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5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug’s efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug’s DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

Summary Inflammatory bowel diseases (IBDs) are genetically complex and exhibit significant inter-patient heterogeneity in disease presentation and therapeutic response. Here, we show that mouse models of IBD exhibit variable responses to inhibition of MK2, a pro-inflammatory serine/threonine kinase, and that MK2 inhibition suppresses inflammation by targeting inflammatory monocytes and neutrophils in murine models. Using a computational approach (TransComp-R) that allows for cross-species comparison of transcriptomic features, we identified an IBD patient subgroup that is predicted to respond to MK2 inhibition, and an independent preclinical model of chronic intestinal inflammation predicted to be non-responsive, which we validated experimentally. Thus, cross-species mouse-human translation approaches can help to identify patient subpopulations in which to deploy new therapies., Graphical abstract, Highlights • MK2 kinase inhibition shows variable efficacy in different IBD mouse models • TCT and TNFΔARE mice express distinct inflammatory and MK2-responsive genes • “Response to MK2i” signature is enriched in monocytes and neutrophils • Cross-species modeling identifies patient groups potentially responsive to MK2i, Immunology; Computational bioinformatics; Systems biology; Transcriptomics

Made available in DSpace on 2020-12-10T17:27:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-01 AES Tiete/ANEEL Triploidization is a technique to produce sterile fish on a large scale. In the spotted catfish Pimelodus maculatus, sterile fish serve as a host of germ cell transplantation for subsequent reconstitution of endangered catfish species. This study aimed to establish an efficient protocol for triploidization in P. maculatus using temperature shock. In this study, we tested the efficacy of heat shock treatments (37 degrees C, 38 degrees C, and 39 degrees C) applied at 2 min post-fertilization (mpf) for the duration of 2 min. Intact embryos served as diploid control. Ploidy status was confirmed by flow cytometry, characterization of erythrocytes nuclei, and karyotyping. Developmental stages were verified at cleavage, blastula, gastrula, somite, and hatching stages, as well as the percentages of normal and abnormal larvae. No significant difference was observed between the temperature treatments for percentage of unfertilized oocytes (P = 0.1834), 2-cell stage (P = 0.2348), blastula (P = 0.2972), hatching (P = 0.0668), and abnormal larvae (P = 0.6177). Heat shock decreased the survival at the blastula stage (P = 0.0178), somite (P = 0.0469), and the percentage of abnormal larvae (P = 0.0261). All treatments presented high percentages of triploid individuals, and the highest values were observed for heat shock at 38 degrees C (96.7 %). Based on the results described above, an efficient protocol for triploidization in P. maculatus was established using heat shock (38 degrees C) at 2 mpf for 2 min. Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Sao Paulo State Univ, Inst Biosci, Rua Prof Doutor Antonio Celso Wagner Zanin S-No, BR-18618689 Botucatu, SP, Brazil Sao Paulo State Univ, Inst Biosci, Rua Prof Doutor Antonio Celso Wagner Zanin S-No, BR-18618689 Botucatu, SP, Brazil AES Tiete/ANEEL: 4690000174

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti–PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti–PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti–PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti–PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti–PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. Significance: These findings show that pan-inhibition of TAM receptors in combination with anti–PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

Chronic inflammation increases the risk for colorectal cancer through a variety of mechanisms involving the tumor microenvironment. MAPK-activated protein kinase 2 (MK2), a major effector of the p38 MAPK stress and DNA damage response signaling pathway, and a critical regulator of pro-inflammatory cytokine production, has been identified as a key contributor to colon tumorigenesis under conditions of chronic inflammation. We have previously described how genetic inactivation of MK2 in an inflammatory model of colon cancer results in delayed tumor progression, decreased tumor angiogenesis, and impaired macrophage differentiation into a pro-tumorigenic M2-like state. The molecular mechanism responsible for the impaired angiogenesis and tumor progression, however, has remained contentious and poorly defined. Here, using RNA expression analysis, assays of angiogenesis factors, genetic models,in vivomacrophage depletion and reconstitution of macrophage MK2 function using adoptive cell transfer, we demonstrate that MK2 activity in macrophages is necessary and sufficient for tumor angiogenesis during inflammation-induced cancer progression. We identify a critical and previously unappreciated role for MK2-dependent regulation of the well-known pro-angiogenesis factor CXCL-12/SDF-1 secreted by tumor associated-macrophages, in addition to MK2-dependent regulation of Serpin-E1/PAI-1 by several cell types within the tumor microenvironment.

Inflammatory bowel disease (IBD) is a chronic and debilitating disorder that has few treatment options due to a lack of comprehensive understanding of its molecular pathogenesis. We used multiplexed mass spectrometry to collect high-content information on protein phosphorylation in two different mouse models of IBD. Because the biological function of the vast majority of phosphorylation sites remains unknown, we developed Substrate-based Kinase Activity Inference (SKAI), a methodology to infer kinase activity from phosphoproteomic data. This approach draws upon prior knowledge of kinase-substrate interactions to construct custom lists of kinases and their respective substrate sites, termed kinase-substrate sets that employ prior knowledge across organisms. This expansion as much as triples the amount of prior knowledge available. We then used these sets within the Gene Set Enrichment Analysis framework to infer kinase activity based on increased or decreased phosphorylation of its substrates in a dataset. When applied to the phosphoproteomic datasets from the two mouse models, SKAI predicted largely non-overlapping kinase activation profiles. These results suggest that chronic inflammation may arise through activation of largely divergent signaling networks. However, the one kinase inferred to be activated in both mouse models was mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 or MK2), a serine/threonine kinase that functions downstream of p38 stress-activated mitogen-activated protein kinase. Treatment of mice with active colitis with ATI450, an orally bioavailable small molecule inhibitor of the MK2 pathway, reduced inflammatory signaling in the colon and alleviated the clinical and histological features of inflammation. These studies establish MK2 as a therapeutic target in IBD and identify ATI450 as a potential therapy for the disease.

Chronic inflammation is a major risk factor for colorectal cancer. The p38/MAPKAP Kinase 2 (MK2) kinase axis controls the synthesis of proinflammatory cytokines that mediate both chronic inflammation and tumor progression. Blockade of this pathway has been previously reported to suppress inflammation and to prevent colorectal tumorigenesis in a mouse model of inflammation-driven colorectal cancer, by mechanisms that are still unclear. Here, using whole-animal and tissue-specific MK2 KO mice, we show that MK2 activity in the myeloid compartment promotes tumor progression by supporting tumor neoangiogenesis in vivo. Mechanistically, we demonstrate that MK2 promotes polarization of tumor-associated macrophages into protumorigenic, proangiogenic M2-like macrophages. We further confirmed our results in human cell lines, where MK2 chemical inhibition in macrophages impairs M2 polarization and M2 macrophage-induced angiogenesis. Together, this study provides a molecular and cellular mechanism for the protumorigenic function of MK2.

Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract that has limited treatment options. To gain insight into the pathogenesis of chronic colonic inflammation (colitis), we performed a multiomics analysis that integrated RNA microarray, total protein mass spectrometry (MS), and phosphoprotein MS measurements from a mouse model of the disease. Because we collected all three types of data from individual samples, we tracked information flow from RNA to protein to phosphoprotein and identified signaling molecules that were coordinately or discordantly regulated and pathways that had complex regulation in vivo. For example, the genes encoding acute-phase proteins were expressed in the liver, but the proteins were detected by MS in the colon during inflammation. We also ascertained the types of data that best described particular facets of chronic inflammation. Using gene set enrichment analysis and trans-omics coexpression network analysis, we found that each data set provided a distinct viewpoint on the molecular pathogenesis of colitis. Combining human transcriptomic data with the mouse multiomics data implicated increased p21-activated kinase (Pak) signaling as a driver of colitis. Chemical inhibition of Pak1 and Pak2 with FRAX597 suppressed active colitis in mice. These studies provide translational insights into the mechanisms contributing to colitis and identify Pak as a potential therapeutic target in IBD., Crohn's and Colitis Foundation of America (Research Fellowship), National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant 1122374), Institute for Collaborative Biotechnologies (W911NF-09-0001)

DNA damaging chemotherapy is a cornerstone of current front‐line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo‐resistant disease; therefore, adjuvant treatments that synergize with DNA‐damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide‐nucleic acid complex that facilitates tumor‐specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex‐mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high‐grade serous OC to cytotoxic chemotherapy by blocking p38/MK2‐dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC., National Institutes of Health (U.S.) (Grant R01-ES015339), National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant 1F32EB017614), National Science Foundation (U.S.) (Grant GFRP 1122374), National Cancer Institute (U.S.) (Grant P30-CA14051), National Science Foundation (U.S.) (Grant DMR-0819762)

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of retinoic acid receptor alpha (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack or RARα resulted in increased KLF4+ goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased reg3g, reduced luminal bacterial detection and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

MAPKAP Kinase-2 (MK2) is a serine/threonine protein kinase that is activated by p38MAPK in response to a variety of stresses including DNA damage, osmotic shock, thermal injury, and inflammation/innate immune signaling downstream of TLR4 activation. MK2 controls the synthesis of many pro-inflammatory cytokines, modulates the actin cytoskeleton in stressed cells, and is responsible for maintaining both the G1/S and G2/M cell cycle checkpoints in p53-defective tumor cells after genotoxic damage. We recently created a “Cre-reversible" MK2 knock-out mouse model that allows the function of MK2 within either the tumor cells or within the tumor microenvironment to be studied independently. We found that MK2-deficient tumor cells in an MK2 wild-type stroma were dramatically sensitized to the anti-tumor effects of cisplatinum and doxorubicin (Morandell et al., Cell Reports, 2013). Here, we report that loss of MK2 in the tumor stroma with preservation of MK2 in tumor cells results in an aggressive tumor-promoting phenotype in an immuno-competent transplant K-RasG12D/p53-/- model of NSCLC. A similar tumor aggressive phenotype was observed in Tie2-Cre-driven endothelial and bone marrow-specific MK2-knockout mice compared to wild-type controls. Tumor-bearing mice with MK2-null stroma display increased total levels of MIP-1α, G-CSF, GM-CSF and IL-6 in the lung. Myeloperoxidase immunohistochemistry reveals enhanced tumor infiltration by neutrophils in these stromal MK2-deficient animals, consistent with the cytokine profile. In contrast with loss of MK2 in the stroma, direct activation of the p38MAPK/MK2 pathway in the tumor microenvironment by weekly intra-tracheal LPS exposure dramatically reduces tumor formation in an autochthonous K-RasG12D/p53-/- murine NSCLC model. Collectively, these results suggest that MK2 signaling in the endothelium and/or immune cells suppresses NSCLC tumor progression and modulates the inflammatory milieu in the tumor microenvironment. Citation Format: Ganapathy Sriram, Lucia Suarez-Lopez, Michael B. Yaffe. Loss of MK2 in tumor stroma promotes aggressive tumor development in an advanced stage lung cancer model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B32.

Activation of the small GTPase RHOA has strong oncogenic effects in many tumour types, although its role in colorectal cancer remains unclear. Here we show that RHOA inactivation contributes to colorectal cancer progression/metastasis, largely through the activation of Wnt/β-catenin signalling. RhoA inactivation in the murine intestine accelerates the tumorigenic process and in human colon cancer cells leads to the redistribution of β-catenin from the membrane to the nucleus and enhanced Wnt/β-catenin signalling, resulting in increased proliferation, invasion and de-differentiation. In mice, RHOA inactivation contributes to colon cancer metastasis and reduced RHOA levels were observed at metastatic sites compared with primary human colon tumours. Therefore, we have identified a new mechanism of activation of Wnt/β-catenin signalling and characterized the role of RHOA as a novel tumour suppressor in colorectal cancer. These results constitute a shift from the current paradigm and demonstrate that RHO GTPases can suppress tumour progression and metastasis.

Human SMC2 is part of the condensin complex, which is responsible for tightly packaging replicated genomic DNA prior to segregation into daughter cells. Engagement of the WNT signaling pathway is known to have a mitogenic effect on cells, but relatively little is known about WNT interaction with mitotic structural organizer proteins. In this work, we described the novel transcriptional regulation of SMC2 protein by direct binding of the β-catenin·TCF4 transcription factor to the SMC2 promoter. Furthermore, we identified the precise region in the SMC2 promoter that is required for β-catenin-mediated promoter activation. Finally, we explored the functional significance of down-regulating SMC2 protein in vivo. Treatment of WNT-activated intestinal tumor cells with SMC2 siRNA significantly reduced cell proliferation in nude mice, compared with untreated controls (p = 0.02). Therefore, we propose that WNT signaling can directly activate SMC2 transcription as a key player in the mitotic cell division machinery. Furthermore, SMC2 represents a new target for oncological therapeutic intervention.

Activation of the small GTPase RHOA has strong oncogenic effects in many tumor types, although its role in colorectal cancer remains unclear. We show that RHOA inactivation contributes to colorectal cancer progression/metastasis, largely through the activation of Wnt/β-catenin signaling. RhoA inactivation in the murine intestine accelerates the tumorigenic process and in human colon cancer cells leads to the redistribution of β-catenin from the membrane to the nucleus and enhanced Wnt/β-catenin signaling, resulting in increased proliferation, invasion and de-differentiation. In mice, RHOA inactivation contributes to colon cancer metastasis and reduced RHOA levels were observed at metastatic sites compared to primary human colon tumors. Therefore, we have identified a new mechanism of activation of Wnt/β-catenin signaling and characterized the role of RHOA as a novel gene with tumor suppressor activity in colorectal cancer. These results constitute a shift from the current paradigm and demonstrate that RHO GTPases can suppress tumor progression and metastasis. Citation Format: Paulo Rodrigues, Irati Macaya, Sarah Bazzocco, Elena Andretta, Rocco Mazzolini, Higinio Dopeso, Silvia Mateo-Lozano, Josipa Bilic, Fernando Cartón-García, Rocio Nieto, Lucia Suárez-López, Elsa Afonso, Stefania Landolfi, Javier Hernandez-Losa, Kazuto Kobayashi, Santiago Ramón y Cajal, Josep Tabernero, Niall C. Tebbutt, John M. Mariadason, Simo Schwartz Jr, Diego Arango. RHOA inactivation enhances Wnt signaling and promotes colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2015-2058