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Common treatment for venous leg wounds includes topical wound dressings with compression. At each dressing change, wounds are debrided and washed; however, the effect of the washing procedure on the wound microbiome has not been studied. We hypothesized that wound washing may alter the wound microbiome. To characterize microbiome changes with respect to wound washing, swabs from 11 patients with chronic wounds were sampled before and after washing, and patient microbiomes were characterized using 16S rRNA sequencing and culturing. Microbiomes across patient samples prior to washing were typically polymicrobial but varied in the number and type of bacterial genera present. Proteus and Pseudomonas were the dominant genera in the study. We found that washing does not consistently change microbiome diversity but does cause consistent changes in microbiome composition. Specifically, washing caused a decrease in the relative abundance of the most highly represented genera in each patient cluster. The finding that venous leg ulcer wound washing, a standard of care therapy, can induce changes in the wound microbiome is novel and could be potentially informative for future guided therapy strategies.

An optimized, well-tested and validated targeted genomic sequencing-based high-throughput assay is currently not available ready for routine biodefense and biosurveillance applications. Earlier, we addressed this gap by developing and establishing baseline comparisons of a multiplex end-point Polymerase Chain Reaction (PCR) assay followed by Oxford Nanopore Technology (ONT) based amplicon sequencing to real time PCR and customized data processing. Here, we expand upon this effort by identifying the optimal ONT library preparation method for integration into a novel software platform ONT-DART (ONT-Detection of Amplicons in Real-Time). ONT-DART is a dockerized, real-time, amplicon-sequence analysis workflow that is used to reproducibly process and filter read data to support actionable amplicon detection calls based on alignment metrics, within sample statistics, and no-template control data. This analysis pipeline was used to compare four ONT library preparation protocols using R9 and Flongle (FL) flow cells. The two 4-Primer methods tested required the shortest preparation times (5.5 and 6.5 h) for 48 libraries but provided lower fidelity data. The Native Barcoding and Ligation methods required longer preparation times of 8 and 12 h, respectively, and resulted in higher overall data quality. On average, data derived from R9 flow cells produced true positive calls for target organisms more than twice as fast as the lower throughput FL flow cells. These results suggest that utilizing the R9 flowcell with an ONT Native Barcoding amplicon library method in combination with ONT-DART platform analytics provides the best sequencing-based alternative to current PCR-based biodetection methods.

The early COVID-19 pandemic has been characterized by rapid global spread. In the United States National Capital Region, over 2,000 cases were reported within three weeks of its first detection in March 2020. We aimed to use genomic sequencing to understand the initial spread of SARS-CoV-2, the virus that causes COVID-19, in the region. By correlating genetic information to disease phenotype, we also aimed to gain insight into any correlation between viral genotype and case severity or transmissibility.We performed whole genome sequencing of clinical SARS-CoV-2 samples collected in March 2020 by the Johns Hopkins Health System. We analyzed these regional SARS-CoV-2 genomes alongside detailed clinical metadata and the global phylogeny to understand early establishment of the virus within the region.We analyzed 620 samples from the Johns Hopkins Health System collected between March 11-31, 2020, comprising 37.3% of the total cases in Maryland during this period. We selected 143 of these samples for sequencing, generating 114 complete viral genomes. These genomes belong to all five major Nextstrain-defined clades, suggesting multiple introductions into the region and underscoring the diversity of the regional epidemic. We also found that clinically severe cases had genomes belonging to all of these clades.We established a pipeline for SARS-CoV-2 sequencing within the Johns Hopkins Health system, which enabled us to capture the significant viral diversity present in the region as early as March 2020. Efforts to control local spread of the virus were likely confounded by the number of introductions into the region early in the epidemic and interconnectedness of the region as a whole.

Background Skin, the largest organ of the human body by weight, hosts a diversity of microorganisms that can influence health. The microbial residents of the skin are now appreciated for their roles in host immune interactions, wound healing, colonization resistance, and various skin disorders. Still, much remains to be discovered in terms of the host pathways influenced by skin microorganisms, as well as the higher-level skin properties impacted through these microbe-host interactions. Towards this direction, recent efforts using mouse models point to pronounced changes in the transcriptional profiles of the skin in response to the presence of a microbial community. However, there is a need to quantify the roles of microorganisms at both the individual and community-level in healthy human skin. In this study, we utilize human skin equivalents to study the effects of individual taxa and a microbial community in a precisely controlled context. Through transcriptomics analysis, we identify key genes and pathways influenced by skin microbes, and we also characterize higher-level impacts on skin processes and properties through histological analyses. Results The presence of a microbiome on a 3D skin tissue model led to significantly altered patterns of gene expression, influencing genes involved in the regulation of apoptosis, proliferation, and the extracellular matrix (among others). Moreover, microbiome treatment influenced the thickness of the epidermal layer, reduced the number of actively proliferating cells, and increased filaggrin expression. Many of these findings were evident upon treatment with the mixed community, but either not detected or less pronounced in treatments by single microorganisms, underscoring the impact that a diverse skin microbiome has on the host. Conclusions This work contributes to the understanding of how microbiome constituents individually and collectively influence human skin processes and properties. The results show that, while it is important to understand the effect of individual microbes on the host, a full community of microbes has unique and pronounced effects on the skin. Thus, in its impacts on the host, the skin microbiome is more than the sum of its parts.

BackgroundThe early COVID-19 pandemic has been characterized by rapid global spread. In the United States National Capital Region, over 2,000 cases were reported within three weeks of its first detection in March 2020. We aimed to use genomic sequencing to understand the initial spread of SARS-CoV-2, the virus that causes COVID-19, in the region. By correlating genetic information to disease phenotype, we also aimed to gain insight into any correlation between viral genotype and case severity or transmissibility.MethodsWe performed whole genome sequencing of clinical SARS-CoV-2 samples collected in March 2020 by the Johns Hopkins Health System. We analyzed these regional SARS-CoV-2 genomes alongside detailed clinical metadata and the global phylogeny to understand early establishment of the virus within the region.ResultsWe analyzed 620 samples from the Johns Hopkins Health System collected between March 11–31, 2020, comprising 37.3% of the total cases in Maryland during this period. We selected 143 of these samples for sequencing, generating 114 complete viral genomes. These genomes belong to all five major Nextstrain-defined clades, suggesting multiple introductions into the region and underscoring the diversity of the regional epidemic. We also found that clinically severe cases had genomes belonging to all of these clades.ConclusionsWe established a pipeline for SARS-CoV-2 sequencing within the Johns Hopkins Health system, which enabled us to capture the significant viral diversity present in the region as early as March 2020. Efforts to control local spread of the virus were likely confounded by the number of introductions into the region early in the epidemic and interconnectedness of the region as a whole.

Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4+ T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5’ noncoding regions. Silencing DAP5 in isolated human naive CD4+ T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation., The differentiation of naive T cells to immune suppressing induced regulatory T (iTreg) cells requires TGF-beta-1 and downregulation of mTORC1 activity, which inhibits mRNA translation. Here the authors show that iTreg cell differentiation uses an alternate mRNA translation mechanism involving translation factors DAP5 and eIF3d.

Hematopoietic stem cells (HSCs) require highly regulated rates of protein synthesis, but it is unclear if they or lineage-committed progenitors preferentially recruit transcripts to translating ribosomes. We utilized polysome profiling, RNA sequencing, and whole-proteomic approaches to examine the translatome in LSK (Lin-Sca-1+c-Kit+) and myeloid progenitor (MP; Lin-Sca-1-c-Kit+) cells. Our studies show that LSKs exhibit low global translation but high translational efficiencies (TEs) of mRNAs required for HSC maintenance. In contrast, MPs activate translation in an mTOR-independent manner due, at least in part, to proteasomal degradation of mTOR by the E3 ubiquitin ligase c-Cbl. In the near absence of mTOR, CDK1 activates eIF4E-dependent translation in MPs through phosphorylation of 4E-BP1. Aberrant activation of mTOR expression and signaling in c-Cbl-deficient MPs results in increased mature myeloid lineage output. Overall, our data demonstrate that hematopoietic stem and progenitor cells (HSPCs) undergo translational reprogramming mediated by previously uncharacterized mechanisms of translational regulation.

// Gizelka David-West 1,4,* , Amanda Ernlund 2,* , Abhilash Gadi 2 and Robert J. Schneider 2,3 1 Division of Gynecologic Oncology, New York University School of Medicine, New York, NY, USA 2 Department of Microbiology, New York University School of Medicine, New York, NY, USA 3 Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA 4 New York Medical College, Westchester Medical Center, Hawthorne, NY, USA * These authors have contributed equally to this work Correspondence to: Robert J. Schneider, email: Robert.schneider@nyumc.org Keywords : mTOR; ovarian cancer; platinum resistance; translational regulation Received: October 31, 2017 Accepted: July 08, 2018 Published: September 04, 2018 Abstract Platinum resistance is a major cause of treatment failure and mortality in epithelial ovarian cancer. mTORC1/2 inhibitors, which impair mRNA translation, can re-sensitize resistant ovarian cancer cells to platinum chemotherapy but the mechanism remains poorly described. Using platinum-resistant OVCAR-3 cells treated with the selective mTORC1/2 inhibitor INK128/MLN128, we conducted genome-wide transcription and translation studies and analyzed the effect on cell proliferation, AKT-mTOR signaling and cell survival, to determine whether carboplatin resistance involves selective mRNA translational reprogramming, and whether it is sensitive to mTORC1/2 inhibition. Gene ontology and Ingenuity Pathway Analysis (IPA) were used to categorize gene expression changes into experimentally authenticated biochemical and molecular networks. We show that carboplatin resistance involves increased mTORC1/2 signaling, resulting in selective translation of mRNAs involved in DNA damage and repair responses (DDR), cell cycle and anti-apoptosis (survival) pathways. Re-sensitization of ovarian cancer cell killing by carboplatin required only modest mTORC1/2 inhibition, with downregulation of protein synthesis by only 20-30%. Genome-wide transcriptomic and translatomic analyses in OVCAR-3 cells revealed that the modest downregulation of global protein synthesis by dual mTORC1/2 inhibition is associated with greater selective inhibition of DDR, cell cycle and survival mRNA translation, which was confirmed in platinum-resistant SKOV-3 cells. These data suggest a clinical path to re-sensitize platinum resistant ovarian cancer to platinum chemotherapy through partial inhibition of mTORC1/2, resulting in selective translation inhibition of DDR and anti-apoptosis protective mRNAs.

This abstract was withdrawn by the authors.

Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis originating in hematopoietic/stem progenitor cells characterized by impaired differentiation and the accumulation of immature blasts in the bone marrow. While genetic, transcriptional, and epigenetic alterations have been comprehensively characterized in human AML, few studies have assessed post-transcriptional and translational regulatory mechanisms in leukemic stem cells (LSCs). We previously identified a novel LSC marker, CD99, that enriches for LSCs among leukemic blasts. We now show that CD99high LSCs express low levels of transcripts encoding ribosomal proteins and exhibit reduced translation compared to non-LSC blasts as assessed by OP-Puro incorporation assays and sucrose gradient polysome profiling. This pattern recapitulates reductions in translation in normal hematopoietic stem cells (HSCs) compared to committed progenitors, suggesting common mechanisms regulating translation in normal and malignant hematopoietic cells. To examine global post-transcriptional and translational regulation in HSCs, we performed polysome profiling, RNA-sequencing and whole-proteomic analysis of HSC-enriched cells (Lin-Sca-1+c-Kit+; LSK) and committed myeloid progenitors (MPs; Lin-Sca-1-c-Kit+). While MP cells exhibit higher levels of global translation than LSK cells, LSK cells exhibit higher translational efficiencies (TEs) of transcripts required for HSC maintenance and self-renewal, and MP cells show increased TEs of genes associated with differentiation. Although HSC-enriched LSK cells exhibit lower levels of global translation than MP cells, they show activation of mTOR signaling and high TEs of mTOR dependent transcripts. In contrast, even though MP cells exhibit higher levels of translation than HSCs, Western blot analysis confirms that they lack significant mTOR protein expression or signaling. Indeed, mTOR expression in MP cells is rescued by proteasome inhibitors, and its expression is restored in the absence of the E3 ubiquitin ligase, c-Cbl, demonstrating its regulation by proteasomal degradation. Confirming that mTOR signaling is not required for MP function, mTOR inhibition with rapamycin in vivo or Torin1 ex vivo did not affect MP translation or differentiation. To determine if similar mTOR-independent mechanisms of translational activation are also present in malignant hematopoiesis, we analyzed CD99high LSCs and CD99low non-LSC AML blasts and observed higher mTOR signaling and lower global translation in LSCs, as well as near-absence of mTOR protein expression in non-LSCs, recapitulating the differences observed in LSK versus MP cells. Collectively, these findings establish the presence of developmental stage-specific mechanisms of translational regulation mediated by mTOR in both normal and malignant hematopoiesis. This abstract is also being presented as Poster B30. Citation Format: Christina C. Spevak, Harold K. Elias, Lavanya Kannan, Gaelle Martin, Shanmugapriya Selvaraj, William S. Eng, Amanda Ernlund, Vinagolu K. Rajasekhar, Carolien M. Woolthuis, Robert J. Schneider, Christopher Y. Park. Developmentally regulated mTOR degradation in normal and malignant hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR06.

Regulatory T cells (Tregs) maintain immune system homeostasis in the organism. In the tumor microenvironment, however, Tregs can hinder the antitumor immune response. Treg maturation in peripheral sites (pTregs) requires inhibition of protein kinase mTORC1, which paradoxically blocks mRNA translation. mTORC1 phosphorylates (inactivates) 4E-BP1, a negative regulator of the translation initiation factor eIF4E. When mTORC1 is active, 4E-BP1 is hyperphosphorylated and releases eIF4E, which can then bind the mRNA cap structure and promote eIF4F complex assembly and ribosome recruitment. Inhibition of mTORC1 results in hypo-phosphorylation (activation) of 4E-BP1, which then binds and sequesters eIF4E, preventing its interaction with the cap and ribosome association to the mRNA. Using human CD4+ T cells differentiated in vitro, we show that mTORC1 inhibition strongly impairs protein synthesis, yet with exposure to TGFbeta is essential to mediate iTreg differentiation and immune suppression activity. We found that TGFbeta reprograms the T-cell transcriptome, while mTORC1 inhibition reprograms the translatome (genome-wide translation signature), mediating Treg differentiation. Genome-wide transcription and translation profiling identified TGFbeta-upregulated mRNAs resistant to mTORC1 inhibition, in part due to their 5'-untranslated regions, that induce Treg differentiation and immune-suppression activity. These include canonical pTreg fate-determining mRNAs FOXP3, CTLA-4, CD101 and CD103, among others. Inhibition of mTORC1 alone, or TGFbeta alone, induces iTregs with poor immune-suppression activity. Treg differentiation is therefore mediated by an mTORC1-independent privileged mRNA translation mechanism that converts activated CD4+ T cells to Tregs. These findings are quite relevant in the tumor microenvironment, where mTORC1-inhibiting conditions such as hypoxia and deprivation of nutrients and energy are present. Investigation of molecular targets acting in this mTORC1/eIF4E-independent translation of Treg mRNAs is ongoing. These targets could be drugged to selectively inhibit Treg differentiation in the tumor microenvironment, thus allowing effector cells to mount the response against cancer cells. Citation Format: Viviana Volta, Amanda Ernlund, Abhilash Gadi, Amanda Valeta-Magara, Columba de la Parra, Preeyam Patel, Robert J. Schneider. mTOR inhibition promotes differentiation of human regulatory T cells via privileged mRNA translation [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A26.

While translation initiation of most mammalian mRNAs is mediated by a 5' cap structure that binds eIF4E, inactivation/silencing of eIF4E, or quantitative sequestration of eIF4E by 4E-BPs through mTORC1 inhibition, does not impair translation of many capped mRNAs. This suggests an alternate mechanism may exist for cap-dependent but eIF4E/mTORC1-independent translation. DAP5 (NAT1, eIF4G2, p97) is an eIF4G family member that lacks the N-terminal binding domains for eIF4E and PABP. DAP5 was shown to mediate translation initiation through internal ribosome entry sites (IRES elements). We recently showed that DAP5 utilizes the cap binding activity of eIF3d to facilitate cap-dependent translation of a large number of capped mRNAs, making it a previous unknown major mechanism of cap-dependent but eIF4E/mTORC1-independent mRNA translation. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factor and cell receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair, oncogenesis, translation initiation, and cancer cell metastasis. Up to 20% of cellular mRNAs utilize DAP5-eIF3d for cap-dependent mRNA translation. Mass spectrometry and in vivo cross-linking studies found that eIF3d is a direct binding partner of DAP5 but does not bind strongly to eIF4GI or II family members that bind eIF4E. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E and mTORC1-independent capped-mRNA translation. Given the prevalence of oncogenic and survival mRNAs directed in translation by DAP5-eIF3d, we analyzed the NCI human tissue genome cancer atlas (TCGA) and found that high expression of DAP5 mRNA is strongly associated with estrogen receptor-negative (ER-) breast cancer metastasis and poor survival. We therefore investigated the role of DAP5 in preclinical mouse models of highly metastatic ER- breast cancer. Reduction of DAP5 expression by ~70% almost eliminated metastasis to the lung and significantly increased survival with no impact on primary tumor growth. These and other studies indicate that the DAP5-eIF3d complex is a significant alternate mechanism for cap-dependent, eIF4E/mTORC1-independent mRNA translation despite lacking eIF4E and PABP binding, that DAP5 directs translation of many capped mRNAs that are involved in cell stress and survival pathways, is essential for promoting breast cancer metastasis, and that most mRNAs translated by DAP5 do not utilize an IRES. Citation Format: Columba de la Parra, Amanda Ernlund, Amandine Alard, Kelly Ruggles, Beatrix Ueberheide, Olga Katsara, Robert J. Schneider. An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA13.

Maturation of regulatory T cells (Tregs) in peripheral sites is known to require TGFβ exposure and inhibition of protein kinase mTORC1. It is well known that mTOR inhibition is associated with repression of cap-dependent mRNA translation, which represents the major mechanism for protein synthesis, leaving unanswered how Tregs carry-out essential translation for development and immune suppression activity. To answer this question, we performed genome-wide transcription and translation profiling in CD4+CD127dim/− CD25+Tregs derived from anti-CD3/CD28-activated human naïve CD4+ T cells, treated with the mTORC1 inhibitor RAD001 and/or TGFβ. We found that TGFβ activated both Treg differentiation and immune suppression genes, while mTORC1 inhibition selectively blocked translation of most T cell mRNAs except those induced by TGFβ, including FOXP3, CTLA-4, CD101 or CD103, locking in Treg lineage commitment and immune suppression function. These canonical Treg fate-determining mRNAs were resistant to mTORC1 inhibition, an effect mediated in part by their 5′-untranslated regions through an alternate form of appears to be cap-dependent, eIF4E-independent mRNA translation. In conclusion, TGFβ transcriptional reprogramming together with mTORC1-independent translational reprogramming enable a privileged translation mechanism by which activated CD4+ T cells become Tregs.

Prior studies in numerous biological systems have shown that alterations in mRNA expression frequently fail to predict changes in protein expression. This may be due to many regulatory mechanisms that occur post-transcriptionally including mRNA recruitment to ribosomes, translational initiation, ribosome processivity, and protein stability, among others. Indeed, several examples of selective translation of mRNAs has been described both in malignant and normal cells. To determine the extent and potential impact of translational reprogramming on early hematopoietic development, we performed an integrated analysis of total RNA, polysome RNA, and whole proteome data generated from HSC-enriched LSK (Lin-Sca-1+c-Kit+) and MP (Lin-Sca-1-c-Kit+) cells from mouse. Our studies revealed that although LSK cells show lower global translation than MPs, they exhibited significantly higher translational efficiency (TE = polysome/total RNA abundance) of mRNAs supporting processes required for HSC maintenance (e.g. glycolysis, fatty acid metabolism, oxidative phosphorylation, mTOR signaling) (Fig 1A). Additionally, integrated analysis of proteomic and RNA expression data showed that, TE changes better predicted protein expression changes for these pathways, than total RNA expression (Fig1B). Biochemical characterization of MP cells revealed markedly decreased mTOR protein expression and signaling in MP cells, especially in GMP and MEP. This is mediated through proteasomal degradation of mTOR protein. An E3 ligase prediction algorithm, identified c-Cbl as a potential candidate, targeting mTOR, which was confirmed by demonstrating the aberrant expression of mTOR in MPs in c-Cbl KO mice. In vitro and in vivo mTOR inhibition studies confirm that the MPN-like phenotype of c-Cbl KO mice, is due to aberrant activation of mTOR signaling in committed myeloid progenitors. Intriguingly, despite decreased expression of mTOR protein in MP cells, 4E-BP1, a known target of mTOR, was still phosphorylated at Ser-65- a critical step for initiating cap-dependent translation. Through a combination of prediction algorithms and candidate gene experimental approaches, we show that the critical phosphorylation event at Ser-65 is mediated by , as immunoprecipitation studies show physical association between CDK1 and 4E-BP, and pharmacological inhibition of CDK1 activity, reduced 4E-BP P-Ser-65 levels. Overall, our data provide the first comprehensive characterization of the translatome in early hematopoiesis and demonstrated that the LSK to MP transition is characterized by significant translational reprogramming. This is, in part, mediated by the activation of a unique, mTOR-independent pathway to activate cap-dependent translation through the concerted action of c-Cbl and CDK1 to induce degradation of mTOR and phosphorylate 4E-BP to activation translation, respectively. Abrogation of the downregulation of mTOR signaling in myeloid progenitors, results in expansions of numerous myeloid lineages including neutrophils, monocytes and platelets (Fig 1C). Thus, our studies demonstrate the importance of proper translational reprogramming in early hematopoiesis. Figure legend. (A) Heatmap showing pathways significantly enriched in LSK and or MP cells based on TE. (B) Comparison of TE to protein expression in LSK cells for genes involved in the indicated biological processes (Blue dots: mRNAs that showed an anticorrelation between total RNA and protein expression; Red dots: mRNAs that showed a positive correlation between total RNA and protein expression). (C) Model for translational reprogramming in early hematopoiesis. Despite lower rates of global translation, LSK cells show preferential translation of mRNAs sensitive to mTOR inhibition and required for HSC maintenance. In contrast, in highly translating MP cells, loss of mTOR expression is mediated by the E3 ubiquitin ligase c-Cbl. When c-Cbl is deleted and mTOR protein is aberrantly expressed, this results in increased mature myeloid output. In the absence of mTOR, eIF4E-cap-dependent translation is maintained through the action of CDK1, which phosphorylates the S65 residue of 4E-BP1 to release eIF4E. Figure Disclosures No relevant conflicts of interest to declare.

Background Translatomics data, particularly genome-wide ribosome profiling and polysome profiling, provide multiple levels of gene regulatory information that can be used to assess general transcription and translation, as well translational efficiency. The increasing popularity of these techniques has resulted in multiple algorithms to detect translational regulation, typically distributed in the form of command line tools that require a basic level of programming ability. Additionally, due to the static nature of current software, dynamic transcriptional and translational comparative analysis cannot be adequately achieved. In order to streamline hypothesis generation, investigators must have the ability to manipulate and interact with their data in real-time. Results To address the lack of integration in current software, we introduce RIVET, Ribosomal Investigation and Visualization to Evaluate Translation, an R shiny based graphical user interface for translatomics data exploration and differential analysis. RIVET can analyze either microarray or RNA sequencing data from polysome profiling and ribosome profiling experiments. RIVET provides multiple choices for statistical analysis as well as integration of transcription, translation, and translational efficiency data analytics and the ability to visualize all results dynamically. Conclusions RIVET is a user-friendly tool designed for bench scientists with little to no programming background. RIVET facilitates the data analysis of translatomics data allowing for dynamic generation of results based on user-defined inputs and publication ready visualization. We expect RIVET will allow for scientists to efficiently make more comprehensive data observations that will lead to more robust hypothesis regarding translational regulation. Electronic supplementary material The online version of this article (10.1186/s12864-018-5166-z) contains supplementary material, which is available to authorized users.

Background Cancers induce gene expression alterations in stroma surrounding tumors that supports cancer progression. However, it is actually not at all known the extent of altered stromal gene expression enacted by tumors nor the extent to which altered stromal gene expression penetrates the stromal tissue. Presently, post-surgical “tumor-free” stromal tissue is determined to be cancer-free based on solely on morphological normality—a criteria that has not changed in more than 100 years despite the existence of sophisticated gene expression data to the contrary. We therefore investigated the extent to which breast tumors alter stromal gene expression in three dimensions in women undergoing mastectomy with the intent of providing a genomic determination for development of future risk of recurrence criteria, and to inform the need for adjuvant full-breast irradiation. Methods and findings Genome-wide gene expression changes were determined in histopathologically normal breast tissue in 33 women undergoing mastectomy for stage II and III primary invasive ductal carcinoma at serial distances in three dimensions from the tumor. Gene expression was determined by genome-wide mRNA analysis and subjected to metagene mRNA characterization. Tumor-like gene expression signatures in stroma were identified that surprisingly transitioned to a plastic, normalizing homeostatic signature with distance from tumor. Stroma closest to tumor displayed a pronounced tumor-like signature enriched in cancer-promoting pathways involved in disruption of basement membrane, cell migration and invasion, WNT signaling and angiogenesis. By 2 cm from tumor in all dimensions, stromal tissues were in transition, displaying homeostatic and tumor suppressing gene activity, while also expressing cancer supporting pathways. Conclusions The dynamics of gene expression in the post-tumor breast stroma likely co-determines disease outcome: reversion to normality or transition to transformation in morphologically normal tissue. Our stromal genomic signature may be important for personalizing surgical and adjuvant therapeutic decisions and risk of recurrence.

The majority of breast cancers expresses the estrogen receptor (ER+) and is treated with anti-estrogen therapies, particularly tamoxifen in premenopausal women. However, tamoxifen resistance is responsible for a large proportion of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen-sensitive and tamoxifen-resistant breast cancer cells, a tamoxifen-resistant patient-derived xenograft model, patient tumor tissues, and genome-wide transcription and translation studies, we show that tamoxifen resistance involves selective mRNA translational reprogramming to an anti-estrogen state by Runx2 and other mRNAs. Tamoxifen-resistant translational reprogramming is shown to be mediated by increased expression of eIF4E and its increased availability by hyperactive mTOR and to require phosphorylation of eIF4E at Ser209 by increased MNK activity. Resensitization to tamoxifen is restored only by reducing eIF4E expression or mTOR activity and also blocking MNK1 phosphorylation of eIF4E. mRNAs specifically translationally up-regulated with tamoxifen resistance include Runx2, which inhibits ER signaling and estrogen responses and promotes breast cancer metastasis. Silencing Runx2 significantly restores tamoxifen sensitivity. Tamoxifen-resistant but not tamoxifen-sensitive patient ER+ breast cancer specimens also demonstrate strongly increased MNK phosphorylation of eIF4E. eIF4E levels, availability, and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through selective mRNA translational reprogramming

mTOR coordinates growth signals with metabolic pathways and protein synthesis and is hyperactivated in many human cancers. mTOR exists in two complexes: mTORC1, which stimulates protein, lipid, and ribosome biosynthesis, and mTORC2, which regulates cytoskeleton functions. While mTOR is known to be involved in the DNA damage response, little is actually known regarding the functions of mTORC1 compared to mTORC2 in this regard or the respective impacts on transcriptional versus translational regulation. We show that mTORC1 and mTORC2 are both required to enact DNA damage repair and cell survival, resulting in increased cancer cell survival during DNA damage. Together mTORC1 and -2 enact coordinated transcription and translation of protective cell cycle and DNA replication, recombination, and repair genes. This coordinated transcriptional-translational response to DNA damage was not impaired by rapalog inhibition of mTORC1 or independent inhibition of mTORC1 or mTORC2 but was blocked by inhibition of mTORC1/2. Only mTORC1/2 inhibition reversed cancer cell resistance to DNA damage and replicative stress and increased tumor cell killing and tumor control by DNA damage therapies in animal models. When combined with DNA damage, inhibition of mTORC1/2 blocked transcriptional induction more strongly than translation of DNA replication, survival, and DNA damage response mRNAs.

This Expression of Concern relates to the article ‘Suppression of intestinal tumorigenesis in Apc mutant mice upon Musashi-1 deletion’ by Andy R. Wolfe, Amanda Ernlund, William McGuinness, Carl Lehmann, Kaitlyn Carl, Nicole Balmaceda and Kristi L. Neufeld. J. Cell Sci. 2017 130 , 805-813 ([doi

Therapeutic strategies based on a specific oncogenic target are better justified when elimination of that particular oncogene reduces tumorigenesis in a model organism. One such oncogene, Musashi-1 (Msi-1), regulates translation of target mRNAs and is implicated in promoting tumorigenesis in the colon and other tissues. Msi-1 targets include the tumor suppressor adenomatous polyposis coli (Apc), a Wnt pathway antagonist lost in ∼80% of all colorectal cancers. Cell culture experiments have established that Msi-1 is a Wnt target, thus positioning Msi-1 and Apc as mutual antagonists in a mutually repressive feedback loop. Here, we report that intestines from mice lacking Msi-1 display aberrant Apc and Msi-1 mutually repressive feedback, reduced Wnt and Notch signaling, decreased proliferation, and changes in stem cell populations, features predicted to suppress tumorigenesis. Indeed, mice with germline Apc mutations (ApcMin) or with the Apc1322T truncation mutation have a dramatic reduction in intestinal polyp number when Msi-1 is deleted. Taken together, these results provide genetic evidence that Msi-1 contributes to intestinal tumorigenesis driven by Apc loss, and validate the pursuit of Msi-1 inhibitors as chemo-prevention agents to reduce tumor burden.

The journal is retracting ‘Suppression of intestinal tumorigenesis in Apc mutant mice upon Musashi-1 deletion’ by Andy R. Wolfe, Amanda Ernlund, William McGuinness, Carl Lehmann, Kaitlyn Carl, Nicole Balmaceda and Kristi L. Neufeld (2017). [J. Cell Sci. 130, 805-813][1] (doi: 10.1242/

Regulatory T-cells (Tregs) maintain immune system homeostasis in the organism. In the tumor micro-environment, however, Tregs can hinder antitumor immune response. Treg maturation in peripheral sites (pTregs) requires inhibition of protein kinase mTORC1, which paradoxically blocks mRNA translation. mTORC1 phosphorylates (inactivates) 4E-BP1, a negative regulator of the translation initiation factor eIF4E. When mTORC1 is active, 4E-BP1 is hyperphosphorylated and releases eIF4E, which can then bind the mRNA cap structure and promote eIF4F complex assembly and ribosome recruitment. Inhibition of mTORC1 results in hypo-phosphorylation (activation) of 4E-BP1, which then binds and sequesters eIF4E, preventing its interaction with the cap and ribosome association to the mRNA. Using human CD4+ T-cells differentiated in vitro, we show that mTORC1 inhibition strongly impairs protein synthesis, yet with exposure to TGFbeta, is essential to mediate iTreg differentiation and immune suppression activity. We found that TGFbeta reprograms the T-cell transcriptome, while mTORC1 inhibition reprograms the translatome (genome-wide translation signature), mediating Treg differentiation. Genome-wide transcription and translation profiling identified TGFbeta-upregulated mRNAs resistant to mTORC1 inhibition, in part due to their 5’-untranslated regions, that induces Treg differentiation and immune suppression activity. These include canonical pTreg fate-determining mRNAs FOXP3, CTLA-4, CD101 and CD103, among others. Inhibition of mTORC1 alone or TGFbeta alone induces iTregs with poor immune suppression activity. Treg differentiation is therefore mediated by an mTORC1-independent privileged mRNA translation mechanism that converts activated CD4+ T-cells to Tregs. These findings are quite relevant in the tumor microenvironment, where mTORC1-inhibiting conditions such as hypoxia and deprivation of nutrients and energy are present. Investigation of molecular targets acting in this mTORC1/eIF4E-independent translation of Treg mRNAs is ongoing. These targets could be drugged to selectively inhibit Treg differentiation in the tumor microenvironment, thus allowing effector cells to mount the response against cancer cells. Citation Format: Viviana Volta, Amanda Ernlund, Columba De la Parra, Abilash Gadi, Amanda Valeta-Magara, Robert J. Schneider. Exploiting mTORC1-independent protein synthesis in Tregs to boost antitumor immune response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A222.

Breast cancer is the most commonly diagnosed malignancy among women in the U.S. and the second leading cause of cancer-related deaths in women. The estrogen receptor-positive (ER+) breast cancer subtype comprises the majority (70-80%) of breast cancer cases and the majority of late recurrences. Anti-estrogen therapy with tamoxifen remains a cornerstone of adjuvant therapy for ER+ breast cancers in the premenopausal setting, but resistance is inevitable for a third of patients, leading to metastatic disease, which is responsible for the majority of breast cancer deaths. Our work demonstrates that tamoxifen resistance in ER+ breast cancer involves selective mRNA translation mediated by hyperactivation of mTOR and MNK1, two kinases that play major roles in mRNA translation. The hyperactivation of mTOR and MNK1 converges on eukaryotic translation initiation factor 4E (eIF4E) to increase its availability and mRNA selectivity. Using tamoxifen-sensitive and -resistant ER+ cells, we conducted genome-wide transcriptomic and translatomic analyses +/- silencing of eIF4E. We identified key genes responsible for tamoxifen resistance, whose mRNAs are selectively translationally upregulated with increased levels of eIF4E. RUNX2 is a key mRNA that is selectively translationally upregulated, which we show provides resistance by opposing estrogenic signaling and possibly phenotypically reprogramming the cell. Silencing of RUNX2 strongly resensitizes the cells to tamoxifen. We also show that RUNX2 impairs cellular proliferation and cell survival of previously drug-resistant cells in the presence of tamoxifen. Silencing of key translationally upregulated mRNAs, such as RUNX2, can partially restore endocrine therapy sensitivity. Using a tamoxifen-resistant ER+ PDX model, and patient-derived sensitive and resistant tumor tissues (no recurrence or recurrence within 5 years), we validated the hyperactivation of mTOR and MNK1, the overexpression of eIF4E, and selective mRNA translation in tamoxifen resistance. We hypothesize that selection for increased expression of RUNX2 and other selectively translated mRNAs promotes a more “basal-like,” estrogen-independent phenotype. We aim to determine how tamoxifen resistance translationally reprograms breast cancer cells to a different breast cancer phenotype (e.g., cancer stem cell-like, basal-like, luminal with disrupted ER signaling). Citation Format: Sofia Bakogianni, Phillip Geter, Amanda Ernlund, Rezina Arju, Abhilash Gadi, Robert J. Schneider. Translational reprogramming of breast cancer and its role in tamoxifen resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5886.

This abstract was not presented at the symposium.

Metastasis is the cause of 90% of cancer-related deaths. Selective mRNA translation has been found to be crucial for breast cancer development, progression and metastasis; however, there is a poor biological and mechanistic understanding of the role of translation in these processes. Our work and others have pioneered an understanding of translational regulation in breast cancer. We have previously shown that overexpression of initiation factor eIF4GI in advanced breast cancer cells selectively increases the translation of mRNAs encoding survival, cell cycle inhibition, and DNA damage and repair proteins, among other DNA damage-protective mRNAs. eIF4G consists of three family members: eIF4GI, eIF4GII, and the poorly studied eIF4G homolog, DAP5, the subject of this study. DAP5 lacks the N-terminal domain for eIF4E and PABP binding. DAP5 is therefore suspected to promote eIF4E-independent or IRES-driven translation of mRNAs involved in cell stress and oncogenesis, but its role in metastasis hasn’t been investigated. We analyzed the NCI human tissue genome cancer atlas (TCGA) and found that higher DAP5 mRNA expression is strongly associated with estrogen receptor negative breast cancer metastasis with lower overall survival. We then engineered a highly metastatic breast cancer cell line MDA-MB-231TR to express dox-inducible shRNA to DAP5 or a non-silencing (NS) control. Tumors were developed in clinically relevant orthotopic mouse models, with and without DAP5 silencing by addition of dox to the drinking water. Remarkably, reduction of DAP5 expression by ~70% had no impact on primary tumor growth but fully eliminated metastasis to the lung. Genome-wide transcriptome and translatome analysis of DAP5 and in vivo ultraviolet-crosslinking and high-throughput sequencing (HITS-CLIP) identified specific mRNAs that interact directly with DAP5. A significant fraction of DAP5 mRNA targets are involved in cell death and survival, cell proliferation, cell mobility, DNA repair and translation initiation and do not have IRESs, suggesting a novel role for DAP5 in a non-classical translation initiation process upon cellular stress and metastasis. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis by a unique mechanism for metastasis-specific translation initiation and provides new concepts for therapeutic strategies involving translational regulation. Citation Format: Columba de la Parra, Amandine Alard, Amanda Ernlund, Robert J. Schneider. Translation initiation factor DAP5 plays an essential role in translational control of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2017-4481

Most colorectal cancers are thought to be initiated by mutation of the tumor suppressor Adenomatous polyposis coli gene (APC). APC mutations that result in loss of function lead to deregulation of Wnt signaling and inappropriate proliferation within intestinal crypts. Understanding APC regulation within normal intestinal cells is important for developing methods to restore function in a pathological state. Using cultured human colonocytes, we have previously shown that translation of APC can be blocked by the RNA binding protein, Musashi 1 (MSI1) and that MSI1 is a Wnt target. We propose that a double negative feedback loop between APC and MSI1 functions in maintenance of intestinal cell homeostasis. To explore the MSI1/APC interaction in vivo, we analyzed intestinal tissue from MSI1 knock-out mice (Msi1-/-). We found higher levels of APC and less Wnt signal activation in intestines from the Msi1-/- mice. There were also more enterocytes within the intestinal crypts of Msi1-/-mice, indicating an increase in differentiation. These three observations provide confirmation of the proposed APC/MSI1 double negative feedback loop in vivo. Evidence from cultured human cells and mouse models supports a role for an APC/MSI1 double negative feedback loop in the response of intestinal cells to Wnt signaling. However, the underlying mechanisms remain to be determined. Proteins that bind to a specific motif in the 3’UTR of target mRNA can induce translation inhibition or promotion as well as stabilization or destabilization of the mRNA. We already established that MSI1 blocks APC translation; however, the effect of MSI1 on APC mRNA stability was unknown. Here, we show that MSI1 binding to the 3’ UTR of APC mRNA stabilizes the message. In contrast, Numb, another established MSI1 target, does not appear to be stabilized by MSI1 binding. If MSI1 bound to the 3’UTR of APC mRNA stabilizes the message, but inhibits translation, then something likely leads to MSI1 release when APC translation resumes. One possibility is that MSI1 protein is inherently unstable. In this case, MSI1 binds to APC mRNA and blocks translation but is rapidly degraded and therefore releases APC mRNA for translation unless additional MSI1 is made. Because MSI1 is a Wnt target, a cell stimulated by Wnt ligand should continually replace the MSI1, therefore APC mRNA translation would remain blocked until the Wnt signal is removed. Our data showing that MSI1 has a relatively long half-life does not support this model. Current work is aimed to explore whether MSI1 release from APC mRNA is regulated by post-translational modifications or other protein-protein interactions. In conclusion, by acting as a Wnt antagonist, APC blocks MSI1 transcription. On the other hand, MSI1 binds to and stabilizes APC mRNA while also blocking APC translation. The MSI/APC double negative feedback loop appears important for maintaining homeostasis of intestinal epithelial cells. Citation Format: Andy R. Wolfe, Erick Spears, Amanda Ernlund, Kristi L. Neufeld. Musashi 1 stabilizes and blocks translation of Adenomatous Polyposis Coli mRNA in intestinal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2013-1110

The immunological composition of the primary cancer, metastatic sites, and stromal tissue determine cancer progression and treatment response. Breast cancers produce cytokines and chemokines that attract and polarize immune cells in a manner that promotes disease progression and metastasis. For example, macrophages are polarized to anti-inflammatory type II tumor activating macrophages (TAMs), and CD4+ T cells into tumor promoting, immune suppressing T regulatory cells (Tregs). Studies in mice and humans show that Tregs also are developed when inhibiting the kinase mTOR (mammalian target of rapamycin) via a poorly understood mechanism. mTOR forms two complexes in the cell, mTORC1 and mTORC2, which regulate multiple metabolic processes. In particular, mTORC1 is inhibited by the immunosuppressant rapamycin and stimulates the translation of mRNAs involved in cell growth and proliferation. Protein synthesis is a highly regulated process involving general mRNA and selective mRNA translation. Our hypothesis is that mTOR downregulation alters lymphocyte gene expression by favoring the translation of specific mRNAs required for Treg differentiation. We generated human Tregs induced in culture (iTregs) by mTORC1 inhibition through RAD001 (everolimus), a rapalog used as an anti-cancer drug. By contrast, mTORC1/2 dual inhibitor PP242 blocked the growth of all the lymphocytes, pointing to a specific role of mTORC1 in Treg differentiation. In fact, we found that the proliferation of highly suppressive iTregs requires co-treatment with RAD001 and the cytokine TGFβ. Murine models do not require TGFβ, suggesting that caution should be used in extending findings from mouse to human Treg studies. Protein synthesis analysis in double-treated (RAD001+TGFβ), control-treated, RAD001- and TGFβ-treated cells shows that translation is greatly inhibited in the double-treated cells. Genome-wide translation profiling of mRNAs associated with actively translating ribosomes confirmed that only a selective pool of specific mRNAs is translated in the iTreg population. These specific mRNAs may be recruited to ribosome via an mTOR-independent mechanism involving PAIP2, and eIF4G adapter protein, and the ribosomal protein S25. Our work indicates the importance of selective translational regulation as an additional determinant of gene expression regulating T cell fate. We suggest that iTreg development is facilitated by selective translation of specific mRNAs whose recruitment to the ribosome is augmented when mTORC1 is inhibited. These findings also suggest that cancer treatments causing an improper balance of mTORC1 inhibition might attenuate the anti-tumor immune response through development of Tregs in the tumor microenvironment