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Bacterial persisters are a quasidormant subpopulation of cells that are tolerant to antibiotic treatment. The combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an antipersister strategy that is aimed at reducing recurrent Pseudomonas aeruginosa infections by enhancing the killing of P. aeruginosa persisters. Stationary-phase cultures of P. aeruginosa were used to generate persister cells. A range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions, including a range of pH values and in the presence of azithromycin or cystic fibrosis (CF) patient sputum. In addition, 96-well dish biofilm and colony biofilm assays were performed, and the cytotoxicity of the tobramycin-fumarate combination was determined utilizing a lactate dehydrogenase (LDH) assay. Enhanced killing of up to 6 orders of magnitude of P. aeruginosa persisters over a range of CF isolates, including mucoid and nonmucoid strains, was observed for the tobramycin-fumarate combination compared to killing with tobramycin alone. Furthermore, significant fumarate-mediated potentiation was seen in the presence of azithromycin or CF patient sputum. Fumarate also reduced the cytotoxicity of tobramycin-treated P. aeruginosa to human epithelial airway cells. Finally, in mucoid and nonmucoid CF isolates, complete eradication of P. aeruginosa biofilm was observed in the colony biofilm assay due to fumarate potentiation. These data suggest that a combination of tobramycin with fumarate as an antibacterial potentiator may be an attractive therapeutic for eliminating recurrent P. aeruginosa infections in CF patients through the eradication of bacterial persisters.

Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To “humanize” this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy. Keywords: alpha-synuclein; iPS cell; Parkinson’s disease; stem cell; mRNA translation; RNA-binding protein; LRRK2; VPS35; vesicle trafficking; yeast

SummaryHSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.

In Huntington's disease (HD), polyglutamine expansions in the huntingtin (Htt) protein cause subtle changes in cellular functions that, over-time, lead to neurodegeneration and death. Studies have indicated that activation of the heat shock response can reduce many of the effects of mutant Htt in disease models, suggesting that the heat shock response is impaired in the disease. To understand the basis for this impairment, we have used genome-wide chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) to examine the effects of mutant Htt on the master regulator of the heat shock response, HSF1. We find that, under normal conditions, HSF1 function is highly similar in cells carrying either wild-type or mutant Htt. However, polyQ-expanded Htt severely blunts the HSF1-mediated stress response. Surprisingly, we find that the HSF1 targets most affected upon stress are not directly associated with proteostasis, but with cytoskeletal binding, focal adhesion and GTPase activity. Our data raise the intriguing hypothesis that the accumulated damage from life-long impairment in these stress responses may contribute significantly to the etiology of Huntington's disease., National Institutes of Health (U.S.) (Grant R24 DK-090963), National Institutes of Health (U.S.) (Grant R01-GM089903), National Institutes of Health (U.S.) (Grant P30-ES002109), National Science Foundation (U.S.) (Award DB1-0821391)

The cellular environment is a complex and dynamic system of functional molecules. These molecules interact, either transiently or stably, in pathways, which remain poorly mapped despite extensive study. High-throughput experimental methods have allowed us to fill some of the gaps in pathway knowledge, but have created new algorithmic challenges associated with the integration of large, disparate sources of data. Here, we present different algorithmic approaches to the integration of heterogeneous datasets. The article examines algorithms that analyze networks with a single data source, methods that analyze networks with more complex data types, and finally algorithms that capture hierarchical flow of biological information. We also examine a critical issue that has been largely ignored in most network approaches: To what extent can gene expression data be used as a proxy for protein levels? While most studies conflate these two, it is now abundantly clear from high-throughput analyses that the correlation between these data is very poor. We close by speculating on where a more realistic modeling of different types of data will lead the field.

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules—TGF-β and SDF1—play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications., Alexander and Margaret Stewart Trust, Kathy and Curt Marble Cancer Research Fund

Introduction Ribosome biogenesis is commonly up-regulated to satisfy the increased anabolic demands associated with malignant transformation and tumor growth. Many different oncogenic signaling pathways converge on the ribosome to increase translational flux. Despite the detailed understanding of ribosome regulation in cancer, it is not clear whether the net translational activity of the ribosome can itself regulate transcriptional programs that support and promote the malignant state. Methods To investigate the transcriptional effects of modulating translational activity in malignant cells, we used integrated chemical and genetic approaches, including a gene signature–based genetic and chemical screen of more than 600,000 gene expression profiles (LINCS database) and an independent, reporter-based chemical screen of more than 300,000 compounds. A lead compound was tested in several cell-lines unified by their increased dependence on HSF1 activation for growth and survival, and in an in vivo cancer model. Results Inhibiting translation led to large changes in the transcriptome. The single most enriched category consisted of genes regulated by the heat-shock transcription factor, HSF1. The most down-regulated mRNA was HSPA8 , which encodes the constitutive HSP70 chaperone that helps to fold nascent polypeptides. The expression of many other genes that HSF1 coordinates to support cancer were also strongly affected. HSF1 protein levels were unchanged, but HSF1 DNA occupancy was nearly eliminated. Inhibition of the HSF1-regulated gene expression program is thus a dominant transcriptional effect elicited by inhibiting protein translation. Using a gene signature of HSF1 inactivation to query the LINCS database revealed a strong connection between HSF1 inactivation and perturbations that inhibit protein translation, including a broad spectrum of chemical and genetic interventions that target the ribosome, eukaryotic initiation factors (eIFs), aminoacyl tRNA synthetases, and upstream signaling/regulatory pathways that control translation. Our high-throughput small-molecule screen identified rocaglamide A, an inhibitor of translation initiation, was the strongest inhibitor of HSF1 activation. An analog of this compound, RHT, increased thioredoxin-interacting protein (TXNIP) mRNA and protein levels and decreased glucose uptake and lactate production. Cell-based cancer models characterized by high dependence on HSF1 activation for growth and survival were highly sensitive to RHT, as were cells derived from diverse hematopoietic malignancies. RHT had a strong antitumor effect—with marked inhibition of HSF1 activity and glucose uptake—against xenografted acute myeloid leukemia cells. Discussion The ribosome functions as a central information hub in malignant cells: Translational flux conveys information about the cell’s metabolic status to regulate the transcriptional programs that support it. Multiple unbiased chemical and genetic approaches establish HSF1 as a prime transducer of this information, centrally poised to regulate the transcription of genes that support protein folding, biomass expansion, anabolic metabolism, cellular proliferation, and survival. Targeting translation initiation may offer a strategy for reversing HSF1 activation, disabling metabolic and cytoprotective pathways in malignant cells. HSF1 at the crossroads of protein translation and metabolism. ( Left ) Cancers activate an HSF1-regulated transcriptional program to adapt to the anabolic demands of relentless biomass expansion. Glucose uptake increases, and expression of TXNIP, an inhibitor of glucose uptake, drops. ( Right ) Down-regulating translation with rocaglate scaffold initiation inhibitors reverses cancer-associated HSF1 activation. Glucose uptake drops as TXNIP levels rise.

In Huntington’s disease (HD), polyglutamine expansions in the huntingtin (Htt) protein cause subtle changes in cellular functions that, over-time, lead to neurodegeneration and death. Studies have indicated that activation of the heat shock response can reduce many of the effects of mutant Htt in disease models, suggesting that the heat shock response is impaired in the disease. To understand the basis for this impairment, we have used genome-wide chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) to examine the effects of mutant Htt on the master regulator of the heat shock response, HSF1. We find that, under normal conditions, HSF1 function is highly similar in cells carrying either wild-type or mutant Htt. However, polyQ-expanded Htt severely blunts the HSF1-mediated stress response. Surprisingly, we find that the HSF1 targets most affected upon stress are not directly associated with proteostasis, but with cytoskeletal binding, focal adhesion and GTPase activity. Our data raise the intriguing hypothesis that the accumulated damage from life-long impairment in these stress responses may contribute significantly to the etiology of Huntington’s disease.

SummaryHeat-Shock Factor 1 (HSF1), master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival, and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their nontransformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, many are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications.

Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells.

The SAM-T04 method for predict- ing protein structures uses a single protocol across the entire range of targets, from comparative model- ing to new folds. This protocol is similar to the SAM-T02 protocol used in CASP5, but has improve- ments in the iterative search for similar sequences in finding and aligning templates, in creating frag- ment libraries, in generating protein conforma- tions, and in scoring the conformations. The auto- matic procedure made some improvements over simply selecting an alignment to the highest-scoring template, and human intervention made substantial improvements over the automatic procedure. The main improvements made by human intervention were from adding constraints to build (or retain) -sheets and from splitting multidomain proteins into separate domains. The uniform protocol was moderately successful across the entire range of target difficulty, but was somewhat less successful than other approaches in CASP6 on the compara- tive modeling targets. Proteins 2005;Suppl 7:135-142.

The heat-shock response is a powerful transcriptional program which acts genome-wide, not only to restore the normal protein folding through the induction of heat shock proteins (HSP), but to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by the Heat Shock Factor 1 (HSF1) transcription factor. We have previously shown that HSF1 plays a fundamental role in tumorigenesis, by promoting the survival and malignance of tumor cells, both in tissue culture and in mouse models of cancer [1]. Recently we demonstrated that HSF1 exerts its role by activating a unique transcriptional program in the cancer cells, that is distinct from the one activated during heat shock [2]. In breast cancer and several other types of carcinoma, we found that high HSF1 protein levels and activation of the HSF1-dependent transcriptional program are associated with poor clinical outcome [3]. Here we show that HSF1 is activated not only in the tumor cells, but also in the stromal cells infiltrating the tumor. Examining human patient samples, we find immunohistochemical evidence for activation of HSF1 in the stroma. Using mouse xenograft models and in vitro co-culture we show that HSF1 in the stroma supports tumor cell growth. Finally, expression profiling and analysis of the DNA binding pattern of HSF1 in tumors and in cell culture indicates that stromal HSF1 supports tumorigenesis by activating a unique, stroma-specific transcriptional program. Taken together, our data suggests that HSF1 acts in the cancer cells and in the stroma to activate distinct, yet complimentary transcriptional programs that will facilitate tumor initiation, progression and metastasis. References 1. Dai, C., et al., Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell, 2007. 130(6): p. 1005-18. 2. Mendillo, M.L., et al., HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers. Cell, 2012. 150(3): p. 549-62. 3. Santagata, S., et al., High levels of nuclear heat-shock factor 1 (HSF1) are associated with poor prognosis in breast cancer. Proc Natl Acad Sci U S A, 2011. 108(45): p. 18378-83. Citation Format: Ruth Scherz-Shouval, Sandro Santagata, Martina Koeva, Luke Whitesell, Susan Lindquist. Cell autonomous and nonautonomous activities of heat shock factor 1 support tumor initiation, progression, and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A23.

HSF1 is best known for its role as the master transcriptional regulator of the evolutionarily conserved heat-shock response. In mice, Hsf1 knock-outs dramatically reduce susceptibility to malignant transformation and tumor formation, and markedly increased survival in cancers driven by both oncogenic and tumor suppressor mutations. Likewise, RNAi-mediated knockdown markedly reduces the growth and survival of human cell lines established from cancers driven by a diversity of genetic lesions. The transcriptional network that HSF1 coordinates during heat-shock is known, but is unknown in malignancy. We compare HSF1 function in isogenic breast cancer cells of high and low malignant potential. HSF1 orchestrates a far-reaching transcriptional program that is dependent on transformation and the degree of malignancy. The oncogenic HSF1 program differs markedly from the classical heat-shock response. It is enriched for genes involved in a myriad of cellular processes known to be important in malignancy, including transcription, translation, glucose metabolism and cellular adhesion. It includes only a particular subset of heat-shock protein genes, and many of these are regulated in a manner that differs from their regulation by heat-shock. We also find that HSF1 is overexpressed and activated in a large subset of all conventionally defined classes of breast cancer. Moreover, tumors with high expression of the oncogenic HSF1 transcriptional network are strongly associated with poor outcome as monitored by metastasis and death. Our findings suggest that the oncogenic HSF1 activation program is rooted in fundamental aspects of tumor biology and will prove a powerful new tool in the clinical management of patients with breast cancer and, likely, other malignancies as well. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-09.

The "stemness" hypothesis states that different types of stem cells share a core set of mechanisms that regulate the shared stem cell properties of self-renewal and multi-lineage potential. Previous attempts to identify genes required for core stem cell function across stem cell types using transcriptional profiling have identified few such genes. We hypothesized that functional redundancy and tissue-specific expression of functionally redundant homologs mask common stem cell mechanisms. Using an unbiased, genome-wide computational screen of many publicly available mouse stem cell profiling experiments, we tested for shared differential expression across different stem cell types accounting for the possible tissue-specific expression of gene homologs. We found 103 evolutionarily related groups of homologous genes with reproducible, statistically significant, cell type diverse and stem cell-specific upregulation in multiple stem cell types. Shared homolog groups include previously identified self-renewal genes in the Myc, Myb, Chd and Cip/KIP families, as well as genes newly implicated in stem cell function. Our results suggest that different stem cells express distinct repertoires of genes that are functionally synonymous and point to specific examples of functional redundancy in pathways controlling cell adhesion, quiescence, and gene silencing. Genes within these homolog families are prime candidate regulators of conserved stemness mechanisms and may play critical roles as stem cell markers.

Background Understanding gene function and genetic relationships is fundamental to our efforts to better understand biological systems. Previous studies systematically describing genetic interactions on a global scale have either focused on core biological processes in protozoans or surveyed catastrophic interactions in metazoans. Here, we describe a reliable high-throughput approach capable of revealing both weak and strong genetic interactions in the nematode Caenorhabditis elegans. Results We investigated interactions between 11 'query' mutants in conserved signal transduction pathways and hundreds of 'target' genes compromised by RNA interference (RNAi). Mutant-RNAi combinations that grew more slowly than controls were identified, and genetic interactions inferred through an unbiased global analysis of the interaction matrix. A network of 1,246 interactions was uncovered, establishing the largest metazoan genetic-interaction network to date. We refer to this approach as systematic genetic interaction analysis (SGI). To investigate how genetic interactions connect genes on a global scale, we superimposed the SGI network on existing networks of physical, genetic, phenotypic and coexpression interactions. We identified 56 putative functional modules within the superimposed network, one of which regulates fat accumulation and is coordinated by interactions with bar-1(ga80), which encodes a homolog of β-catenin. We also discovered that SGI interactions link distinct subnetworks on a global scale. Finally, we showed that the properties of genetic networks are conserved between C. elegans and Saccharomyces cerevisiae, but that the connectivity of interactions within the current networks is not. Conclusions Synthetic genetic interactions may reveal redundancy among functional modules on a global scale, which is a previously unappreciated level of organization within metazoan systems. Although the buffering between functional modules may differ between species, studying these differences may provide insight into the evolution of divergent form and function.