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Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING-41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. We report that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function. Using western blots, we found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, we discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFβ signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN-γ, TNF-α, and TRAIL production. Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1/L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations. To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1 beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC.

Background Cigarette smoke exposure increases the risk of developing Acute Respiratory Distress Syndrome (ARDS) after a number of disparate insults 1-3. It is not known why some at risk individuals develop ARDS, while others do not. Based on our previous finding that smoke-exposed AKR mice were more susceptible to lipopolysaccharides (LPS)-induced Acute Lung Injury (ALI) than C57BL/6 mice4, we investigated strain-dependent lung transcriptomic responses to cigarette smoke exposure. Methods Eight week old male AKR and C57BL/6 mice were exposed to 3 weeks of room air (RA) or cigarette smoke (CS) for 6 hours per day, 4 days per week, followd by intra-tracheal instillation of LPS or normal saline (NS) and microarray analysis of lung homogenate gene expression. Other groups of AKR and C57 mice were similarly exposed to RA or CS for 6 weeks, followed by evaluation of lung mechanics, pathology, and gene expression. Results Transcriptomic analyses of lung homogenates show distinct strain dependent lung transcriptional responses to CS and LPS, with AKR mice having larger numbers of genes affected than similarly treated C57 mice, congruent with strain differences in physiologic and inflammatory parameters previously observed in LPS-induced ALI after CS priming. These results suggest that genetic differences may underlie differing susceptibility of smokers to ARDS and emphysema. Conclusions Strain-based differences in gene transcription contribute to CS and LPS induced lung injury. Implications There may be a genetic basis for smoking related susceptibility to lung injury. Clinicians should consider cigarette smoke exposure as a risk factor for ALI and ARDS.

Inhibition of GSK-3 using small-molecule elraglusib has shown promising preclinical antitumor activity. Using in vitro systems, we found that elraglusib promotes immune cell-mediated tumor cell killing, enhances tumor cell pyroptosis, decreases tumor cell NF-κB-regulated survival protein expression, and increases immune cell effector molecule secretion. Using in vivo systems, we observed synergy between elraglusib and anti-PD-L1 in an immunocompetent murine model of colorectal cancer. Murine responders had more tumor-infiltrating T-cells, fewer tumor-infiltrating Tregs, lower tumorigenic circulating cytokine concentrations, and higher immunostimulatory circulating cytokine concentrations. To determine the clinical significance, we utilized human plasma samples from patients treated with elraglusib and correlated cytokine profiles with survival. Using paired tumor biopsies, we found that CD45+ tumor-infiltrating immune cells had lower expression of inhibitory immune checkpoints and higher expression of T-cell activation markers in post-elraglusib patient biopsies. These results introduce several immunomodulatory mechanisms of GSK-3 inhibition using elraglusib, providing a rationale for the clinical evaluation of elraglusib in combination with immunotherapy.Statement of significancePharmacologic inhibition of GSK-3 using elraglusib sensitizes tumor cells, activates immune cells for increased anti-tumor immunity, and synergizes with anti-PD-L1 immune checkpoint blockade. These results introduce novel biomarkers for correlations with response to therapy which could provide significant clinical utility and suggest that elraglusib, and other GSK-3 inhibitors, should be evaluated in combination with immune checkpoint blockade.

The RNA lariat debranching enzyme is the sole enzyme responsible for hydrolyzing the 2′–5′ phosphodiester bond in RNA lariats produced by the spliceosome. Here we test the ability of Dbr1 to hydrolyze branched RNAs (bRNAs) which contain a 2′–5′-phosphorothioate linkage, a modification commonly used to resist degradation. We attempted to co-crystallize a phosphorothioate branched RNA (PS-bRNA) with wild-type Entamoeba histolytica Dbr1 (EhDbr1) but observed in-crystal hydrolysis of the phosphorothioate bond. The crystal structure revealed EhDbr1 in a product-bound state, with the hydrolyzed 2′–5′ fragment of the PS-bRNA mimicking the binding mode of the native bRNA substrate. These findings suggest that product inhibition may contribute to the kinetic mechanism of Dbr1. We show that Dbr1 enzymes cleave phosphorothioate linkages at rates ~10,000-fold more slowly than native phosphate linkages. This new product-bound crystal structure offers atomic details which can aid inhibitor design. Dbr1 inhibitors could be therapeutic or investigative compounds for human diseases such as HIV, ALS, cancer, and viral encephalitis.

Colorectal cancer (CRC) caused over 900,000 deaths worldwide in 2020. A majority of late-stage CRC patients are treated with 5-fluorouracil (5-FU) combined with either irinotecan (CPT-11), oxaliplatin, or both. Despite their widespread use, the mechanisms of efficacy and toxicity of these drugs remain incompletely understood. While previous work has investigated cellular responses to these agents individually, we directly compare the transcriptomic and cytokine profiles of HCT116 wild-type and p53−/− colorectal cancer cells treated with these drugs and report pan-drug, drug-specific, drug class-specific, p53-independent, and p53-dependent signatures. We observed downregulation of histone genes by 5-FU (that significantly correlates with improved survival in CRC patients) and upregulation of FOS and ATF3 by oxaliplatin (which may contribute to peripheral neuropathy). BTG2 was identified as a top gene upregulated by all four drugs, suggesting its critical role in the cellular response to chemotherapy in CRC. Soluble TRAILR2 (death receptor 5; DR5) is a decoy receptor for TRAIL, an apoptosis-inducing cytokine. TRAILR2 was down-regulated by oxaliplatin and 5-FU, was not affected by CPT-11, and was increased by cisplatin. There was an increase in IL-8 by oxaliplatin and increase in ferritin by cisplatin which may contribute to cancer cell survival. Novel drug-specific mechanisms of efficacy or toxicity identified in these signatures may be targeted with combination therapies or development of new targeted therapies. Together, the findings here contribute to our understanding of the molecular bases of efficacy and toxicity of chemotherapeutic agents often used for treatment of GI cancer such as CRC.

While the bone marrow (BM) microenvironment is significantly remodelled in acute myeloid leukaemia (AML), molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma. We assessed the effect of anthracycline- and cytarabine-based induction chemotherapy on both haematopoietic and non-haematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis. We compared matched human core BM biopsies at diagnosis and 2 weeks after cytarabine- and anthracycline-based induction therapy in responders (5% blasts present after treatment) and non-responders (≥5% blasts present after treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2 (SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1) and osteonectin (SPARC) was observed in non-responders. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimised therapeutic strategies for AML.

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase with key roles in myriad biological processes such as tumor progression, and inhibition of GSK-3 using the small molecule elraglusib has shown promising preclinical antitumor activity in multiple tumor types. Our preclinical experiments showed that elraglusib treatment increased tumor cell PD-L1 expression, downregulated angiogenic and immunosuppressive signaling pathways, and increased anti-tumor immune responses in vitro and in vivo. Subsequent studies showed that several circulating factors were predictive of response to PD-1/PD-L1 blockade and GSK-3 inhibition in a murine model of colorectal cancer. To determine the translational relevance of our prior results we evaluated tumor biopsies and plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib (NCT03678883). Plasma samples were collected from patients at baseline and 24 hours post-IV administration of elraglusib and were analyzed using Luminex technology. Paired FFPE tumor biopsies from patients with colorectal or pancreatic cancer before and after treatment were selected to analyze the tumor microenvironment using NanoString GeoMx DSP technology. The region of interest (ROI) selection strategy focused on mixed tumor and immune cell segments and ROIs were segmented using panCK+ and CD45+ morphology stains. Cytokine analysis revealed that elevated baseline plasma levels of IL-1 beta and reduced levels of VEGF correlated with improved progression-free survival (PFS) and overall survival (OS). PFS was also found to be positively correlated with elevated plasma levels of immunostimulatory analytes such as Granzyme B, IFN-gamma, and IL-2 at 24 hours post-treatment with elraglusib. CD45+ tumor-infiltrating immune cells had lower expression of VISTA, PD-1, and IDO-1 inhibitory checkpoint proteins and higher expression of OX40L and B7-H3 stimulatory checkpoint proteins in post-treatment biopsies as compared to pre-treatment biopsies. Moreover, time-on-study length negatively correlated with CD39 expression in PanCK+ segments and positively correlated with CD163 expression in CD45+ segments. This ongoing study, to our knowledge, represents the first digital spatial analysis of tumor biopsies from patients treated with elraglusib. These novel circulating biomarkers of response to GSK-3 inhibition could provide significant clinical utility and the spatial proteomics data may give us insights into the immunomodulatory mechanisms of GSK-3 inhibition. Citation Format: Kelsey E. Huntington, Christoph Schorl, Shaolei Lu, Daniel Newhouse, Benedito A. Carneiro, Wafik S. El-Deiry. Multiplex digital spatial profiling (DSP) of proteins in the tumor microenvironment in response to GSK-3 inhibition by 9-ING-41 (elraglusib) correlates with novel immunostimulatory effects observed in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5636.

Extracellular vesicles (EVs) are excreted from all cells in the human body and are heterogeneous lipid bilayer particles containing proteins, RNA, DNA, and other cargo. T cells are primary players of the adaptive immune system and have a significant role in anti-cancer immunotherapies. Tumor derived EVs have diverse effects on host cells including modulating the immune response. In this study, we investigate the role acute myeloid leukemia (AML) derived EVs have on modulation of gene expression levels in three different T cell populations (CD8+, CD4+, and CD4 + CD39 + T cells). AML derived EVs modulate gene expression levels in all three cell populations with transcripts associated with major immunoregulatory pathways being significantly altered. Genes whose expression were significantly upregulated or downregulated were analyzed for gene ontogeny category expression and for the impact of the changes on specific immunoregulatory pathways. CD8 + T cells were modulated to a larger extent than other T cell populations and gene expression levels associated with proliferation and differentiation were influenced by AML-derived EVs.

Background Mesenchymal stem cell–derived extracellular vesicles (EVs) promote angiogenesis in the ischemic myocardium. This study examines the difference in vascular density, myocardial perfusion, molecular signaling, and gene expression between normal diet (ND) and high fat diet (HFD) groups at baseline and following intramyocardial injection of EVs. Methods and Results Intact male Yorkshire swine fed either an ND (n=17) or HFD (n=14) underwent placement of an ameroid constrictor on the left circumflex coronary artery. Subsequently, animals received either intramyocardial injection of vehicle‐saline as controls; (ND‐controls n=7, HFD‐controls, n=6) or EVs; (ND‐EVs n=10, HFD‐EVs n=8) into the ischemic territory. Five weeks later, myocardial function, perfusion, vascular density, cell signaling, and gene expression were examined. EVs improved indices of myocardial contractile function, myocardial perfusion, and arteriogenesis in both dietary cohorts. Interestingly, quantification of alpha smooth muscle actin demonstrated higher basal arteriolar density in HFD swine compared with their ND counterparts; whereas EVs were associated with increased CD31‐labeled endothelial cell density only in the ND tissue, which approached significance. Levels of total endothelial nitric oxide synthase, FOXO1 (forkhead box protein O1) , transforming growth factor‐β, phosphorylated VEGFR2 (vascular endothelial growth factor receptor 2), and phosphorylated MAPK ERK1/ERK2 (mitogen‐activated protein kinase) were higher in ischemic myocardial lysates from ND‐controls compared with HFD‐controls. Conversely, HFD‐control tissue showed increased expression of phosphorylated endothelial nitric oxide synthase, phosphorylated FOXO1, VEGFR2, and MAPK ERK1/ERK2 with respect to ND‐controls. Preliminary gene expression studies indicate differential modulation of transcriptional activity by EVs between the 2 dietary cohorts. Conclusions HFD produces a profound metabolic disorder that dysregulates the molecular mechanisms of collateral vessel formation in the ischemic myocardium, which may hinder the therapeutic angiogenic effects of EVs.

The tumor microenvironment regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. Increased expression of type X collagen α-1 (ColXα1) in tumor-associated stroma correlates with poor pathologic response to neoadjuvant chemotherapy in estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Evaluation of ColXα1 expression patterns suggests a potential connection with elastin fibers. To investigate the possible interaction between ColXα1 and elastin, we evaluated the expression of ColXα1 in relation to elastin fibers in normal breast tissue, ductal carcinoma in situ, and invasive breast carcinomas at cellular and subcellular levels. Our findings demonstrate that ColXα1 colocalizes with elastin in invasive breast cancer-associated stroma by immunohistochemistry, immunofluorescence, and electron microscopy. In 212 invasive breast carcinomas, this complex was aberrantly and selectively expressed in tumor extracellular matrix in 79% of ER+/HER2-, 80% of ER+/HER2+, 76% of ER-/HER2+, and 58% of triple negative breast cancers. In contrast, ColXα1 was generally absent, while elastin was present perivascularly in normal breast tissue. ColXα1 and elastin were coexpressed in 58% of ductal carcinoma in situ (DCIS) in periductal areas. In mass-forming DCIS with desmoplastic stroma, the complex was intensely expressed in periductal areas as well as within the tumor-associated stroma in all cases. Our data suggest that the breast carcinoma neoplastic process may involve aberrant expression of ColXα1 and elastin in the tumor microenvironment emerging early at the DCIS stage. Enrichment of these complexes in tumor-associated stroma may represent a stromal signature indicative of intrinsic differences between breast cancers. These findings shed light on investigation into the role of aberrant collagen complex expression in tumorigenesis and tumor progression which may be leveraged in therapeutic and theranostic applications.

Exposure to excess retinoic acid (RA) disrupts the development of the mammalian testicular seminiferous cord. However, the molecular events surrounding RA-driven loss of cord structure have not previously been examined. To investigate the mechanisms associated with this adverse developmental effect, fetal rat testes were isolated on gestational day 15, after testis determination and the initiation of cord development, and cultured in media containing all-trans RA (ATRA; 10(−8) to 10(−6) M) or vehicle for 3 days. ATRA exposure resulted in a concentration-dependent decrease in the number of seminiferous cords per testis section and number of germ cells, assessed by histopathology and immunohistochemistry. Following 1 day of culture, genome-wide expression profiling by microarray demonstrated that ATRA exposure altered biological processes related to retinoid metabolism and gonadal sex determination. Real-time RT-PCR analysis confirmed that ATRA enhanced the expression of the key ovarian development gene Wnt4 and the antitestis gene Nr0b1 in a concentration-dependent manner. After 3 days of culture, ATRA-treated testes contained both immunohistochemically DMRT1-positive and FOXL2-positive somatic cells, providing evidence of disrupted testicular cell fate maintenance following ATRA exposure. We conclude that exogenous RA disrupts seminiferous cord development in ex vivo cultured fetal rat testes, resulting in a reduction in seminiferous cord number, and interferes with maintenance of somatic cell fate by enhancing expression of factors that promote ovarian development.

Heterogeneity in the p53 response to traditional chemotherapy is recognized but its significance has yet to be exploited in the clinic. Further investigation could provide insight into the relative importance of individual p53 target genes and identify unique characteristics of specific drugs, guiding therapy selection. Wild-type or p53 null HCT-116 cells were treated with oxaliplatin, cisplatin, CPT-11 (irinotecan), or 5-Fluorouracil (5-FU) at an IC50 dose for 8 hours, in triplicate. Total cellular RNA was isolated and Clariom™ D microarrays were used to measure changes in RNA expression relative to untreated cells. TAC software was used to calculate fold-changes and statistical significance. Western blot was used to confirm treatment-induced increase in p53 and to further investigate p53 target heterogeneity at the protein level. The top 100 p53-dependent genes upregulated (FC>1.5) by each drug were identified, which revealed BTG2 as the top upregulated gene after oxaliplatin, cisplatin, and 5-FU treatment. Eight genes appeared in all four lists (GPR87, FAS, TP53INP1, DCP1B, POLH, PDGFA, MDM2, and PHLDA3) but variations in magnitude of change across drugs were noted for several including GPR87, FAS, TP53INP1, and MDM2. Moreover, heterogeneity in the magnitude of change of p53 targets was observed by western blot, which also showed differential upregulation of p21 and DR5 across drug treatment conditions. The lists contained numerous genes that were unique to each treatment, with oxaliplatin regulating the highest number of unique genes (31/100). Several transcripts outside of the top 100 were also identified as unique in each drug treatment including BAX, which showed slight preferential upregulation by 5-FU. Evaluation of the p53-independent transcriptional regulation revealed a strikingly significant and unique regulation of histone modifications by 5-FU compared to the other treatments. Beyond the specific genes mentioned, variation in the magnitude of change and identification of the unique targets after each drug treatment highlights potentially important mechanistic differences between the chemotherapeutic drugs. The appearance of eight transcripts in the top 100 upregulated genes for all four drug treatments indicates their importance as well as the contribution of the p53 response to the molecular effects of chemotherapy. We are currently further evaluating the relevance of these genes and their unique contributions to the action of the chemotherapeutic drugs. Citation Format: Lindsey Carlsen, Lanlan Zhou, Liz J. Hernández Borrero, Arunasalam Navaraj, Shengliang Zhang, Christoph Schorl, Wafik S. El-Deiry. The transcriptional response in colorectal cancer cells varies across four clinically used chemotherapeutic drugs in terms of gene identity, magnitude of change, and p53 dependence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 999.

Background: Acute Myeloid Leukemia (AML) has a five-year survival rate of 25% and its high mortality is linked to poor response to treatment and relapse. Our understanding of the molecular mechanisms controlling relapse and AML progression is limited. Animal models indicate that AML cells significantly modulate their bone marrow microenvironment inducing gradual loss of endosteal and vascular niches, both playing critical roles in support and maintenance of normal hematopoiesis. The goal of this study was to determine microenvironmental factors driving the gradual retraction of endosteal and vascular niches directly in the AML core bone marrow biopsies, and assess the treatment effect on hematopoietic and non-hematopoietic cells. Methods: Transcriptomics and histopathologic evaluations of matched human AML core bone marrow biopsies obtained at diagnosis (n=12) and day 14 post-induction therapy (n=12) with daunorubicin and cytarabine (7+3) were performed. Based on post-treatment frequency of blasts in the AML bone marrow aspirate, patients were classified as responders ( 5% blasts). Three of 6 responders (3 men, 3 women, average age 59 yrs) had normal karyotype, and three of 6 non-responders (1 man, 5 women, average age 52.6 yrs), had normal karyotype. RNA was isolated from the core bone marrow biopsies and subjected to Clariom D Human Affymetrix arrays. Transcriptomics data were analyzed using Affymetrix Transcriptome Analysis Console with LIMMA R package and Gene Set Enrichment Analysis (GSEA). H&E stained bone marrow biopsy slides were subjected to blinded histopathological assessment. Results: Transcriptomic data analysis of responder vs. non-responder samples at diagnosis indicated significant loss of transcripts associated with heme metabolism (HBB, HBD, GYPE, CA1) suggesting decrease in frequency of erythroid progenitors (Fig.A). Trends of decreased frequency of erythroid progenitors were noted in both bone marrow biopsies and aspirates of diagnostic non-responder samples (Fig.B). Decreased frequency of lymphoid cells was also noted (Fig.B). Interestingly, while post-treatment we noted a relative increase in frequencies of lymphoid cells in both responder and non-responder samples, the increase was more prominent in responders (Fig.B). Trilineage hematopoiesis appeared affected more in diagnostic and post-treatment responder samples. Transcriptome analyses of diagnostic vs. post-treatment responder samples indicated significant increase in transcripts associated with activity within endosteal niche (SPARC, SPP1, DCN, VCAN, BGN) and significant loss of transcripts associated with DNA replication (TOP2, HELLS, E2F8) (Fig.C), the latter was consistent with treatment-related loss of cellularity. Only modest increase in SPARC, SPP1 or BGN levels and no significant decrease in DNA-replication associated transcripts were noted in non-responder post-treatment samples (Fig.1D). These data indicate greater loss of AML cells and greater activity within the endosteal niche in responder in comparison to non-responder samples. Finally, analyses performed on post-treatment responder vs. non-responder samples showed significant decrease in SPARC, SPP1, DCN, VCAN, BGN in non-responder post-treatment samples (Fig.E, F). Endosteal niche in histopathologic evaluation at diagnosis was generally unremarkable in both responder and non-responder samples with only rare osteoblasts present. In contrast, post-treatment, we found an elevated number of osteoblasts in responders in comparison to non-responder samples (Fig.G, H). Conclusions: Transcriptomic and histopathologic analyses of AML bone marrow biopsies procured at diagnosis and post-treatment from responder or non-responders indicate inverse correlation between the activity of endosteal niche and levels of transcripts involved in osteoblast maturation and homeostasis. Significant suppression of mesenchymal/osteoblast component of the niche is observed in non-responder samples. To our knowledge this is a first report showing the correlation between levels of osteopontin (SPP1), osteonectin (SPARC) and biglycan (BGN) and response to chemotherapy directly in the AML core bone marrow biopsies. Our data suggest that osteo-stimulatory factors could be used to achieve better therapeutic outcomes in AML. Disclosures No relevant conflicts of interest to declare.

We examined the precise localization of dimethylated histone three lysine four (H3K4me2) in mature rat sperm. Within nonintergenic-enriched regions, half of the DNA peaks associated with H3K4me2 retention fell in gene bodies and the other half in promoter regions. The most significant peaks near annotated DNA regions in the composite data included loci known to be associated with RNA metabolism, cell cycle regulation, and spermatogenesis. Regions associated with differential retention of H3K4me2 within gene bodies were significantly enriched for housekeeping gene and cell-cycle functionality. Proximal promoter-associated peaks were enriched for viral reproduction and cell cycle regulation genes, while Promoter1k and Promoter3k peaks were enriched for RNA metabolism functions. Further, homeobox- and kruppel-like factor motifs were among the most significantly enriched de novo and known motifs discovered within gene-associated H3K4me2 peaks. Motif analysis and native chromatin immunoprecipitation followed by sequencing (nChIP-seq) peak calling indicated an instructive role for retained paternal histones in the epigenetic regulation of early embryonic development in the rat.

The underlying mechanism of normal lung organogenesis is not well understood. An increasing number of studies are demonstrating that extracellular vesicles (EVs) play critical roles in organ development by delivering microRNAs (miRNA) to neighboring and distant cells. miRNAs are important for fetal lung growth; however, the role of miRNA-EVs (miRNAs packaged inside the EVs) during fetal lung development is unexplored. The aim of this study was to examine the expression of miRNA-EVs in MLE-12, a murine lung epithelial cell line subjected to mechanical stretch in vitro with the long-term goal to investigate their potential role in the fetal lung development. Both cyclic and continuous mechanical stretch regulate miRNA differentially in EVs released from MLE-12 and intracellularly, demonstrating that mechanical signals regulate the expression of miRNA-EVs in lung epithelial cells. These results provide a proof-of-concept for the potential role that miRNA-EVs could play in the development of fetal lung.

Background Preterm birth is a significant clinical problem and an enormous burden on society, affecting one in eight pregnant women and their newborns. Despite decades of research, the molecular mechanism underlying its pathogenesis remains unclear. Many studies have shown that preterm birth is associated with health risks across the later life course. The “fetal origins” hypothesis postulates that adverse intrauterine exposures are associated with later disease susceptibility. Our recent studies have focused on the placental epigenome at term. We extended these studies to genome-wide placental DNA methylation across a wide range of gestational ages. We applied methylation dependent immunoprecipitation/DNA sequencing (MeDIP-seq) to 9 placentas with gestational age from 25 weeks to term to identify differentially methylated regions (DMRs). Results Enrichment analysis revealed 427 DMRs with nominally significant differences in methylation between preterm and term placentas (p

Gammaherpesviruses are oncogenic pathogens that persist in ~95% of the adult population. Cellular metabolic pathways have emerged as important regulators of many viral infections, including infections by gammaherpesviruses that require several lipid synthetic pathways for optimal replication. Liver X receptors (LXRs) are transcription factors that are critical regulators of cellular fatty acid and cholesterol synthesis pathways. Not surprisingly, LXRs are attractive therapeutic targets in cardiovascular disease. Here we describe an antiviral role for LXRs in the context of gammaherpesvirus infection of primary macrophages. We show that type I interferon increased LXR expression following infection. Surprisingly, there was not a corresponding induction of LXR target genes. Rather, LXRs suppressed the expression of target genes, leading to decreased fatty acid and cholesterol synthesis, two metabolic pathways that support gammaherpesvirus replication. This report defines LXR-mediated restriction of cholesterol and lipid synthesis as an intrinsic metabolic mechanism to restrict viral replication in innate immune cells., IMPORTANCE Fatty acid and cholesterol synthesis pathways of the host play important roles in diverse biological systems. Importantly, these two metabolic pathways are also usurped by a number of viruses to facilitate viral replication. In this report, we show that suppression of these pathways by liver X receptors in primary macrophages creates an intrinsic antiviral state that attenuates gammaherpesvirus replication by limiting viral access to the two metabolic pathways.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using “Cre-loxP”-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration., Chondrogenesis: SHP2 regulates skeletal cell fate The enzyme SHP2 is critical for the differentiation of skeletal cells and could be a pharmacological target for bone and cartilage regeneration. Osteo-chondroprogenitor cells arise from stem cells in the bone marrow and can differentiate into either osteoblasts (bone-producing cells) or chondrocytes (cartilage cells). A team headed by Wentian Yang at Brown University, Rhode Island found that mice deficient in SHP2 had increased cartilage mass and decreased ossification. As a result of SHP2 deficiency, abnormal cartilage growth developed at sites where mineralized bone would normally form. The team’s findings suggest that SHP2-deficient osteo-chondroprogenitor cells become chondrocytes, not osteoblasts. The authors believe that the ability to therapeutically manipulate cell fate by regulating SHP2 activity offers a new means of controlling cartilage formation in patients with various cartilage-related disorders, including tumors and osteoarthritis.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.

Microchimerism has generally been shown to protect against cancer (Gilmore et al. in Exp Hematol 36(9):1073-1077, 2008). The mechanism of how this occurs is an area of intense study, as it may lead to new cancer treatments. The leading theory is that microchimeric cells perform immune surveillance by directly fighting cancerous cells and that they also act as stem cells, repairing damaged tissue (Khosrotehrani et al. in JAMA 292:75-80, 2004). However, there is conflicting evidence to support this theory. Several small studies have found few microchimeric cells in tumor tissue (Gadi in Breast Cancer Res Treat 121(1):241-244, 2010; Cirello et al. in Int J Cancer 126:2874-2878, 2010), while another study contradicted these findings by showing microchimeric cells clustered around tumor tissue (O'Donoghue et al. in Reprod Biomed Online 16:382-390, 2008). To date, we have designed the largest and broadest study to investigate this question of whether microchimeric cells really do cluster at tumor tissue. We analyzed 245 samples from a broad range of cancer types. Using PCR for the male chromosome marker TSPY1, we identified only 12 out of 245 samples with microchimerism for a rate of 4.9% (95% confidence interval 2.2-7.6%). Five of these samples were confirmed using Y fluorescence in situ hybridization. This rate of 4.9% microchimerism is the lowest reported in any study. The low percentage of microchimerism observed in our broad study suggests that microchimeric cells do not invade tumors to fight off neoplasm.

Bone morphogenetic proteins (BMPs) function in most tissues but have cell type-specific effects. Given the relatively small number of BMP receptors, this exquisite signaling specificity requires additional molecules to regulate this pathway's output. The receptor tyrosine kinase MuSK (muscle-specific kinase) is critical for neuromuscular junction formation and maintenance. Here, we show that MuSK also promotes BMP signaling in muscle cells. MuSK bound to BMP4 and related BMPs with low nanomolar affinity in vitro and to the type I BMP receptors ALK3 and ALK6 in a ligand-independent manner both in vitro and in cultured myotubes. High-affinity binding to BMPs required the third, alternatively spliced MuSK immunoglobulin-like domain. In myoblasts, endogenous MuSK promoted BMP4-dependent phosphorylation of SMADs and transcription of Id1, which encodes a transcription factor involved in muscle differentiation. Gene expression profiling showed that MuSK was required for the BMP4-induced expression of a subset of genes in myoblasts, including regulator of G protein signaling 4 (Rgs4). In myotubes, MuSK enhanced the BMP4-induced expression of a distinct set of genes, including transcripts characteristic of slow muscle. MuSK-mediated stimulation of BMP signaling required type I BMP receptor activity but was independent of MuSK tyrosine kinase activity. MuSK-dependent expression of Rgs4 resulted in the inhibition of Ca(2+) signaling induced by the muscarinic acetylcholine receptor in myoblasts. These findings establish that MuSK has dual roles in muscle cells, acting both as a tyrosine kinase-dependent synaptic organizing molecule and as a BMP co-receptor that shapes BMP transcriptional output and cholinergic signaling.

Chromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole-genome tiling microarrays of activation and repressive chromatin marks, whole-genome transcriptional microarrays and single-cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single-cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome-wide approaches such as whole-genome chromatin immunoprecipitation and transcriptional studies in conjunction with single-cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.

The mammalian ovary is unique in that its reproductive life span is limited by oocyte quantity and quality. Oocytes are recruited from a finite pool of primordial follicles that are usually exhausted from the ovary during midadult life. If regulation of this pool is perturbed, the reproductive capacity of the ovary is compromised. TAF4B is a gonad-enriched subunit of the TFIID complex required for female fertility in mice. Previous characterization of TAF4B-deficient ovaries revealed several reproductive deficits that collectively result in infertility. However, the etiology of such fertility defects remains unknown. By assaying estrous cycle, ovarian pathology, and gene expression changes in young Taf4b-null female mice, we show that TAF4B-deficient female mice exhibit premature reproductive senescence. The rapid decline of ovarian function in Taf4b-null mice begins in early postnatal life, and follicle depletion is completed by 16 wk of age. To uncover differences in gene expression that may underlie accelerated ovarian aging, we compared genome-wide expression profiles of 3-wk-old, prepubescent Taf4b-null and wild-type ovaries. At 3 wk of age, decreased gene expression in Taf4b-null ovaries is similar to that seen in aged ovaries, revealing several molecular signatures of premature reproductive senescence, including reduced Smc1b. One significantly reduced transcript in the young TAF4B-null ovary codes for MOV10L1, a putative germline-specific RNA helicase that is related to the Drosophila RNA interference protein, armitage. We show here that Mov10l1 is expressed in mouse oocytes and that its expression is sensitive to TAF4B level, linking TAF4B to the posttranscriptional control of ovarian gene expression.

Fluorescence activated cell sorting (FACS) analysis has become a standard tool to analyze cell cycle distributions in populations of cells. These methods require relatively large numbers of cells, and do not provide optimal resolution of the transitions between cell cycle phases. In this report we describe in detail complementary methods that utilize the incorporation of nucleotide analogs combined with microscopic examination. While often more time consuming, these protocols typically require far fewer cells, and allow accurate kinetic assessment of cell cycle progression. We also describe the use of a technique for the synchronization of adherent cells in mitosis by simple mechanical agitation (mitotic shake-off) that eliminates physiological perturbation associated with drug treatments.

Introduction: Previously(Lassaletta et al,PMC3943541)we showed that Rapamycin treatment of normal pigs subjected to an acute myocardial ischemia-reperfusion injury (IRI) was associated with increase myocardial necrosis and refractory left ventricle (LV) defibrillations. In an effort to better understand rapamycin effects we applied a non-bias genomic analysis to determine if microRNAs were modified by rapamycin treatment in this pre-clinical animal model of IRI. Hypothesis: We hypothesized that microRNA expression were modified by rapamycin-treated and would identify novel and meaningful pharmacological mechanisms related to its detrimental effects on reperfusion injury. Methods: Young male Yorkshire swine (16-19 kg) received either no drug (controls, n=5) or were given 4mg of oral rapamycin daily (n=5). After two weeks of treatment, all animals underwent median sternotomy and the mid-LAD was occluded for 60 minutes followed by 120 minutes of reperfusion. RNA was extracted from LV non-ischemic and ischemic areas from each group and processed (according to the manufacture′s specifications at Brown University Genomic Core facility) for microRNA (miRNA) expression analysis using a Affymetrix GeneChip miRNA3.0 Array containing over 25,000 probe sets of which 257 miRNA probes of domestic pigs. Data were processed using MetaCore version 5.0 (GeneGo, St Joseph, MI) for both statistical (FDR: p Results: miRNA regulated in 5 animals per group were annotated for human orthologs. A total of 23 miRNAs were differentially expressed. Interestingly, all miRNAs were down-regulated in the rapamycin-treated animals. Of those, nine had human orthologs: miR 181c-3p was highly represented followed by miR323a-5p, miR1245b-3p, miR4324, miR331-5p, miR491-5p and miR490-3p. Conclusions: Recent studies show that miRNA 181c regulates myocardial mitochondrial function through cytochrome c oxidase and complex IV gene expressions (PMCID: PMC4014556)and regulates Glucose-regulated protein (GRP78)/BiP [an endoplasmic reticulum stress (ER) chaperone](PMID: 24696166). Hence It is likely that rapamycin effects (detrimental or not) are associated with mitochondrial function and ER stress response.

Human fetal lung xenografts display an unusual pattern of non-sprouting, plexus-forming angiogenesis that is reminiscent of the dysmorphic angioarchitecture described in bronchopulmonary dysplasia (BPD). The aim of this study was to determine the clinicopathological correlates, growth characteristics and molecular regulation of this aberrant form of graft angiogenesis.Fetal lung xenografts, derived from 12 previable fetuses (15 to 22 weeks' gestation) and engrafted in the renal subcapsular space of SCID-beige mice, were analyzed 4 weeks posttransplantation for morphology, vascularization, proliferative activity and gene expression.Focal plexus-forming angiogenesis (PFA) was observed in 60/230 (26%) of xenografts. PFA was characterized by a complex network of tortuous nonsprouting vascular structures with low endothelial proliferative activity, suggestive of intussusceptive-type angiogenesis. There was no correlation between the occurrence of PFA and gestational age or time interval between delivery and engraftment. PFA was preferentially localized in the relatively hypoxic central subcapsular area. Microarray analysis suggested altered expression of 15 genes in graft regions with PFA, of which 7 are known angiogenic/lymphangiogenic regulators and 5 are known hypoxia-inducible genes. qRT-PCR analysis confirmed significant upregulation of SULF2, IGF2, and HMOX1 in graft regions with PFA.These observations in human fetal lungs ex vivo suggest that postcanalicular lungs can switch from sprouting angiogenesis to an aberrant intussusceptive-type of angiogenesis that is highly reminiscent of BPD-associated dysangiogenesis. While circumstantial evidence suggests hypoxia may be implicated, the exact triggering mechanisms, molecular regulation and clinical implications of this angiogenic switch in preterm lungs in vivo remain to be determined.

Definitive localization of c-Myc within the nucleus is important to fully understand the regulation and function of this oncoprotein. Studies of c-Myc distribution, however, have produced conflicting results. To overcome technical challenges inherent in c-Myc cytology, we use here three methods to visualize c-Myc and in addition examine the impact of proteasome inhibition. EYFP or HA-tagged Myc was reintroduced by stable transfection into myc null diploid rat fibroblasts, replacing endogenous Myc with tagged Myc expressed at or near normal levels. This tagged Myc is shown to functionally replace the endogenous Myc by restoration of normal cell morphology and growth rate. We were able to confirm key findings using antibodies to the endogenous c-Myc and/or its partner, Max. Contrary to some published reports, by all three methods the c-Myc protein in rat fibroblasts distributes predominantly throughout the nucleus in a dispersed granular pattern, avoiding the nucleolus. Importantly, however, several findings provide evidence for an unanticipated relationship between c-Myc and PML nuclear bodies, which is enhanced under conditions of proteasome inhibition. Evidence of Max concentration within PML bodies is shown both with and without proteasome inhibition, strengthening the relationship between PML bodies and Myc/Max. Some accumulation of Myc and Max in nucleoli upon proteasome inhibition is also observed, although co-localization of ubiquitin was only seen with PML bodies. This work provides a comprehensive study of c-Myc distribution and also presents the first evidence of a relationship between turnover of this oncoprotein and PML nuclear bodies, known to break down in certain cancers.

c-myc is an important protooncogene whose misregulation is believed to causally affect the development of numerous human cancers. c-myc null rat fibroblasts are viable but display a severe (two- to threefold) retardation of proliferation. The rates of RNA and protein synthesis are reduced by approximately the same factor, whereas cell size remains unaffected. We have performed a detailed kinetic cell cycle analysis of c-myc −/− cells by using several labeling and synchronization methods. The majority of cells (>90%) in asynchronous, exponential phase c-myc −/−cultures cycle continuously with uniformly elongated cell cycles. Cell cycle elongation is due to a major lengthening of G1 phase (four- to fivefold) and a more limited lengthening of G2phase (twofold), whereas S phase duration is largely unaffected. Progression from mitosis to the G1 restriction point and the subsequent progression from the restriction point into S phase are both drastically delayed. These results are best explained by a model in which c-Myc directly affects cell growth (accumulation of mass) and cell proliferation (the cell cycle machinery) by independent pathways.

Primary myelofibrosis (PMF) is an age-related myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, increasingly ineffective hematopoiesis, vascular and inflammatory complications, and progression to acute myeloid leukemia (AML) in 20% of cases. PMF is the rarest and the most severe of MPNs, with unfavorable prognosis and challenging treatment. Constitutive activation of the JAK-STAT pathway is an essential contributor to the pathogenesis of PMF. Nonetheless, clinically available JAK inhibitors do not show consistent disease-modifying effects at the molecular and malignant stem cell levels, underscoring the need to identify other targetable pathogenic mechanisms in PMF and related MPNs. We recently described a new signaling cascade involved in the maintenance of the malignant phenotype of stem cells in chronic myeloid leukemia that affects their adhesiveness, interaction with the microenvironment and resistance to imatinib (Chorzalska et al., Leukemia 2014). These phenotypic changes were caused by loss of Abelson interactor-1 (Abi-1) which regulates integrin α4 and the Src family kinases (SFKs) signaling pathways. To further delineate how organ-specific absence of Abi-1 affects hematopoiesis and hematopoietic stem cells, we generated a conditional, inducible bone marrow-specific Abi-1 transgenic mouse knockout model. For this purpose, we crossed conditional Abi-1(fl/fl) knockout mice with transgenic Tg(Mx1-cre+/-) mice (B6.Cg-Tg(Mx1-cre+ 1Cgn/J) that express Cre recombinase under the control of the polyinosinic:polycytidylic acid (poly(I:C)) or interferon-inducible Mx1 promoter. Obtained Abi-1(fl/fl);Tg(Mx1- cre(+)) animals were subjected to bone marrow-specific Abi-1 deletion induced by intraperitoneal administration of poly(I:C), resulting in organ-specific Abi-1 knockout (Abi-1 KO). Abi-1(fl/fl);Tg(Mx1- cre(-)) animals subjected to the same poly(I:C) treatment (Abi-1 WT) were used as controls in all experiments (Figure 1A). We confirmed the absence of Abi-1 protein in the Abi-1 KO bone marrow by immunoblotting (Figure 1B). Abi-1 KO mice developed deranged hematopoiesis with progressive anemia, thrombocythemia, megakaryocytosis, and myeloid hyperplasia (Figure 1C). They also developed progressive splenomegaly with 1.4x, 2.4x, and 4.8x enlargement of the spleen at 4, 28, and 50 weeks after recombination, respectively, and progressive femur pallor (Figure 1D) with bone marrow fibrosis confirmed by trichrome and silver staining (Figure 1D). Using fluorescence-activated cell sorting, we determined that Abi-1 KO resulted in more than two-fold expansion of the most primitive long-term hematopoietic stem cells (Lin-CD34-Sca-1+c-Kit+ CD135-) in the bone marrow and 3.75-fold increase of LSK cells (Lin- Sca-1+c-Kit+) in the peripheral blood compared with controls (Figure 1E). Increased activities of the SFKs and NFκB were observed in Abi-1 KO bone marrow, and confirmed in human CD34+ cells isolated from the marrow of patients with PMF or AML (Figure 1F,G). Furthermore, CD34+ hematopoietic progenitors and granulocytes from PMF patients, as well as CD34+ hematopoietic progenitors from AML patients, showed decreased levels of ABI1 transcript (Figure 1H). Taken together, our results indicate that a targeted deactivation of Abi-1 in the bone marrow causes expansion of hematopoietic stem cells, myeloid hyperplasia with thrombocytosis and overproduction of collagen and reticulin fibers. The Abi-1 KO murine phenotype meets the Mouse Models of Human Cancers Consortium criteria for a myeloproliferative neoplasm with features resembling human PMF (Kogan et al., Blood 2002). These data indicate that loss of Abi-1 is sufficient for initiation of bone marrow fibrosis, and that Abi-1-SFKs-NFκB cross-talk may represent a new molecular target for the treatment of PMF. Bone marrow-specific, conditional Abi-1 KO animals represent a new model of PMF offering a unique opportunity to interrogate the molecular details of the Abi-1-driven mechanism that leads to the development of stem cell-derived MPNs, and to elucidate potential novel therapeutic approaches that directly address their pathogenesis. Disclosures Olszewski: Bristol-Myers Squibb: Consultancy; Genentech: Research Funding; TG Therapeutics: Research Funding. Reagan:Alexion: Honoraria; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Understanding the genetic contribution(s) to the risk of preterm birth may lead to the development of interventions for treatment, prediction and prevention. Twin studies suggest heritability of preterm birth is 36-40%. Large epidemiological analyses support a primary maternal origin for recurrence of preterm birth, with little effect of paternal or fetal genetic factors. We exploited an "extreme phenotype" of preterm birth to leverage the likelihood of genetic discovery. We compared variants identified by targeted sequencing of women with 2-3 generations of preterm birth with term controls without history of preterm birth. We used a meta-genomic, bi-clustering algorithm to identify gene sets coordinately associated with preterm birth. We identified 33 genes including 217 variants from 5 modules that were significantly different between cases and controls. The most frequently identified and connected genes in the exome library were IGF1, ATM and IQGAP2. Likewise, SOS1, RAF1 and AKT3 were most frequent in the haplotype library. Additionally, SERPINB8, AZU1 and WASF3 showed significant differences in abundance of variants in the univariate comparison of cases and controls. The biological processes impacted by these gene sets included: cell motility, migration and locomotion; response to glucocorticoid stimulus; signal transduction; metabolic regulation and control of apoptosis.

Background The characterization of miRNAs and their target mRNAs involved in regulation of the immune process is an area of intense research and relatively little is known governing these processes in allergic inflammation. Here we present novel findings defining the miRNA and mRNA transcriptome in eosinophilic esophagitis (EoE), an increasing recognized allergic disorder. Methods Esophageal epithelial miRNA and mRNA from five paired biopsies pre- and post-treatment with glucocorticosteroids were profiled using Taqman and Affymetrix arrays. Validation was performed on additional paired biopsies, untreated EoE specimens and normal controls. Differentially regulated miRNAs and mRNAs were generated, within which miRNA-mRNA target pairs with high predicted confidence were identified. Results Compared to the post-glucocorticoid treated esophageal mucosa, of all the 377 miRNA sequences examined, 32 miRNAs were significantly upregulated and four downregulated in the pre-treated biopsies. MiR-214 was the most upregulated (150 fold) and miR-146b-5b, 146a, 145, 142-3p and 21 were upregulated by at least 10 fold. Out of 12 miRNAs chosen for validation by qRT-PCR, five (miR-214, 146b-5p, 146a, 142-3p and 21) were confirmed and 11 shared the same trend. When the expression of the 12 miRNAs in the EoE mucosa was compared to unrelated normal mucosa, six (miR-214, 146b-5p, 146a, 21, 203, and 489) showed similar significant changes as in the paired samples and 10 of them shared the same trend. In the same five pairs of samples used to profile miRNA, 311 mRNAs were down-regulated and 35 were up-regulated in pre-treated EoE mucosa. Among them, 164 mRNAs were identified as potential targets of differentially regulated miRNAs. Further analysis revealed that immune-related genes, targeted and non-targeted by miRNAs, were among the most important genes involved in the pathogenesis of EoE. Conclusions Our findings add to the accumulating body of data defining a regulatory role for miRNA in immune and allergic processes.

A major challenge in translating the positive effects of dietary restriction (DR) for the improvement of human health is the development of therapeutic mimics. One approach to finding DR mimics is based upon identification of the proximal effectors of DR life span extension. Whole genome profiling of DR in Drosophila shows a large number of changes in gene expression, making it difficult to establish which changes are involved in life span determination as opposed to other unrelated physiological changes. We used comparative whole genome expression profiling to discover genes whose change in expression is shared between DR and two molecular genetic life span extending interventions related to DR, increased dSir2 and decreased Dmp53 activity. We find twenty-one genes shared among the three related life span extending interventions. One of these genes, takeout, thought to be involved in circadian rhythms, feeding behavior and juvenile hormone binding is also increased in four other life span extending conditions: Rpd3, Indy, chico and methuselah. We demonstrate takeout is involved in longevity determination by specifically increasing adult takeout expression and extending life span. These studies demonstrate the power of comparative whole genome transcriptional profiling for identifying specific downstream elements of the DR life span extending pathway.

The mammalian ovary is unique in that its reproductive life span is limited by oocyte quantity and quality. Oocytes are recruited from a finite pool of primordial follicles that are usually exhausted from the ovary during midadult life. If regulation of this pool is perturbed, the reproductive capacity of the ovary is compromised. TAF4B is a gonad-enriched subunit of the TFIID complex required for female fertility in mice. Previous characterization of TAF4B-deficient ovaries revealed several reproductive deficits that collectively result in infertility. However, the etiology of such fertility defects remains unknown. By assaying estrous cycle, ovarian pathology, and gene expression changes in young Taf4b-null female mice, we show that TAF4B-deficient female mice exhibit premature reproductive senescence. The rapid decline of ovarian function in Taf4b-null mice begins in early postnatal life, and follicle depletion is completed by 16 wk of age. To uncover differences in gene expression that may underlie accelerated ovarian aging, we compared genome-wide expression profiles of 3-wk-old, prepubescent Taf4b-null and wild-type ovaries. At 3 wk of age, decreased gene expression in Taf4b-null ovaries is similar to that seen in aged ovaries, revealing several molecular signatures of premature reproductive senescence, including reduced Smc1b. One significantly reduced transcript in the young TAF4B-null ovary codes for MOV10L1, a putative germline-specific RNA helicase that is related to the Drosophila RNA interference protein, armitage. We show here that Mov10l1 is expressed in mouse oocytes and that its expression is sensitive to TAF4B level, linking TAF4B to the posttranscriptional control of ovarian gene expression.

In hematological malignancies, quiescent leukemic stem cells are responsible for persistence of minimal residual disease and relapse. We have recently identified a new signaling pathway that is significantly dysregulated in imatinib mesylate (IM) resistant leukemic cells (Chorzalska et all, Leukemia in press). A key player in this pathway is Abelson interactor-1 (Abi-1). Abi-1 was originally identified as Abl kinase associating protein that was later confirmed to be one of the Bcr-Abl interactors. Abi-1 was recently shown to interact directly with α4 integrin, which controls lodging of hematopoietic and leukemic stem cells (HSCs/LSCs) in the bone marrow microenvironment. We have recently obtained evidence that Abi-1 plays a role in signaling cross-talk between Bcr-Abl and α4 integrin. We have found that loss of Abi-1 leads to increased adhesion and quiescence, resulting in increased chemoresistance of leukemic CD34+ progenitor cells. Comparison of Abi-1 (ABI-1) and α4 integrin (ITGA4) gene expression in relapsing Bcr-Abl positive CD34+ progenitor cells demonstrated a reduction in Abi-1 and an increase in α4 integrin mRNA in the absence of Bcr-Abl mutations. This inverse correlation between Abi-1 and α4 integrin expression, as well as linkage to elevated phospho-Akt and phospho-Erk signaling, was confirmed in imatinib mesylate (IM) resistant leukemic cells. These results indicate that the α4-Abi-1 signaling pathway may mediate acquisition of the drug resistant phenotype of leukemic cells. Based on our findings, we hypothesize that chemoresistance arises as a consequence of dysregulation of a pathway involving α4 integrin and Abi-1, and is mediated through a previously unknown mechanism that is independent of oncogene activity. Citation Format: Anna Chorzalska, Ibrahem Salloum, Philip Marjon, Diana Treaba, Christoph Schorl, Morgan John, Christine R. Bryke, John Reagan, Eric Winer, Adam J. Olszewski, Patrycja M. Dubielecka. New Abelson interactor-1(Abi-1)-driven mechanism of acquired drug resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B08.

7107 Background: In hematological malignancies, quiescent leukemic stem cells are responsible for persistence of minimal residual disease and relapse. Emerging evidence points to the involvement of...

Abnormalities of the p53 tumor suppressor gene are the single most common molecular abnormality seen in human cancer. Considerable evidence indicates that the product of this gene has critical roles in coordinating the response of cells to a diverse range of environmental stresses. At present, there is a gamut of biochemical properties and interactions ascribed to p53, but the in vivo physiological relevance of many of these remains uncertain. The development of clinical applications and novel therapeutic strategies utilizing our knowledge of p53 is contingent upon bridging the gap between rigorous biochemistry and holistic in vivo studies.