retinal ganglion cell circuitry.

Neural circuits associated with retinal ganglion cells have long been used as models for investigating the mechanisms that govern circuit development and function. Similar to neurons in the brain, retinal ganglion cells are subdivided into distinct classes based upon their morphology, physiology, and patterns of connectivity. Newly developed transgenic tools in which individual classes of retinal ganglion cells are labeled with reporter proteins have recently provided a method to study the development of their class-specific circuitry. Here, we examine a single class of intrinsically photosensitive retinal ganglion cells and discuss their class-specific circuitry, as well as the cellular and molecular mechanisms that govern assembly of this circuitry.

The anti-CD20 mAb LFB-R603 interrupts the dysregulated NF-κB/Snail/RKIP/PTEN resistance loop in B-NHL cells: Role in sensitization to TRAIL apoptosis

LFB-R603 is a chimeric anti-CD20 mouse/human monoclonal antibody with a human IgG1 constant (Fc) region. In comparison to rituximab, LFB-R603 exhibits a high affinity to the Fcγ RIII (CD16) and a potent in vitro antibody-dependent cellular cytotoxicity (ADCC). We examined several experimental designs for the biological anti-tumor activity of LFB-R603 as well as its sensitizing activity to apoptosis in resistant non-Hodgin's B-cell lymphoma (B-NHL) in comparison to rituximab. Treatment of the B-NHL cell line Ramos with LFB-R603 was not toxic at the concentrations used and induced cell aggregation following culture at 24 and 48 h similarly to rituximab. Hence, we hypothesized that LFB-R603 may signal the tumor cells and modify intracellular survival/anti-apoptotic pathways. Treatment of Ramos cells with LFB-R603 inhibited the constitutively active NF-κB survival pathway, followed by significant potentiation of TRAIL-mediated apoptosis. We examined the underlying molecular mechanism by which the LFB-R603-mediated NF-κB inhibition results in the reversal of tumor cell resistance to TRAIL. We hypothesized that downstream gene products regulated by NF-κB that are involved in the resistance may be involved in LFB-R603-mediated sensitization. We found that LFB-R603 inhibited NF-κB activation and the anti-apoptotic factor Snail and induced the pro-apoptotic factor RKIP. The direct roles of Snail and RKIP modulation by LFB-R603 in the reversal of B-NHL resistance to TRAIL were examined by knocking down Snail and overexpressing RKIP in Ramos cells, respectively. Both approaches increased significantly the cell sensitivity to TRAIL apoptosis. In addition to changes observed in the expression levels of Snail and RKIP, Ramos cells treated with LFB-R603 induced the expression of PTEN along with inhibition of the PI3K-AKT activated pathway. PTEN silencing in Ramos cells pretreated with LFB-R603 and TRAIL inhibited TRAIL apoptosis; thus, demonstrating that PTEN induction by LFB-R603 has a direct role in tumor cell sensitization to TRAIL apoptosis. Several of the findings obtained in the experimental designs with LFB-R603 were superior to those obtained with rituximab. Overall, the findings demonstrate that LFB-R603 interferes with the dysregulated NF-κB/Snail/RKIP/PTEN/AKT resistance circuitry in B-NHL cells. Further, the findings suggest that LFB-R603 may sensitize tumor cells to various apoptotic stimuli including cytotoxic ligands such as TRAIL and chemotherapeutic drugs.

Endogenous Expression of Matriptase in Neural Progenitor Cells Promotes Cell Migration and Neuron Differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.

On, Off, On, Off: The Circuitry of Insulin-Releasing Cells

On, Off, On, Off: The Circuitry of Insulin-Releasing Cells

Small-molecule-mediated G-quadruplex isolation from human cells

Nucleic acids containing stretches of tandem guanines can fold into four-stranded structures called G-quadruplexes. The existence of such sequences in genomic DNA suggests the occurrence of these motifs in cells, with potential implications in a number of biological processes relevant to cancer. Small molecules have proven to be valuable tools to dissect cell circuitry. Here, we describe a synthetic small molecule derived from an N,N'-bis(2-quinolinyl)pyridine-2,6-dicarboxamide, which is designed to mediate the selective isolation of G-quadruplex nucleic acids. The methodology was successfully applied to a range of DNA and RNA G-quadruplexes in vitro. We demonstrate the general applicability of the method by isolating telomeric DNA-containing G-quadruplex motifs from cells. We show that telomeres are targets for the probe, providing further evidence of the formation of G-quadruplexes in human cells.

Determining the specificity of protein–DNA interactions

Proteins, such as many transcription factors, that bind to specific DNA sequences are essential for the proper regulation of gene expression. Identifying the specific sequences that each factor binds can help to elucidate regulatory networks within cells and how genetic variation can cause disruption of normal gene expression, which is often associated with disease. Traditional methods for determining the specificity of DNA-binding proteins are slow and laborious, but several new high-throughput methods can provide comprehensive binding information much more rapidly. Combined with in vivo determinations of transcription factor binding locations, this information provides more detailed views of the regulatory circuitry of cells and the effects of variation on gene expression.

Abstract IA2-2: Targeting redundancy in RAS-PI3K signaling in NSCLC

Abstract Cytotoxic chemotherapy has formed the backbone of cancer treatment for decades. The clinical use of such agents was established using dosing strategies that favored efficacy (optimal tumor regression) coupled with minimal toxicity. Conceptually, little regard was given to mechanisms governing survival circuitry, or the genetic alterations in a given tumor, which we now appreciate, are important prognosticators of clinical response for both cytotoxic drugs and therapies that target aberrant signaling pathways involved in cancer pathogenesis. Pharmacogenomic profiling is focused on characterizing genetic changes in tumor cells that drive malignant growth and render cells ‘addicted’ to certain pathways for survival and growth. By identifying these genes and the pathways in which they participate, one can devise therapeutic strategies to disable them. An underappreciated fact in cancer therapeutics is that cancer cells respond to and counteract the effects of any drugs that threaten their survival. Signaling pathways have evolved with innate adaptive abilities to form a regulatory circuitry via positive and negative feedback. These adaptive properties present in normal cells are exploited further in cancer genomes due to their genomic instability and the resulting plasticity. The MAPK-RAS-PI3K signaling network is oncogenic in many human malignancies, including NSCLC, though somatic mutations in PI3K are rare in this disease. Efforts to therapeutically target MAPK dysfunction have been focused on RAF and RAS, since their high frequency of mutations in human tumors renders these druggable targets (activating point mutations in K-RAS occur in approximately 30% of lung adenocarcinomas). Therapeutic strategies to inhibit oncogenic RAS alone have not been successful since these have focused on disrupting effectors downstream of the canonical RAS pathway, without perturbing other RAS effectors. One such RAS- binding protein is type I PI3K, which is required for RAS-driven tumorigenesis: therefore, the PI3K / mTOR component is an important target in RAS-mutant tumors. Therapies directed at MEK, a downstream RAS effector, have been in therapeutic development and shown to exhibit optimal singleagent activity in RAF-mutant tumors. However, the enthusiasm for their clinical application in lung cancer is moderated by the low prevalence of RAF mutations/amplifications in the disease and the appreciation that there is significant redundancy in the MAPK-PI3K pathway that mediates acute resistance to these agents. Therefore, like most targeted therapies, the optimal use for this drug will be in combination. The plasticity of cancer cell circuitry is an important consideration in the rational design of drug combinations, particularly those targeting this signaling network. By characterizing the mechanisms that RAS and RAF mutant cancer cells use to escape MEK-inhibitor therapy, one can use combinations of drugs that target addicted pathways and also prevent cancer cells activating secondary pathways to evade death. We have focused on strategies to counteract these mechanisms of resistance and exploit them therapeutically. An example of this is using MEK inhibitors with rapamycin, which is synergistic in a diverse range of NSCLC cell lines and xenograft models. We have identified proteins in the RAS-PI3K network that moderate the therapeutic response to the MEK-rapamycin combination. In addition, other combinations of MEK inhibitors with drugs that target the estrogen receptor and erbB receptor tyrosine kinases, respectively, will be discussed. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA2-2.