Abstract The most common adult brain tumor is Glioblastoma Multiforme (GBM), an extremely aggressive cancer with only scant treatment options. Even with standard of care most patients present with a recurrence and the median survival is only circa 15 months. The need, therefore, for new therapeutic targets and treatment options is pressing. Here we describe here a multipronged approach to identifying said targets. We present an established methodology for the isolation and culture of patient derived GBM samples that retain the “stem-like” fraction thought to underlie resistance and recurrence. Furthermore we show genomically that these samples represent specific subtypes of the disease yet still form distinct groups in unbiased clustering analysis. Thus we have multiple representative patient derived cultures that are suitable for our drug discovery and chemical biology analyses. Using a process we term Chemical Biology Fingerprinting (CBF) we utilize small focused, and clinically relevant, chemical collections in order to identify patterns of chemovulnerabilities across multiple samples. This allows an unbiased yet cancer relevant sub-stratification and the identification of agents, and therefore targets, which may be relevant for GBM patient subtypes. Indeed our use of the highly annotated NCI CTD2 Informer Set of chemicals allows ready drug-to-target mapping and facilitates data sharing across the CTD2 network. Moreover, already defined subgroups can be clustered to find agents, or groups of agents, that show selective activity against traditional classifications (e.g. proneural, mesenchymal etc.). Finally our strategy is permissive for the identification of “exceptional responders”. That is, individual patient samples that respond to a specific drug whilst most samples are refractory. In sum we demonstrate generation of patient derived models and identify specific, and novel, drugs that may be relevant for specific GBM subtypes. Supported by NIH U01CA168397 Citation Format: Darren Finlay, Pedro Aza-Blanc, Harshil Dhruv, Alexey Eroshkin, Craig Hauser, Jeff Kiefer, Seungchan Kim, Tao Long, Robert G. Oshima, Sen Peng, Gil Speyer, Michael Berens, Kristiina Vuori. Novel target discovery for glioblastoma using chemical biology fingerprinting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1142. doi:10.1158/1538-7445.AM2017-1142

BACKGROUND: The goal of genomics enabled medicine is to understand how specific molecular pathology of an individual tumor informs therapy tailored to specific vulnerabilities. Exploiting large datasets of human glioblastoma (GBM) and relevant, molecularly-profiled preclinical models enables an iterative learning-loop by which such profiling can produce hypotheses of effective, targeted treatment. Genomic, transcriptomic, and epigenomic profiling of glioblastoma enables depiction of driver-passenger relationships of molecular events among subgroupings of cases of the disease. The present study queries genomic profiles of GBM (TCGA) as well as a panel of patient-derived xenograft (PDX) models. Predictions of targets, and targeted drugs effective against those targets are tested empirically using short-term cultures of GBM PDX models. METHODS: Datamining of TCGA GBM combined with similar genomic profiling of a panel of orthotopic GBM PDX models establishes a data warehouse for discovery of subsets of the disease. These subsets, defined by driver-passenger relationships, partition GBM cases into discreet clusters, which enable follow-on bioinformatic queries for nodes of vulnerability. Short-term cultures of GBM PDX models afford testing cell proliferation, survival, migration, invasion, and differentiation responses to a collection of annotated chemical entities characterized as inhibition a wide spectrum of cancer-related targets (n = 600), establishing a “chemical biology fingerprint” of GBMs of distinct contexts. The chemical biology fingerprints can be mapped against the genomic profiling to find and prioritize candidate therapeutic targets matched to the molecular profile. RESULTS: Human GBM profiling bins tumors into molecular contexts based on driver-passenger relationships; GBM PDX models reside in the same or analogous contexts as primary tumor samples. Within such contexts, biochemical signaling and pathway functions portray associated distinct prioritized candidate targets, which align to varying degrees with Chemical Biology Fingerprints (CBFs). CONCLUSIONS: Multi-omics characterization of human GBM uncovers subclusters of the disease with distinct and unique chemovulnerabilities. Single- and multi-agent studies will afford matching effective interventions tailored to individual tumors. Supported by NIH NCI U01CA168397. SECONDARY CATEGORY: Tumor Biology.

Recent evidence supports a role for EphB receptor tyrosine kinases as tumour suppressors in colorectal and prostate cancer. However, it is unclear how these receptors inhibit cancer cell tumorigenicity — an activity that is highly unusual for a family of receptor tyrosine kinases. Here, we report that the EphB4 receptor can behave as a tumour suppressor in a mouse xenograft model of breast cancer when stimulated by its ligand, ephrin-B2. In breast cancer cells, EphB4 activates an antioncogenic pathway involving Abl family tyrosine kinases and the Crk adaptor protein. This Abl–Crk pathway inhibits breast cancer cell viability and proliferation in addition to motility and invasion, and also downregulates the pro-invasive matrix metalloprotease, MMP-2. Consistent with these effects, EphB4 and the Abl–Crk pathway are constitutively active in non-transformed mammary epithelial cells. These findings identify a novel Eph receptor signalling pathway with tumour-suppressor activity and predict that therapeutic intervention to activate EphB4 signalling will inhibit tumour progression.

The ErbB2 (Neu) receptor tyrosine kinase is frequently over-expressed in human breast cancers, and this phenotype correlates with a poor clinical prognosis. We examined the effects of the mammalian target of rapamycin inhibitor, rapamycin, on mammary tumorigenesis in transgenic mice bearing an activated ErbB2 (NeuYD) transgene in the absence or presence of a second transgene encoding vascular endothelial growth factor (VEGF). Treatment of NeuYD or NeuYD X VEGF mice with rapamycin dramatically inhibited tumor growth accompanied by a marked decrease in tumor vascularization. Two key events that may underlie the antitumor activity of rapamycin were decreased expression of ErbB3 and inhibition of hypoxia-inducible factor-1-dependent responses to hypoxic stress. Rapamycin exposure caused only a modest inhibition of the proliferation of tumor-derived cell lines in standard monolayer cultures, but dramatically inhibited the growth of the same cells in three-dimensional cultures, due in part to the induction of apoptotic cell death. These studies underscore the therapeutic potential of mammalian target of rapamycin inhibitors in ErbB2-positive breast cancers and indicate that, relative to monolayer cultures, three-dimensional cell cultures are more predictive in vitro models for studies of the antitumor mechanisms of rapamycin and related compounds.

The Neu (ErbB2, HER2) member of the epidermal growth factor receptor family is implicated in many human breast cancers. We have tested the importance of increased angiogenic signaling in the NeuYD [mouse mammary tumor virus (MMTV)-Neu(ndl)-YD5] mammary tumor model. Transgenic mice expressing vascular endothelial growth factor (VEGF)(164) from the MMTV promoter were generated. These mice expressed VEGF(164) RNA and protein at 20- to 40-fold higher levels throughout mammary gland development but exhibited normal mammary gland development and function. However, in combination with the NeuYD oncogene, VEGF(164) expression resulted in increased vascularization of hyperplastic mammary epithelium and dramatic acceleration of tumor appearance from 111 to 51 days. Gene expression profiling also indicated that the VEGF-accelerated tumors were substantially more vascularized and less hypoxic. The preferential vascularization of early hyperplastic portions of mammary epithelia in NeuYD;MMTV-VEGF animals was associated with NeuYD RNA expression, disorganization of the tight junctions, and overlapping transgenic VEGF expression. NeuYD;MMTV-VEGF(164) bigenic, tumor-bearing animals resulted in an average of 10 tumor cell colonies/lung lodged within vascular spaces. No similar lung colonies were found in control NeuYD mice with similar tumor burdens. Overall, these results demonstrate the angiogenic restriction of early hyperplastic mammary lesions. They also reinforce in vivo the importance of activated Neu in causing disorganization of mammary luminal epithelial cell junctions and provide support for an invasion-independent mechanism of metastasis.

The Ets family of transcription factors in mouse or humans is comprised of around 27 unique family members that contain an evolutionarily conserved DNA-binding domain called the Ets domain. The Ets family includes both transcriptional activators and repressors. The normal cellular Ets transcription factors have been implicated as mediators of a wide range of cellular processes, including oncogenic transformation. This chapter provides an overview of the Ets family, and describes each of the multiple lines of evidence that Ets transcription factors are mediators of cellular transformation. This evidence includes: (a) cancers resulting from Ets factor overexpression or chromosomal translocations that generate fusion proteins containing Ets factor domains; (b) signaling from oncogenes to Ets factors; (c) expression correlation of Ets factors with tumor formation; (d) reversal of cellular transformation by dominant inhibitory Ets constructs; (e) delayed tumor development after genetic disruption of an Ets factor; and (f) the potential role of many Ets target genes in transformation. A better understanding of the role of Ets factors and their target genes in cancer should provide the basis for more specific novel therapeutic approaches for the treatment of cancers.

Ets2 is a widely expressed Ets family transcription factor which is activated by Ras signaling and has been reported to transform fibroblasts. Expression of a dominant inhibitory Ets2 construct consisting of just the Ets2 DNA binding domain (Ets2DBD), reverses Ras transformation of NIH3T3 cells and the transformed characteristics of several human tumor cells. However, the Ets2DBD may interfere with multiple Ets family members. We have now utilized cell lines with a disrupted ets2 gene to determine whether Ets2 is required to mediate oncogenic signaling. Expression of the Ets2DBD in an Ets2-deficient cell line dramatically inhibited Ets-dependent (but not AP-1-dependent) reporter gene expression, revealing that the Ets2DBD does inhibit additional Ets family members. The transformation efficiency of Ets2-deficient cell lines by oncogenic Ras or Neu/ErbB-2 was similar to that of control cells in several in vitro assays, and was not enhanced by re-expression of Ets2. Finally, overexpression of Ets2 was not sufficient to induce focus formation in NIH3T3 cells, nor to enhance transformation by oncogenic Ras. Thus, Ets2 is not an essential mediator of Ras or Neu/ErbB-2 transformation in these cells. Our results illustrate the importance of utilizing specific approaches for analyzing the function of individual members of large gene families.

The Ets2 transcription factor is regulated by mitogen-activated protein (MAP) kinase phosphorylation of a single threonine residue. We generated by gene targeting a single codon mutation in Ets2 substituting Ala for the critical Thr-72 phosphorylation site (Ets2A72), to investigate the importance of MAP kinase activation of Ets2 in embryo and tumor development. Ets2(A72/A72) mice are viable and develop normally. However, combining the Ets2A72 allele with a deletion mutant of Ets2 results in lethality at E11.5 and shows that Ets2A72 is a hypomorphic allele. Mammary tumors caused by transgenic polyomavirus middle T antigen, activated Neu(Erbb2), or the combination of Neu and transgenic VEGF (Neu; VEGF-25) were all restricted in Ets2(A72/A72) females. The Ets2(A72/A72) restriction on Neu; VEGF-25 tumor growth was associated with increased p21Cip1 expression. The size of tumors transplanted into fat pads of mice with Ets2 targeted alleles was correlated directly with Ets2 activity and fewer stromal cells expressing matrix metalloproteinase 9 (MMP-9). Decreased MMP-3 and MMP-9 mRNAs were confirmed in Ets2(A72/A72) macrophages. Activation of Ets2 at Thr-72 acts in the stroma, downstream of vascular endothelial growth factor production, in part through the regulation of macrophage proteases to support the progression of Neu- and polyomavirus middle-T-initiated mammary tumors.

Parathyroid hormone-related protein (PTHrP) promotes the metastatic potential and proliferation of breast cancer cells, and acts anti-apoptotically. In invasive MDA-MB-231 breast cancer cells, transforming growth factor beta-regulated PTHrP synthesis is mediated by an Ets1/Smad3-dependent activation of the PTHrP P3 promoter. In the present study, we studied the regulation of PTHrP expression in non-invasive, Ets1-deficient and transforming growth factor beta-resistant MCF-7 cells. We found PMA to be a strong stimulator of P3-dependent PTHrP expression in MCF-7 cells. Mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase 1 (MEK-1)/ERK1/2 inhibitor PD98059 interfered with this activity. Promoter studies revealed that the PMA effect depended on the Ets and stimulating protein-1 (Sp1)-binding sites. Of several Ets factors tested, Ets2, but not Ese-1, Elf-1 or Ets1, supported the PMA-dependent increase in promoter activity. PD98059 and a threonine to alanine mutation of the ERK1/2-responsive Ets2 phosphorylation site at position 72 inhibited the Ets2/PMA effect. Activated protein kinase C (PKC) epsilon could mimic PMA by stimulating the P3 promoter alone or in co-operation with Ets2 in an MEK-1/ERK1/2-dependent manner. Activated PKC alpha, although capable of co-operating with Ets2, failed to induce transcription from the P3 promoter on its own. The Ets2/PKalpha synergistic effect was neither sensitive to PD98059 nor to Thr(72)/Ala(72) mutation. PMA neither increased the expression of Sp1 nor modulated the transcriptional activity of Sp1. However, it induced the displacement of a yet unknown factor from the Sp1-binding site, which may result in Sp1 recruitment to the promoter. Our results suggest an ERK1/2-dependent Ets2/PKC epsilon synergism to be involved in PTHrP expression in MCF-7 breast cancer cells.

Interfering with Ets transcription factor function reverses multiple aspects of the transformed phenotype of mouse or human tumor cells. However, the unknown number of individual Ets factors expressed in any cellular context and the similar DNA binding specificities of Ets family members complicates the identification of those that mediate transformation. By utilizing quantitative PCR assays for 25 mouse Ets factors, we analyzed the expression of essentially the entire Ets family in normal mammary tissue, mammary-related cell lines, and mammary tumors. In normal mammary tissue, 24 Ets factors were expressed. Even clonal derived cell lines expressed 14-20 Ets members. The most abundant Ets factor mRNAs measured in normal mammary tissue were Elk4, Elf1, and Ets2. Subtractive analysis of mammary tissue identified which Ets factors were predominantly expressed in the myeloid/lymphoid or epithelial cell compartments. Comparison of Ets factor expression in normal mammary tissue and mammary tumors identified significantly elevated expression of Pse/PDEF, Ese2/Elf5, Ese3/Ehf, TEL/Etv6, and Elf2/NERF in mammary tumors and confirmed previously reported alterations in expression of Ese1/Elf3 and the PEA3 subfamily. Expression of 13 Ets target genes, implicated in various aspects of tumor progression, was also analyzed. Altered expression of particular Ets target genes was significantly correlated with particular Ets factors (e.g. maspin and Ese2), suggesting specific in vivo regulatory roles. Together, this comprehensive analysis revealed unexpectedly diverse Ets family gene expression, characterized novel Ets factor changes in mammary tumors, and implicated specific Ets factors in the regulation of multiple genes involved in mammary tumor progression.

The candidate tumor suppressor ING1 was identified in a genetic screen aimed at isolation of human genes whose expression is suppressed in cancer cells. It may function as a negative growth regulator in the p53 signal transduction pathway. However, its molecular mechanism is not clear. The ING1 locus encodes alternative transcripts of p47(ING1a), P33(ING1b) and p24(ING1c). Here we report differential association of protein products of ING1 with the mSin3 transcriptional corepressor complex. p33(ING1b) associates with Sin3, SAP30, HDAC1, RbAp48, and other proteins, to form large protein complexes, whereas p24(ING1c) does not. The ING1 immune complexes are active in deacetylating core histones in vitro, and p33(ING1b) is functionally associated with HDAC-1-mediated transcriptional repression in transfected cells, Our data provide basis for a p33(ING1b)-specific molecular mechanism for the function of the ING1 locus.

This chapter describes the mechanism of Ras signaling for the activation of transcription process. Activation of Ras signaling stimulates multiple downstream signaling cascades, many of which converge on the nucleus and alter cellular gene expression. These widespread changes in gene expression are important for regulating normal growth and, when aberrantly regulated, for mediating oncogenests. Many of the changes in gene expression mediated by Ras are the result of signaling through multiple parallel pathways resulting in activation of mitogen activated protein (MAP) kinase family members. One approach is to transiently contransfect oncogenic Ras and a reporter construct consisting of the promoter region of the gene fused to an easily assayed reporter coding sequence (e.g., luciferase). The type of reporter gene analysis described in this chapter utilizes fusion proteins between a heterologous DNA-binding domain from yeast GAL4 and the trans-activation domain of a specific transcription factor. Overall, this type of reporter gene analysis involves transient contransfection of two or three different plasmids into an appropriate cell line.

The Ets family of transcription factors are important downstream targets in cellular transformation, as altering Ets activity has been found to reverse the transformed phenotype of Ras transformed mouse fibroblasts and of several human tumor cell lines. To determine whether Ets factors are important targets in the largely uncharacterized aberrant signaling in prostate cancer, we have altered Ets activity in the prostate tumor cell line PPC-1, by stable expression of either full-length Ets2, or a dominant inhibitor of Ets activity, the Ets2 DNA binding domain (Ets2DBD). Analysis of multiple independent clonal cell lines revealed that expression of either Ets2 or the Ets2DBD inhibited the anchorage-independent growth of PPC-1 cells up to 20-fold. In contrast to our previous findings with Ras-transformed NIH3T3 cells, PPC-1 cell lines expressing either Ets2 or the EtsDBD exhibited slower attached cell growth, increased Ets-dependent gene expression, and up to a 10-fold increase in apoptotic cell death. The p21cip gene was identified as a potential target of altered Ets signaling. Interestingly, the two distinct Ets2 constructs had strikingly different effects on in vitro invasiveness. Expression of the Ets2DBD almost completely blocked PPC-1 cell invasion through Matrigel, whereas over-expression of full-length Ets2 did not inhibit invasion. Overall, these results demonstrate that the balance of Ets factor activity can regulate multiple aspects of the transformed phenotype of PPC-1 prostate tumor cells, including anchorage-independent growth, survival, and invasiveness.

The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappa B. However, the mechanisms by which p75NTR initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75NTR. Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappa B activation with contrasting effects. Coexpression of TRAF2 with p75NTR enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75NTR. TRAF4 also inhibited the NF-KB response, whereas TRAF2 and TRAF6 enhanced p75NTR-induced NF-kappa B activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappa B activation and cell death induction.

Heterozygous female mice carrying a targeted mutation of the Ets2 transcription factor gene were mated with a mouse strain that develops mammary tumors due to the expression of the polyoma virus middle T oncogene. Tumors from females with only one wild-type Ets2 gene were approximately one-half the size of tumors from controls. The smaller size of the tumors was correlated with a more differentiated state of early hyperplastic growths and not to differential growth of the frank tumors or to decreased middle T gene expression. Ets2 may regulate the progression of these aggressive mammary tumors.