Candidate Targets For Therapeutic Intervention Research Articles

Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli

Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention.

Abstract 4371: ERBB4 heterodimers appear to drive BRAFWT melanoma cell lines

Abstract Purpose of The Study: To determine whether heterodimerization of ERBB4 with EGFR or ERBB2 drives the proliferation of BRAF WT melanoma cell lines. Introduction: Metastatic melanomas that possess wild-type (WT) BRAF alleles are just as aggressive as melanomas that harbor gain-of-function (e.g., V600E) BRAF alleles. Moreover, no targets for effective chemotherapeutic intervention (other than immune checkpoint inhibitors) have been identified for these tumors. Thus, our goal is to identify candidate targets for therapeutic intervention in BRAF WT melanomas. ERBB4 (HER4) is a receptor tyrosine kinase that is closely related to the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). We have previously demonstrated that ERBB4 is both sufficient and necessary for the proliferation of BRAF WT melanoma cell lines. However, the constitutively homodimerized ERBB4 Q646C mutant inhibits the proliferation of BRAF WT melanoma cell lines. We have also previously demonstrated that ERBB4-EGFR and ERBB4-ERBB2 heterodimers stimulate proliferation and other oncogenic phenotypes in a variety of tumor model systems. Hence, here we test the hypothesis that ERBB4-EGFR or ERBB4-ERBB2 heterodimers drive BRAF WT melanomas. Experimental Approach: We are testing whether wild-type EGFR or ERBB2 alleles stimulate the proliferation of four different human BRAF WT melanoma cell lines. We are testing whether the dominant-negative EGFR (K721A) or ERBB2 (K753A) mutant alleles inhibit the proliferation of these cell lines. Likewise, we are testing whether EGFR or ERBB2 shRNAs inhibit the proliferation of these cell lines. Finally, we are testing whether EGFR or ERBB2 enables ERBB4 mutant alleles found in BRAF WT melanoma samples to drive greater levels of proliferation in 32D cells than wild-type ERBB4. Unpublished Results: Wild-type ERBB2 causes a significant increase in the proliferation of the MEL-JUSO and IPC-298 BRAF WT female melanoma cell lines. The ERBB2 dominant-negative allele causes a significant decrease in the proliferation of these cell lines. Finally, preliminary data suggest that the EGFR and ERBB2 dominant-negative alleles inhibit the proliferation of the MeWo BRAF WT male melanoma cell line. Preliminary Conclusions: Our data suggest that ERBB2-ERBB4 heterodimers drive two female BRAF WT melanoma cell lines, whereas ERBB2-ERBB4 and EGFR-ERBB4 heterodimers drive a male BRAF WT melanoma cell line. Thus, we predict that FDA-approved ERBB2 inhibitors (and in some cases, EGFR inhibitors) may be effective in ERBB4-dependent, BRAF WT melanomas. Moreover, ERBB4 heterodimerization with ERBB2 or EGFR may account for the gender disparities observed in BRAF WT melanomas. Citation Format: Vipasha Dwivedi, Lauren M. Lucas, Jen Davis, Kaitlyn O'Daniel, Rees H. Cooke, Joelle N. Woggerman, Ella N. Wilson, Maddy N. Ingrao, David J. Riese. ERBB4 heterodimers appear to drive BRAFWT melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4371.

Abstract 1689: Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts

Abstract Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, WNT, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents. Citation Format: John Landua, Ping Gong, Michael T. Lewis. Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1689.

Nkx2.8 promotes chemosensitivity in bladder urothelial carcinoma via transcriptional repression of MDR1

Multidrug resistance gene 1 (MDR1), a key factor contributing to drug insensitivity, has been associated with treatment failure and poor prognoses in various cancers, including bladder urothelial carcinoma (UC). Here we show that positive Nkx2.8 expression was associated with better prognosis of UC patients received chemotherapy. Patients with positive Nkx2.8 expression had promising prognosis from adjuvant chemotherapy. Enforced expression of Nkx2.8 promotes drug sensitivity of UC cells. Mechanistic investigations showed that Nkx2.8 negatively regulated expression of MDR1 by binds directly to the MDR1 promoter and transcriptionally represses MDR1 expression. P-gp inhibitor reversed chemosensitivity inhibition by Nkx2.8 scilencing. In clinical UC specimens, expression of Nkx2.8 inversely correlated with P-gp expression, and UC patients with Nkx2.8 positivity and low P-gp expression displayed the best prognosis. Our findings uncovered a new mechanism of chemosensitivity in UC cells and proposing Nkx2.8-MDR1 axis as a novel candidate target for therapeutic intervention of UC.

Detection of molecular signatures and pathways shared by Alzheimer's disease and type 2 diabetes.

Alzheimer's disease (AD) and type 2 diabetes (T2D) are common in the general elderly population, conferring heavy individual, social, and economic stresses on families and society. Accumulating evidence indicates T2D to be a risk factor for AD. However, the underlying mechanisms for this association are largely unknown. This study aimed to identify the shared molecular signatures between AD and T2D through integrated analysis of temporal cortex gene expression data. Gene Ontology (GO) and pathway enrichment analysis, protein over-representation analysis, protein-protein interaction, DEG-transcription factor interactions, DEG-microRNA interactions, protein-drug interactions, gene-disease association analysis, and protein subcellular localization analysis of the common DEGs were performed. We identified 16 common DEGs between the two datasets, which were mainly enriched in the biological processes of apoptosis, autophagy, inflammation, and hemostasis. We also identified five hub proteins encoded by the DEGs, five central regulatory transcription factors, and six microRNAs. Protein-drug interaction analysis showed C1QB to be associated with different drugs. Gene-disease association analysis revealed that hub genes, SFN and ITGB2, were actively engaged in other diseases. Collectively, these findings provide new insights into shared molecular mechanisms between AD and T2D and provide novel candidate targets for therapeutic intervention.

Tumor-associated macrophages based signaling pathway analysis and hub genes identification in glioma.

Tumor-associated macrophages (TAMs) play a crucial role in the immune response to many malignancies, but the signaling pathways by which the glioma microenvironment cross-talk with TAMs are poorly understood. The aim of this study was to uncover the potential signaling pathways of the regulation of TAMs and identify candidate targets for therapeutic intervention of glioma through bioinformatics analysis.Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) datasets were used to download RNA-Seq data and microarray data of human glioma specimen. Differentially expressed genes (DEGs) between CD68-high samples and CD68-low samples were sorted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs was conducted. Protein-protein interaction (PPI) network were formed to identify the hub genes.The prognostic value of TAMs in glioma patients was confirmed. A total of 477 specific DEGs were sorted. The signaling pathway was identified in pathway enrichment and the DEGs showed prominent representations of immune response networks in glioma. The hub genes including C3, IL6, ITGB2, PTAFR, TIMP1 and VAMP8 were identified form the PPI network and they were all correlated positively with the expression of CD68 and showed the excellent prognostic value in glioma patients.TAMs can be used as a good prognostic indicator in glioma patients. By analyzing comprehensive bioinformatics data, we uncovered the underlying signaling pathway of the DEGs between glioma patients with high and low expression level of CD68. Furthermore, the 6 hub genes identified were closely associated with TAMs in glioma microenvironment and need further investigation.

Identification of a TLR-Induced Four-lncRNA Signature as a Novel Prognostic Biomarker in Esophageal Carcinoma.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of Toll-like receptor (TLR) signaling to control innate immunity, and this regulatory mechanism has recently been implicated in esophageal carcinoma (ESCA). However, a comprehensive analysis of TLR-induced lncRNAs and their roles in diagnosis and prognosis in ESCA is still lacking. In this study, we first investigated the precise relationship between lncRNA perturbations and alteration of TLR signaling by constructing the lncRNA-TLRs co-expression network involved in ESCA, and identified 357 TLR-related lncRNAs. Of them, four TLR-related lncRNAs (AP000696.1, LINC00689, LINC00900, and AP000487.1) are significantly associated with the overall survival (OS) of ESCA patients, and utilizing this four-lncRNA signature is capable of stratifying patients into high-risk and low-risk groups with significantly different OS in the discovery set. Further analysis in different independent patient sets also confirmed the robustness of the prognostic value of the four-TLR-lncRNA signature in predicting the OS of ESCA patients. Moreover, the results of multivariate analysis in different patient sets indicated that the four-TLR-lncRNA signature is an independent factor after adjusted by other clinical factors. Thus, we have identified a TLR-induced four-lncRNA signature, which represents a promising prognosis biomarker for ESCA, and our study might provide new candidate targets for therapeutic intervention via targeting TLR-induced lncRNAs in ESCA patients.

Circulating inflammatory mediators as biomarkers of ocular toxoplasmosis in acute and in chronic infection.

Toxoplasmosis is highly endemic worldwide. In Brazil, depending on the geographical region and socioeconomic status, 40-70% of individuals become seropositive at some point in their lives. A significant proportion of Toxoplasma gondii-chronically infected individuals who are otherwise immunocompetent develop recurrent ocular lesions. The inflammatory/immune mechanisms involved in development of ocular lesion are still unknown and, despite previous investigation, there are no reliable immune biomarkers to predict/follow disease outcome. To better understand the impact of the immune response on parasite control and immunopathology of ocular toxoplasmosis, and to provide insights on putative biomarkers for disease monitoring, we assessed the production of a large panel of circulating immune mediators in a longitudinal study of patients with postnatally acquired toxoplasmosis stratified by the presence of ocular involvement, both at the early acute stage and 6 months later during chronic infection, correlating them with presence of ocular involvement. We found that T. gondii-infected patients, especially during the acute stage of the disease, display high levels of chemokines, cytokines, and growth factors involved in the activation, proliferation, and migration of inflammatory cells to injured tissues. In particular, major increases were found in the IFN-induced chemokines CXCL9 and CXCL10 in T. gondii-infected patients regardless of disease stage or clinical manifestations. Moreover, a specific subgroup of circulating cytokines and chemokines including GM-CSF, CCL25, CCL11, CXCL12, CXCL13, and CCL2 was identified as potential biomarkers that accurately distinguish different stages of infection and predict the occurrence of ocular toxoplasmosis. In addition to serving as predictors of disease development, these host inflammatory molecules may offer promise as candidate targets for therapeutic intervention.

Abstract P4-06-10: Lentiviral signaling reporters uncover tumor-initiating cell heterogeneity in breast cancer patient-derived xenografts

Abstract Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents. Citation Format: John Landua, Ping Gong, Michael Lewis. Lentiviral signaling reporters uncover tumor-initiating cell heterogeneity in breast cancer patient-derived xenografts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-10.

Metabolic Control of Astrocyte Pathogenic Activity via cPLA2-MAVS

Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention.

Abstract 3437: Requirement for YAP1 signaling in myxoid liposarcoma

Abstract Myxoid liposarcomas (MLS) account for 20% of malignant adipocytic tumors and are characterized by a high rate of local recurrence and development of distant metastases in approximately 40% of patients. Most MLS are driven by the FUS-DDIT3 fusion gene encoding an aberrant transcription factor. The mechanisms whereby FUS-DDIT3 mediates sarcomagenesis are incompletely understood, and strategies to selectively target MLS cells remain elusive. In this study, we employed genome-scale RNA interference (RNAi) screening to uncover that human mesenchymal stem cells engineered to express FUS-DDIT3 and MLS cell lines are dependent on YAP1, a transcriptional co-activator and central effector of the Hippo pathway involved in tissue growth and tumorigenesis. Analysis of a large cohort of primary MLS specimens (n=223) revealed that nuclear YAP1 expression was significantly more prevalent in MLS compared to other liposarcoma subtypes. In support of the concept that increased YAP1-mediated transcriptional activity represents an essential feature of MLS development, RNAi-based YAP1 depletion in cultured MLS cells resulted in suppression of cell viability, cell cycle arrest, cellular senescence, and induction of apoptosis accompanied by decreased YAP1 target gene expression, and YAP1-positive primary MLS tumors showed strong expression of YAP1 downstream effectors such as FOXM1 and PLK1. Mechanistically, FUS-DDIT3 promotes YAP1 transcription, nuclear localization, and transcriptional activity and physically associates with YAP1 in the nucleus of MLS cells, pointing to the coordinate establishment of gene expression programs that promote MLS tumorigenesis. Consistent with the hypothesis that a YAP1-directed therapeutic approach could represent a rational strategy to selectively target FUS-DDIT3-expressing MLS cells, pharmacologic inhibition of YAP1 activity with verteporfin suppressed cell viability and YAP1 target gene expression in MLS cell lines, and the growth-inhibitory effects of YAP1 knockdown or verteporfin treatment could be recapitulated in MLS cell line-based xenograft models. Collectively, our data identify dependence on aberrant YAP1 activity as specific liability of FUS-DDIT3-expressing MLS cells, and provide preclinical evidence that YAP1-mediated signal transduction represents a candidate target for therapeutic intervention that warrants further investigation. Citation Format: Marcel Trautmann, Ya-Yun Cheng, Patrizia Jensen, Ninel Azoitei, Ines Brunner, Jennifer Hüllein, Mikolaj Slabicki, Ilka Isfort, Magdalene Cyra, Eva Wardelmann, Sebastian Huss, Bianca Altvater, Claudia Rossig, Susanne Hafner, Thomas Simmet, Anders Ståhlberg, Pierre Åman, Thorsten Zenz, Undine Lange, Thomas Kindler, Claudia Scholl, Wolfgang Hartmann, Stefan Fröhling. Requirement for YAP1 signaling in myxoid liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3437.

Genetic Insights Into Smooth Muscle Cell Contributions to Coronary Artery Disease.

Coronary artery disease is a complex cardiovascular disease involving an interplay of genetic and environmental influences over a lifetime. Although considerable progress has been made in understanding lifestyle risk factors, genetic factors identified from genome-wide association studies may capture additional hidden risk undetected by traditional clinical tests. These genetic discoveries have highlighted many candidate genes and pathways dysregulated in the vessel wall, including those involving smooth muscle cell phenotypic modulation and injury responses. Here, we summarize experimental evidence for a few genome-wide significant loci supporting their roles in smooth muscle cell biology and disease. We also discuss molecular quantitative trait locus mapping as a powerful discovery and fine-mapping approach applied to smooth muscle cell and coronary artery disease-relevant tissues. We emphasize the critical need for alternative genetic strategies, including cis/trans-regulatory network analysis, genome editing, and perturbations, as well as single-cell sequencing in smooth muscle cell tissues and model organisms, under both normal and disease states. By integrating multiple experimental and analytical modalities, these multidimensional datasets should improve the interpretation of coronary artery disease genome-wide association studies and molecular quantitative trait locus signals and inform candidate targets for therapeutic intervention or risk prediction.

S128 BCP‐ AND T‐ALL CELLS HIDE IN DISTINCT NICHES OF THE BONE MARROW

Background:Persistence of miminal residual disease (MRD) is a strong indicator for drug resistance in acute lymphoblastic leukemia (ALL). The contribution of the bone marrow microenvironment to resistance remains incompletely understood. In‐vivo imaging in a murine model of T‐cell ALL (T‐ALL) infirm the existence of distinct predilection sites for leukemia cells in the bone marrow. A recent report based on experiments with patient‐derived‐xenografts (PDX) has suggested a role for a dormant subpopulation in B‐cell precursor ALL (BCP‐ALL), possibly in vicinity to the bone endothelium, to confer chemoresistence.Aims:We here set out to compare the topology of the bone marrow microenvironment in primary patient‐derived BCP‐ and T‐ALL under therapeutic pressure in PDX models.Methods:We established a PDX model of ALL induction chemotherapy with Dexamethasone, Vincristine and Doxorubicin in NOD/scid ILR2gamma null mice and analyzed the distribution of residual ALL cells in relation to bone and vascular structures using 3D confocal immunofluorescence microscopy of different bones at specific timepoints before and after treatment. We used cell‐type specific immunofluorescence staining combined with 3D confocal z‐stack tiling and applied distance analysis algorithms to define the cellular components of the leukemia niche.Results:Analyzing long bones from 5 BCP‐ALL and 3 T‐ALL PDX both at early engraftment (4 or 11 days after transplantation) and at MRD (after induction chemotherapy), we show that BCP‐ALL cells are located in close contact to the extravascular side of endothelial cells of bone marrow sinusoids. BCP‐ALL cells were enriched within a distance below 5 μm from endothelial cells and reproducibly more distant from arterioles and transition zones. In contrast, T‐ALL PDX at early engraftment and after induction chemotherapy were spread over the bone marrow with a marked tendency to cluster close to the endosteum after induction chemotherapy (∼30 % of all T‐ALL cells). We next attempted to identify subsets with slow cell cycle rates using Carboxyfluoresceinsuccinimidylester (CFSE) labelled ALL PDX. In our model of induction chemotherapy, proliferation of BCP‐ as well as T‐ALL cells was transiently decelerated. However, we could not detect any persistence of a reproducible population with reduced proliferative activity, challenging the notion that a dormant ALL subpopulation can be detected by this approach. Moreover, residual CFSE retaining cells after chemotherapy localized next to bone marrow sinusoids for BCP‐ALL and more widespread within the bone marrow for T‐ALL. Finally, we confirm an increase of CFSE retention in distal adipocytic rich yellow bone marrow (e.g. tail vertebra) compared to the red bone marrow of long bones, suggesting differences that may impact on the activity of therapeutic agents targeting rapidly cycling leukemia cells.Summary/Conclusion:Our data reconcile previous reports by revealing robust differences between BCP‐ and T‐ALL with respect to the topology of the leukemia niche in MRD. BCP‐ALL MRD was detected mostly in the perivascular space, while T‐ALL MRD was less specifically distributed with some clusters at the endosteum. This provides the basis to investigate molecular determinants of BCP‐ALL interactions with the vascular compartment, which may leverage new candidate targets for therapeutic intervention.

Prognostic significance of LAPTM4B and p27kip1 expression in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is associated with an aggressive phenotype and poor prognosis, and the lack of druggable markers leads to the unavailability of targeted therapies. Thus, there is an urgent need to identify potential targets for triple-negative breast cancer. In this study, we aimed to explore the expression of LAPTM4B and p27kip1 in triple-negative breast cancer, and its clinical significance. We analyzed the expression and association of LAPTM4B and p27kip1 in human breast cancer databases. To analyze the role of LAPTM4B in the aggressiveness of the human triple-negative breast cancer, the expressions of LAPTM4B were knocked down in MDA-MB-231 and HCC1187 cell lines. Then, cell proliferation, migration and apoptosis were assessed in vitro. Furthermore, the immunohistochemistry examinations of LAPTM4B and p27kip1 expression were performed using surgical specimens from 188 primary triple-negative breast cancer patients. Through analyses of several independent breast cancer cohorts, we found the correlation of the LAPTM4B and p27kip1 expression. Remarkably, the knockdown of LAPTM4B restored p27kip1 expression and inhibited the aggressiveness of breast cancer cells. Meanwhile, the knockdown of p27kip1 relieved the suppression of cell migration. Consistent with the analyses of human breast cancer cohorts, the immunohistochemistry results showed that the expression levels of LAPTM4B and p27kip1 were correlated in 188 triple-negative breast cancer samples (p= 0.019). We also validated that the higher LAPTM4B expression, the lower p27kip1 expression (p= 0.0001), and the LAPTM4B+/p27kip1- subgroup (p< 0.0001) were poor prognostic indicators, as well as the higher histologic grade (p= 0.0001). In the multivariate Cox regression, p27kip1 expression was considered as an independent predictor of survival (p< 0.001). The overexpression of LAPTM4B and the loss of p27kip1 expression are correlated. Meanwhile, the up-regulated expression of LAPTM4B together with the down-regulated expression of p27kip1 could classified a group of breast cancer patients with poor prognosis, consequently considered as a potentially prognostic marker and candidate target for therapeutic intervention of triple-negative breast cancer.

Triglyceride-Rich Lipoproteins.

Plasma triglyceride levels are causally related to an increased risk of cardiovascular disease. Here, the authors summarize their understanding of triglyceride-rich lipoprotein metabolism, previous and newly identified regulators, and their relevance as candidate targets for therapeutic intervention.

Nkx2.8 Inhibits Epithelial-Mesenchymal Transition in Bladder Urothelial Carcinoma via Transcriptional Repression of Twist1.

Epithelial-to-mesenchymal transition (EMT) promotes metastasis, which is the main cause of bladder urothelial carcinoma-related death. Loss of the candidate tumor-suppressor gene Nkx2.8 has been associated with urothelial carcinoma lymph node metastasis. Here, we show that enforced expression of Nkx2.8 is sufficient to inhibit EMT, reduce motility, and blunt invasiveness of urothelial carcinoma cells. Mechanistic investigations showed that Nkx2.8 negatively regulated expression of the EMT inducer Twist1 in urothelial carcinoma cells, at both the level of mRNA and protein accumulation. Nkx2.8 bound directly to the promoter region of this gene and transcriptionally repressed its expression. Twist1 upregulation reversed EMT inhibition by Nkx2.8, restoring the invasive phenotype of urothelial carcinoma cells. In clinical urothelial carcinoma specimens, expression of Nkx2.8 inversely correlated with Twist1 expression, and urothelial carcinoma patients with Nkx2.8 positivity and low Twist1 expression displayed the best prognosis. Our findings highlight the Nkx2.8-Twist1 axis as candidate target for therapeutic intervention in advanced urothelial carcinoma.Significance: These findings highlight a novel EMT signaling axis as a candidate target for therapeutic intervention in advanced urothelial carcinomas. Cancer Res; 78(5); 1241-52. ©2018 AACR.

Abstract 5583: Integrative genomic analyses on pediatric acute myeloid leukemia

Abstract Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide range of response variability to conventional therapy, excessive treatment related toxicity, and an overall poor outcome. The NCI supported Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative provided initial support for large-scale genomics to identify novel disease-associated genomic and epigenomic alterations that could be used to develop novel, targeted therapies for pediatric AML. Patients Diagnostic (Dx), remission (Rm), and relapse (Rel) samples were obtained from over 200 children with AML treated on the most recent Children's Oncology Group clinical trials. Data analyses were performed using Partek Genomics Suite 6.6 and Ingenuity Pathway Analysis. Results Methylation profiling (Illumina HumanMethylation27) identified 603 CpG sites significantly differentially methylated between Dx and Rm. In addition, 514 CpG sites were significantly differentially methylated between matched Rel and Rm (p&amp;lt;0.05 FDR, change in β≥0.25). Pathway analysis using genes associated with these CpG sites identified significant (p&amp;lt;0.01) over-representation of the WNT/β-catenin signaling pathway in Dx vs. Rm and in Rel vs. Rm. Gene expression profiling (Affymetrix Human Gene 1.1 ST) revealed 1,142 significantly differentially expressed between Dx and normal bone marrow obatined from the blood bank. Similarly, there were 1,242 significantly differentially expressed genes between Rel and normal bone marrow (p&amp;lt;0.05 FDR, fold-change≥2.00). Many expression differences were coordinated with methylation changes. Pathway analysis revealed 100 significantly (p&amp;lt;0.05) over-represented pathways in Dx vs. normal, Rel vs. normal, or both. Copy-number data analysis (Affymetrix Genome-Wide Human SNP 6.0) identified 180,379 significantly different regions in 254 Dx vs matched Rm. Similarly, 4,707 regions a significantly different in 49 Rel vs matched Rm (p&amp;lt;0.01). Many regions were distinct to either Dx or Rel groups. Whole-genome sequencing (Complete Genomics, Inc) was performed on 100 cases with matched Dx and Rm pairs and on 86 cases with matched Dx, Rel, and Rm samples. The frequencies of either somatic or germline variants were not significantly different when comparing Dx vs. Rel samples. Pathway analysis compared 1,528 mutated genes in the pediatric (TARGET) cohort with 1,963 mutated genes in adult de novo AML (TCGA cohort). Of the 85 and 80 significantly (p&amp;lt;0.05) over-represented canonical pathways, respectively, there are 44 pathways in common and many could be targeted by existing drugs. Conclusion A combination of genomic approaches have identified genes and pathways that appear deregulated in pediatric AML. Many changes are specific to Dx or Rel sample groups, or to pediatric vs. adult AML. Further study into these genes and pathways may reveal candidate targets for therapeutic intervention. Citation Format: Daniel H. Wai, Rhonda E. Ries, Michael Considine, David W. Lee, Sun-Mei Liu, Nicole M. O'Connor, Oliver Pepper, Ranjan Bista, Eiman Aleem, David O. Azorsa, Tanja M. Davidsen, Toni Farley, Jaime Guidry-Auvil, Jason E. Farrar, Malcolm A. Smith, Daniela S. Gerhard, MIchael F. Ochs, Spyro Mousses, Soheil Meshinchi, Robert J. Arceci. Integrative genomic analyses on pediatric acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2014-5583

Highlights of This Issue

Epithelial ovarian cancer (EOC) is one of the most aggressive forms of cancer, with few effective treatments and a five-year survival of only ∼ 40%. To identify potentially useful therapies, a meta-analysis of multiple siRNA screens was performed, which identified HSP90 as a major hub controlling drug resistance. Ganetespib is a second generation HSP90 inhibitor that is showing considerable promise in clinical trials. In multiple mouse models, ganetespib had single-agent activity and also strongly enhanced the activity of paclitaxel, a standard cytotoxic agent used for EOC treatment, suggesting the potential value of clinical assessment of this drug combination.Integration of transcriptomics and metabolomics is a novel approach to reveal the underlying mechanisms of tumor progression and to identify potential therapeutic targets. Zhang and colleagues have used this approach to investigate the mechanism of disease aggressiveness in pancreatic cancer and showed that an impaired lipolytic network involving four lipases and their metabolites play an important role in the progression of pancreatic cancer and are candidate targets for therapeutic intervention. The article shows that the integration of “omics” data provides a system-level perspective of pancreatic cancer that could facilitate the development of novel treatment strategy.This article reports that epidermal growth factor receptor (EGFR) over-expression is frequently observed in follicular dendritic cell sarcoma (FDC-S). In a patient-derived short term culture, the engagement of surface EGFR by EGF drives the survival and proliferation of tumor cells via MAPK and STAT pathways. Remarkably, EGFR ligands, and Betacellulin (BTC) in particular, are produced in the tumor microenvironment of FDC-S. Lack of somatic mutations of KRAS, NRAS, BRAF, and PI3KCA in FDC-S tumor samples predicts sensitivity to anti-EGFR monoclonal antibody and suggests possible candidates for EGFR blockade in this neoplasm.The drug-metabolizing enzyme and transporter (DMET)-platform is capable of detecting 1,936 genetic variants in 225 metabolizing enzyme and drug transporter genes. de Graan and colleagues developed a genetic prediction model for paclitaxel pharmacokinetics based on the DMET-platform. In the validation set, the model had a 95% sensitivity to identify patients with a low paclitaxel clearance. The positive predictive value was 22%. After multivariate analysis, the model remained associated with low paclitaxel clearance.

A TRP among the astrocytes

TRP channels were first identified as membrane proteins mediating phototransduction in fruit flies. Astrocytes were initially referred to as the silent elements of the nervous system. At the time these discoveries were made, few would have suspected TRP channels and astrocytes could contribute significantly to our understanding of brain signalling. Recent findings, however, put TRP channels and astrocytes in the spotlight, describe their ability to modulate the activity of specific sets of synapses, and raise some interesting questions. What makes astrocytes capable of exerting cell-specific effects on interneuronal signals? How do different synapses respond to changes in astrocytic function and in the local micro-structure of the neuropil? Can astrocytes be considered good candidate targets for therapeutic intervention to treat neurological diseases? Here I discuss the recent developments on TRP channels and astrocytes that have made us aware of the many structural and functional features of synapses that still need to be discovered and that could lead a new avant-garde in decoding the cellular and molecular basis of brain (dys)function.

A Chemical Screen Identifies Class A G-Protein Coupled Receptors As Regulators of Cilia

Normal cilia length and motility are critical for proper cellular function. Prior studies of the regulation of ciliary structure and length have primarily focused on the intraflagellar transport machinery and motor proteins required for ciliary assembly and disassembly. However, several mutants with abnormal length flagella highlight the importance of signaling proteins as well. In this study, an unbiased chemical screen was performed to uncover signaling pathways that are critical for ciliogenesis and length regulation using flagella of the green alga Chlamydomonas reinhardtii as a model. The annotated Sigma LOPAC1280 chemical library was screened for effects on flagellar length, motility, and severing as well as cell viability. Assay data were clustered to identify pathways regulating flagella. The most frequent target found to be involved in flagellar length regulation was the family of dopamine binding G-protein coupled receptors (GPCRs). In mammalian cells, cilium length could indeed be altered with expression of the dopamine D1 receptor. Our screen thus reveals signaling pathways that are potentially critical for ciliary formation, resorption, and length maintenance, which represent candidate targets for therapeutic intervention of disorders involving ciliary malformation and malfunction.