Cell Mol Life Sci Research Articles

IL-34 Exerts Anti-Inflammatory Activities on Keratinocytes and Is Downregulated in Psoriatic-Inflamed Skin

Interleukin-34 (IL-34) is expressed by keratinocytes in the skin and plays a role in Langerhans cell (LC) homeostasis through its binding to the colony-stimulating factor 1 receptor (CSF-1R) ( Greter et al., 2012 Greter M. Lelios I. Pelczar P. Hoeffel G. Price J. Leboeuf M. et al. Stroma-derived interleukin-34 controls the development and maintenance of langerhans cells and the maintenance of microglia. Immunity. 2012; 37: 1050-1060 Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar ; Wang et al., 2012 Wang Y. Szretter K.J. Vermi W. Gilfillan S. Rossini C. Cella M. et al. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. Nat Immunol. 2012; 13: 753-760 Crossref PubMed Scopus (652) Google Scholar ). In recent years, a growing body of literature has reported its overexpression during inflammation in tissues and/or in the serum of patients with rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases or Sjogren’s syndrome, in association with disease severity ( Munoz-Garcia et al., 2021 Munoz-Garcia J. Cochonneau D. Teletchea S. Moranton E. Lanoe D. Brion R. et al. The twin cytokines interleukin-34 and CSF-1: masterful conductors of macrophage homeostasis. Theranostics. 2021; 11: 1568-1593 Crossref PubMed Scopus (31) Google Scholar ). By contrast, some immunoregulatory properties of IL-34 have also been described in other physiopathological contexts, either by controlling the expression of proinflammatory cytokines or by promoting M2 macrophage differentiation and Treg cell functions ( Guillonneau et al., 2017 Guillonneau C. Bézie S. Anegon I. Immunoregulatory properties of the cytokine IL-34. Cell Mol Life Sci. 2017; 74: 2569-2586 Crossref PubMed Scopus (67) Google Scholar ). Although IL-34 is mainly produced in the skin, very few studies have investigated its expression, regulation and role during skin inflammation and it is still unclear whether IL-34 has a beneficial or a deleterious role. Li et al. reported low serum concentrations of IL-34 in psoriasis patients similar to those of healthy controls ( Li et al., 2017 Li J. Liu L. Rui W. Li X. Xuan D. Zheng S. et al. New Interleukins in Psoriasis and Psoriatic Arthritis Patients: The Possible Roles of Interleukin-33 to Interleukin-38 in Disease Activities and Bone Erosions. Dermatology. 2017; 233: 37-46 Crossref PubMed Scopus (67) Google Scholar ), without investigating IL-34 tissue expression. By contrast, Esaki et al. reported a reduced IL-34 expression in skin lesions of atopic dermatitis (AD) patients, suggesting that IL-34 could have immunoregulatory properties in the skin ( Esaki et al., 2015 Esaki H. Ewald D.A. Ungar B. Rozenblit M. Zheng X. Xu H. et al. Identification of novel immune and barrier genes in atopic dermatitis by means of laser capture microdissection. J Allergy Clin Immunol. 2015; 135: 153-163 Abstract Full Text Full Text PDF PubMed Google Scholar ).

PSAT118 Elevated Serum IgA at Onset of Type 1 Diabetes in Children

Abstract IgA is the most produced antibody in the body. There are two major forms of IgA, monomeric serum IgA and dimeric mucosal (secretory) IgA. During infection or inflammation, IgA binding to its antigen induces crosslinking of FcaRI, which activates immune effector cells to carry out their functions and can thus induce pro-inflammatory responses, which is hypothesized to occur when IgA titers are high. (1,2) Elevated serum IgA has been associated with autoimmune diseases (3) including type 1 diabetes (T1D) but its role in disease pathogenesis is not well known. To address this gap in knowledge, we aimed to test the hypothesis that at diagnosis of T1D, children and adolescents with elevated serum IgA have different demographic, clinical and laboratory characteristics than those with normal serum IgA. We analyzed 612 children 9.7 [4.2] (mean [SD]) years old (59% Non-Hispanic White, 20% Hispanic, 16% Black, 5% Other) with new onset T1D diagnosed between 1/2008-2/2012 at a large, academic pediatric hospital. Serum IgA values above age-adjusted normal ranges were defined as elevated serum IgA. Patients with low IgA were excluded because of known association between IgA deficiency and autoimmune disease. Demographic and clinical variables were compared between youth with and without elevated serum IgA. Univariable and multivariable regression models were used to compare baseline characteristics that were associated with elevated serum IgA. A significance level of 0.05 was used. We found that elevated serum IgA was present in 21% (128/612) of the children at T1D onset. By multivariate analysis, compared to children with normal IgA, those with elevated IgA were more likely to be of Hispanic ethnicity (Odds Ratio (OR)=3.27, 95% Confidence Interval (CI)=2.00, 5.35) and had a higher hemoglobin A1c (A1c) (OR=1.13, 95% CI=1.01, 1.26). They also had higher odds of having insulin autoantibody (IAA) positivity at diagnosis (OR=1.65, 95% CI=1.10, 2.45) and lower odds of GAD autoantibody (GADA) positivity (OR= 0.57, 95% CI=0.36, 0.93) (all, p<0.05), while there were no significant associations with age, glucose level or presence of DKA. In sum, at diagnosis of T1D, 21% of youth have elevated serum IgA and this was associated with Hispanic ethnicity, higher A1C and positivity for IAA. Our findings suggest that elevated serum IgA may help identify youth with T1D, islet autoimmunity and insulin deficiency. Further studies are warranted to investigate potential pathogenic correlates and persistence over time. This knowledge may lead to new targeted strategies to preserve or improve beta-cell function in individuals with clinical or pre-clinical T1D. References (1) Hansen IS et al. Cell Mol Life Sci. 2019 Mar;76(6): 1041-1055. PMID: 30498997 (2) Leong KW et al. DNA Cell Biol. 2014 Dec;33(12): 823-9. PMID: 25188736 (3) Kerr MA. Biochem J. 1990 Oct 15;271(2): 285-96. PMID: 2241915 Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Can we talk about myoblast fusion?

Can we talk about myoblast fusion?

Role of Acute Myeloid Leukemia Derived-Extracellular Vesicles in Dysregulating Normal Hematopoiesis

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of hematopoietic stem/progenitor cells (HSPCs). Leukemogenesis is characterized by rapid growth and impaired differentiation of leukemic cells, which progressively occupy and likely alter bone marrow (BM) niche where normal HSPCs reside [Kumar B., et al. Leukemia. 2018]. The homing and adhesion of AML cells to the BM niche is critical for its pathogenesis and involves many adhesion molecules, chemokine receptors, their ligands and signaling cascades. In the healthy context, the CXC chemokine receptor type 4 (CXCR4) expressed on HSPCs and its ligand, stromal derived factor-1 alpha (SDF-1α) produced by BM niche, are key mediators of HSPC-BM niche interaction inducing the homing, quiescence and survival of HSPCs, and the regulation of the HSC pool amount [Sánchez-Aguilera A., et al. Cell Mol Life Sci. 2017]. In AML context, where CXCR4/SDF-1α axis is deregulated, growth, BM niche-homing and adhesion of leukemic cells are altered also inducing AML relapse/resistance [Sánchez-Aguilera A., et al. Cell Mol Life Sci. 2017]. Despite recent improvements in therapeutic approaches, only a fraction of AML patients is cured. Therefore, it is crucial to identify new molecular mechanisms driving the AML pathogenesis. Extracellular vesicles (EVs) are lipidic bi-layer membrane particles released by normal and neoplastic cells. They shuttle a complex molecular cargo which reflects that of the origin cell [Caivano A., et al. Tumor Biol 2015; Caivano A., et al. Cell Oncol 2017] and which can be transferred to target cells altering their biological functions [De Luca L., et al. Oncotarget 2016; De Luca L., et al. Int J Mol Sci. 2017]. In particular, neoplasm derived EVs promote a tumor-supporting environment in non-malignant cells favoring cancer proliferation [Laurenzana I., et al. Stem Cells Int. 2018; De Luca L., et al. Expert Rev Mol Diagn. 2019]. We studied the effect of leukemia derived-EVs (LEVs) on healthy umbilical cord blood HSPCs to explore their potential role in dysregulating normal hematopoiesis. Specifically, the proliferation and differentiation, the quantitative and qualitative colony-forming features and the SDF-1α /CXCR4 mediated-migration of HSPCs induced by LEVs were investigated. EVs were isolated from AML cell lines by centrifugation steps and analyzed for AML surface markers by flow cytometry. Next, they were co-incubated with healthy CD34+ sorted cells for 24 hours. After treatment, cell count, apoptosis, HSPC-specific cluster of differentiation marker (CD10, CD34, CD38, CD45RA, CD90, CD123) expression [Karamitros D., et al. Nat Immunol. 2018], in vitro colony-forming unit cell assay, CXCR4 levels and SDF-1α mediated-migration assay were assessed. Our data showed: i) LEVs displaying specific AML surface markers; ii) a similar number of CD34+ cells in LEV treated-HSPCs, including HSCs, compared with controls; iii) no differences in apoptosis between the two groups; iv) an increase of multipotent progenitor (MPP, p=0.01) associated with a reduction of lymphoid-primed multipotent progenitors (LMPP, p=0.01) in treated cells; v) an increase of common myeloid progenitors (CMP, p=0.01), together with reduction of granulocyte macrophage progenitors (GMP, p=0.01) and megakaryocyte erythroid progenitors (MEP) in treated group; vi) a reduction of colony-forming ability, including erythroid, granulocytic and megakaryocytic colonies (p=0.03), in treated cells; vii) decreased levels of CXCR4 in LEV treated-HSPCs, that were accompanied by reduced HSPC migration mediated by SDF-1α. Altogether, this study suggests that AML derived-EVs can dysregulate the normal hematopoiesis promoting an increase of less differentiated precursors (MPP and CMP) which is associated with a reduction of more committed myeloid progenitors (GMP and MEP) (Figure 1). Furthermore, AML derived-EVs seem to induce less attraction of HSPCs in BM niche by interfering with SDF-1α/CXCR4 axis. Strategies to block EV production and secretion, as well as EV induced-reprogramming, could be novel exciting therapeutic approaches in AML. Disclosures Musto: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Progesterone Effects in the Nervous System.

The sex hormone progesterone is mainly known as a key factor in establishing and maintaining pregnancy. In addition, progesterone has been shown to induce morphological changes in the central and peripheral nervous system by increasing dendrito-, spino-, and synaptogenesis in Purkinje cells (Wessel et al.: Cell Mol Life Sci (2014a) 1723-1740) and increasing axonal outgrowth in dorsal root ganglia (Olbrich et al.: Endocrinology (2013) 3784-3795). These effects mediated mainly by the classical progesterone receptors (PRs) A and B seem to be limited to young neurons. It may be assumed that microRNAs (miRNAs), which are potent regulators of nervous system maturation and degeneration, are also involved in the regulation of progesterone-mediated neuronal plasticity by altering the expression patterns of the corresponding PR A/B receptors (Theis and Theiss: Neural Regen Res (2015) 547-549, Pieczora et al.: Cerebellum (2017) 376-387). This review critically discusses current data on the neuroprotective effect of progesterone and its corresponding receptors in the nervous system, with possible regulatory processes by miRNAs. Preclinical studies on stroke and traumatic brain injury revealed neuroprotective and neuroregenerative effects of progesterone in the treatment of severe neurological diseases in animal models, but have so far failed in humans. In this context, the identification of specific miRNAs that regulate the expression of progesterone and PR could help to exploit the neuroprotective potential of progesterone for the treatment of various neurological disorders. Anat Rec, 302:1276-1286, 2019. © 2019 Wiley Periodicals, Inc.

The Role of Nestin+ Mesenchymal Stem Cells (MSCs) in Bone Marrow Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). cGVHD is a syndrome of variable clinical features resembling autoimmune, involving not only the epithelial target tissues (skin, lung, gastrointestinal tract and liver) but also any other organ system (musculoskeletal, joint, ocular and genital tissues). Bone marrow (BM) suppression without infection is often observed in patients undergoing allo-HSCT as GVHD symptoms appear, suggesting that BM could be a target of GVHD (Yusuke S. et. al. Blood 2010). However, the BM cGVHD is rarely reported and the mechanism for BM cGVHD is still unclear. Nestin+ mesenchymal stem cells (MSCs) are capable of differentiating into cells of multiple cell lineages and involved in the formation of fibrotic lesions (Miaohua M. et. al. Cell Mol Life Sci 2016). To determine whether Nestin+ MSCs play an important role in the development of BM cGVHD. We used two murine models of cGVHD, including MHC-matched BALB/c (H-2d) recipients given B10D2 (H-2d) grafts, and MHC-mismatched BALB/c (H-2d) recipients given C57BL/6 (H-2b) grafts. We observed that BM displayed a significant increase in collagen and reticulin fibers deposition around blood vessels in cGVHD group compared to non-cGVHD group. The decreased absolute numbers of monocytes and neutrophils in the BM indicated the broad BM suppression in the cGVHD group. We further demonstrated that Nestin+MSCs located in perivascular BM niche underwent tremendous expansion in cGVHD group (p<0.001). Nestin+ MSCs in cGVHD group acquired expression of alpha smooth muscle actin (a-SMA), indicating myofibroblast differentiation. Moreover, transforming growth factor- beta1 (TGF-β1) concentrations also increased in cGVHD BM microenvironment (p<0.0001). High concentrations of TGF-β1 induced formation of Nestin+ MSC clusters and more transition from Nestin+ MSCs to a-SMA+ cells (p<0.05). Besides, expanded Nestin+ MSCs were surrounded by many macrophages and expressed high level of TGF-β1 receptor (p<0.001 and p<0.005). Taken together, these results indicate that Nestin+ MSCs participate BM cGVHD development through transition to a-SMA+ cells induced by high TGF-β1 in cGVHD microenvironment. DisclosuresZhang:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding. Yang:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding. Chen:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding.

Cellular Models for the Serpinopathies.

Our current knowledge about the cellular mechanisms underlying serpin-related disorders, the serpinopathies, is predominantly based on studies in cell culture models of disease, particularly for alpha-1 antitrypsin (AAT, SERPINA1) deficiency causing emphysema and the familial encephalopathy with neuroserpin (NS, SERPINI1) inclusion bodies (FENIB). FENIB, a neurodegenerative dementia, is caused by polymerization of NS (Miranda and Lomas, Cell Mol Life Sci 63:709-722, 2006; Roussel BD et al., Epileptic Disor 18:103-110, 2016), while AAT deficiency presents as a result of several divergent mutations in the AAT gene that cause lack of protein synthesis orcomplete intracellular degradation (null variants) or polymer formation (polymerogenic variants) (Lomas et al., J Hepatol 65:413-424, 2016; Greene et al., Nat Rev Dis Primers 2:16051, 2016; Ferrarotti et al. Orphanet J Rare D 9:172, 2014). Both diseases have been extensively modeled in cell culture systems by expressing mutant variants in a variety of ways. Here we describe the methodologies we follow in our cell model systems used to examine serpin disorders.

Abstract 916: Asymmetric cell division regulator prevents hyperproliferation in glioma cell-of-origin

Abstract In the past, we demonstrated that adult oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to self-renew and generate functional cells by unequally distributing active EGFR and the OPC-specific marker NG2. Moreover, premalignant OPC switch from an asymmetric to symmetric, self-renewing division mode at premalignant stages, and exhibit aberrant proliferation and impaired differentiation (Sugiarto S et al Cancer Cell 2011). OPC are the cell-of-origin of glioma in genetically engineered mouse models (Persson A et al Cancer Cell 2010) and OPC-like glioma cells have high tumor-initiating potential in some glioma types. These data contribute to the emerging evidence that asymmetric cell division (ACD) has tumor suppressive activity. Whether the loss of ACD directly promotes tumor initiation is unclear, because the detailed mechanisms of mammalian ACD have not been elucidated (Gomez-Lopez S et al. Cell Mol Life Sci 2014). Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and has been implicated in the asymmetric localization of cell fate determinants in mammalian neural progenitor cells (Klezovitch et al., Genes Dev, 2004). Others have shown also that human Lgl1 is inhibited by PTEN loss, which is common in glioblastoma, and Lgl1 depletion in human glioblastoma cells increases their invasive properties (Gont et al, Oncotarget, 2013 and 2014). Whether Lgl1 restricts proliferation, promotes differentiation and prevents neoplastic transformation of OPC, by regulating their asymmetric division, is unknown. We developed a flow cytometry method to isolate proliferative and differentiation-committed OPC (COP) and showed that Lgl1 expression is elevated in differentiation. Using a Cre-conditional knock-out allele (Klezovitch et al., Genes Dev, 2004), we show that Lgl1 knockout OPC proliferate more and differentiate less when compared with Lgl1 wildtype OPC. Lgl1 depletion increases symmetric, self-renewing divisions and reduces the frequency of ACD. Lgl1 appears to control the expression of several conserved ACD regulators, as revealed by comparative Taqman expression analyses of Lgl1 knockout versus control OPC. Ongoing studies are aimed to investigate the link of Lgl1 depletion, altered ACD regulator expression and observed OPC phenotypes. Data derived from this work are expected to provide further insights into the extent to which defective ACD contributes to neoplastic transformation in all cancers. Citation Format: Mathieu Daynac, Claudia K. Petritsch. Asymmetric cell division regulator prevents hyperproliferation in glioma cell-of-origin [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 916. doi:10.1158/1538-7445.AM2017-916

Signaling via the NFκB system.

The nuclear factor kappa B (NFκB) family of transcription factors is a key regulator of immune development, immune responses, inflammation, and cancer. The NFκB signaling system (defined by the interactions between NFκB dimers, IκB regulators, and IKK complexes) is responsive to a number of stimuli, and upon ligand-receptor engagement, distinct cellular outcomes, appropriate to the specific signal received, are set into motion. After almost three decades of study, many signaling mechanisms are well understood, rendering them amenable to mathematical modeling, which can reveal deeper insights about the regulatory design principles. While other reviews have focused on upstream, receptor proximal signaling (Hayden MS, Ghosh S. Signaling to NF-κB. Genes Dev 2004, 18:2195-2224; Verstrepen L, Bekaert T, Chau TL, Tavernier J, Chariot A, Beyaert R. TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme. Cell Mol Life Sci 2008, 65:2964-2978), and advances through computational modeling (Basak S, Behar M, Hoffmann A. Lessons from mathematically modeling the NF-κB pathway. Immunol Rev 2012, 246:221-238; Williams R, Timmis J, Qwarnstrom E. Computational models of the NF-KB signalling pathway. Computation 2014, 2:131), in this review we aim to summarize the current understanding of the NFκB signaling system itself, the molecular mechanisms, and systems properties that are key to its diverse biological functions, and we discuss remaining questions in the field. WIREs Syst Biol Med 2016, 8:227-241. doi: 10.1002/wsbm.1331 For further resources related to this article, please visit the WIREs website.

Editorial Comment

Editorial Comment

CBIO-29TARGETING A Plk1-CONTROLLED POLARITY CHECKPOINT IN GLIOBLASTOMA MULTIFORME

Stem-like glioma cells, frequently referred to as tumor-propagating cells or cancer stem cells are culprits for recurrence due to their intrinsic resistance to standard therapy and their ability to regrow the parental tumor in xenografts. Similar to normal neural stem cells CD133+ tumor-propagating cells undergo asymmetric, self-sustaining cell divisions (Gomez-Lopez S. et al, Cell Mol Life Sci, 2014, 71(4):575-97). Cancer-associated changes in cell division mode contribute to neoplastic transformation of glioma precursors (Sugiarto S. et al, Cancer Cell, 2011 20(3):328-40). While increased symmetric, self-renewing divisions of glioma precursors correlate with therapy sensitivity, asymmetric self-sustaining divisions potentially maintain the pool of tumor-propagating cells and contribute to therapy evasion. How pharmacological MAPK pathway inhibitors clinically evaluated for the treatment of GBM affect the division mode of tumor-propagating cells is unknown. We investigate CD133+ tumor-propagating cell responses to novel targeted therapies, in specifically, small molecule inhibitors of BRAFV600E, a mutant kinase frequently found in pediatric malignant astrocytoma. Our investigations showed that CD133+ tumor-propagating cells have higher asymmetric divisions than progenitor-like glioma cells. CD133+ stem-like GBM subpopulation exhibit decreased sensitivity to the anti-proliferative effects of MAPK pathway inhibition and show extended G2/M phase. We find that the mitotic checkpoint kinase and polarity regulator Plk1 is more active in CD133+ tumor-propagating cells. Plk1 activity links asymmetric division and mitotic entry. In an orthotopic GBM xenograft model, combined MAPK-pathway and PLK1 inhibition showed increased anti-proliferative effects and cell death frequency towards CD133+ cells beyond that achieved by either inhibitor alone. Ongoing work is investigating if Plk1 controls a polarity checkpoint, the integrity of which is especially important in the therapy–evasive compartment in GBM and that provides a rationale for combination therapy.

Abstract B07: Targeting an MT1-MMP/MMP2 axis in melanoma by a novel MT1-MMP/MMP2 inhibitor

Abstract Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. Matrix metalloproteases (MMPs) are among the proteolytic enzymes responsible for the degradation/ digestion of the extracellular matrix (ECM) and have been associated with the aggressiveness of a variety of cancers including melanoma (1,2). MT1-MMP is a membrane associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion-promoting, pro-tumorigenic MMP in cancer. We have recently demonstrated that MT1-MMP plays a key role in melanoma metastasis (3). MT1-MMP expression increases with disease progression and is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1-MMP in melanoma pathogenesis. Indeed, we showed that MT1-MMP is required for melanoma cells to metastasize, as cells deprived of MT1-MMP fail to form distant metastasis in an orthotopic mouse melanoma model. Mechanistically, we demonstrated for the first time that MT1-MMP affects cell invasion and motility by activating an MMP2-RAC1 signaling axis in melanoma cells (3). These findings imply MT1-MMP/MMP2 represent good molecular targets to thwart melanoma metastasis. However, the targeting of MMPs has not met with very successful stories mostly because the use of broad-spectrum inhibitors have been accompanied by severe side effects, such as musculoskeletal pain and inflammation (4,5) due to the targeting of both the “good” and the “bad” MMPs (6-8). Here we present new data in support of a novel, specific MT1-MMP/MMP2 inhibitor. ND-322 is a slow binding selective inhibitor of MT1-MMP and MMP2 (9). Data in our laboratory indicate ND-322 inhibits cell growth, migration and invasion of several melanoma cell lines similarly to the knock down of MT1-MMP and/or MMP2; it decreases RAC1 activity; and importantly, significantly reduces in vivo tumor growth and metastasis in an orthotopic mouse melanoma model. Preliminary data in vitro also show ND-322 in combination with vemurafenib decreases melanoma cell viability in a synergistic manner. Given these results and considering that this compound is very well tolerated in vivo (10), we believe ND-322 represent a promising novel, safe addition to the current standard of care for melanoma patients with advanced disease. Literature Cited 1 Bartolome, R.A., Ferreiro, S., Miquilena-Colina, M.E., Martinez-Prats, L., Soto- Montenegro, M.L., Garcia-Bernal, D., Vaquero, J.J., Agami, R., Delgado, R., Desco, M., Sanchez-Mateos, P., &amp; Teixido, J. Am J Pathol 174 (2), 602-612 (2009). 2 Moro, N., Mauch, C., &amp; Zigrino, P. Eur J Cell Biol 93 (1-2), 23-29 (2014). 3 Shaverdashvili k., W.P., Ma J., Zhang K., Osman I., Bedogni B. Pigment Cell and melanoma Res In Press (2013). 4 Skiles, J.W., Gonnella, N.C., &amp; Jeng, A.Y. Curr Med Chem 11 (22), 2911-2977 (2004). 5 Drummond, A.H., Beckett, P., Brown, P.D., Bone, E.A., Davidson, A.H., Galloway, W.A., Gearing, A.J., Huxley, P., Laber, D., McCourt, M., Whittaker, M., Wood, L.M., &amp; Wright, A. Ann N Y Acad Sci 878, 228-235 (1999). 6 Zucker, S. &amp; Cao, J. Cancer Biol Ther 8 (24), 2371-2373 (2009). 7 Hua, H., Li, M., Luo, T., Yin, Y., &amp; Jiang, Y. Cell Mol Life Sci 68 (23), 3853-3868 (2011). 8 Dufour, A. &amp; Overall, C.M. Trends Pharmacol Sci 34 (4), 233-242 (2013). 9 Gooyit, M., Lee, M., Schroeder, V.A., Ikejiri, M., Suckow, M.A., Mobashery, S., &amp; Chang, M. J Med Chem 54 (19), 6676-6690 (2011). 10 Cui, J., Chen, S., Zhang, C., Meng, F., Wu, W., Hu, R., Hadass, O., Lehmidi, T., Blair, G.J., Lee, M., Chang, M., Mobashery, S., Sun, G.Y., &amp; Gu, Z. Mol Neurodegener 7, 21 (2012). Citation Format: Khvaramze Shaverdashvili, Poki Wong, Jun Ma, Keman Zhang, Iman Osman, Barbara Bedogni. Targeting an MT1-MMP/MMP2 axis in melanoma by a novel MT1-MMP/MMP2 inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B07.

Alpha1B‐ and alpha2‐Adrenoceptors Mediate Contractions of Mouse and Rat Spleen; Pharmacological and Gene Knock‐out Data

Contractions of rat spleen involve α1- and α2-adrenoceptors, and α1- actions are probably mediated by α1B-adrenoceptors (see Docherty, 2010). In the absence of a truly selective α1B-adrenoceptor antagonist, it is difficult to positively identify the α1-adrenoceptors involved. In this study, we have investigated norepinephrine (NE)-evoked contractions of rat and mouse spleen employing pharmacology and knock-out technology, α1A/D-KO mice in which the only α1-adrenoceptor remaining is the α1B-adrenoceptor. Animals were killed by overdose of CO2 and cervical dislocation. In rat spleen, NE produced contractions with a potency (pEC50) of 5.43±0.06 (n=8, -log M), but NE was more potent in spleen from WT (7.04±0.08, n=6) and KO (7.14±0.05, n=4) mice. Prazosin (0.01 μM) or the α2-adrenoceptor antagonist yohimbine (1 μM) produced only small shifts in NE potency, but the combination of praz and yoh produced 100 fold shift in NE potency in both rat and mouse. In rat spleen, the α1-adrenoceptor agonist phenylephrine (PHE) produced contractions with a potency of 5.70±0.19 (n=6), and the putative α1B-adrenoceptor antagonist cyclazosin (0.1 μM) significantly shifted PHE potency, but the selective α1D-adrenoceptor antagonist BMY7378 (0.3 μM) was ineffective, and the α1A-adrenoceptor antagonist RS100329 exhibited low potency. In α1A/D-KO mice, the effects of praz and yoh were similar to their effects in WT mice. In spleen from both WT and KO mice, prazosin shifted the contractile response to higher concentrations of NE more markedly than the response to low concentrations. In conclusion, the major, if not exclusive, α1-adrenoceptor involved in contractions of mouse, and probably rat, spleen is the α1B-adrenoceptor. Dochery, J.R. (2010). Cell Mol Life Sci 67, 405-417.

Dysregulation of the Let-7/HMGA2 Axis with Methylation of p16 Promoter As a Possible Target of Histone Deacetylase Inhibitor in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal hematological disorders characterized by proliferation of mature blood cells. Recently, several agents that influence epigenetic modifications, such as histone deacetylase inhibitors (HDACi), as well as JAK2 inhibitors, have been investigated for high-risk MPNs. For example, an HDACi, panobinostat has shown significant efficacy including nearly complete response in PMF (Mascarenhas et al, BJH, 2013), but molecular targets of HDACi remain largely unknown. The High Mobility Group AT-hook 2 (HMGA2) is a non-histone chromatin protein that modulates transcriptions of various genes and contributes to chromatin modification and epigenetic regulation including DNA methylation (Fusco et al, Nat Rev Cancer, 2007; Sun et al, PNAS 2013). Let-7 micro RNAs (miRNAs) negatively regulate expression of HMGA2 through 3’UTR of HMGA2 mRNA, although HMGA2 mRNA consists of both the major variant containing 3’UTR with let-7-specific sites (variant 1) and some minor variants without 3’UTR. We previously reported that overexpression of HMGA2 due to transgenic expression of Hmga2 cDNA without 3’UTR caused proliferative hematopoiesis with providing a clonal advantage to hematopoietic stem cells in mice (Ikeda et al, Blood, 2011). We also showed a deregulation of HMGA2 mRNA expression due to reduced let-7 miRNA level in granulocytes from patients with almost all of PMF and over 20% of PV and ET (Harada-Shirado et al, Blood [Abst], 2013), being associated with splenomegaly, elevated serum LDH values, and methylation of p16 promoter. Thus, we hypothesized that HMGA2 may be a candidate gene as a therapeutic target in MPNs. Since association of HDAC with HMGA2 has been reported in cord blood-derived cells (Lee et al, Cell Mol Life Sci, 2011), we here studied effects of the panobinostat on expressions of HMGA2 and let-7 in HMGA2-expressing myeloid cells including PMF-derived CD34+ cells. First, we found significantly higher HMGA2 mRNA levels in CD34+ cells from 2 PMF patients compared with CD34+ cells from 2 healthy individuals (P<0.001), as well as U937 cells compared with HL60 cells (P<0.001). Thus, we used CD34+ cells from one of these 2 PMF patients and U937 cells for further experiments. Interestingly, treatment with panobinostat at the concentration of 40 nM for 8 hours significantly increased expressions of let-7a (P<0.001 and P=0.003, respectively), -7b (P<0.001 and P<0.001), and -7c (P<0.001 and P=0.06) in U937 cells and PMF CD34+ cells, compared with samples without the treatment. In contrast, Western blotting showed clearly reduced expression of HMGA2 protein in U937 cells after the treatment with panobinostat. Moreover, we found that variant 1 of HMGA2 mRNA with 3’UTR was significantly reduced by the treatment with panobinostat, compared with samples without the treatment in both U937 cells and PMF CD34+ cells (P<0.001 and P<0.001, respectively), while expression levels of variant 2 lacking let-7-specific sites were not changed by the treatment. These findings strongly suggested that panobinostat decreased expression of HMGA2 through 3’UTR of HMGA2 mRNA by increasing expressions of let-7 miRNAs. Of note, we next found much higher expression of variant 1 of HMGA2 mRNA than variant 2 in granulocytes from 15 of 17 (88%) MPN patients whose HMGA2 mRNA levels were higher than controls in our previous study (Blood [Abst], 2013). We also assessed if treatment by panobinostat for the dysregulated let-7/HMGA2 axis may be a therapeutic option for MPNs with respect to DNA methylation. Panobinostat treatment substantially reduced the expressions of DNMT1 and DNMT3a as well as HMGA2 proteins, significantly demethylated the p16 promoter (P<0.001), and decreased survival (P<0.001) in U937 cells. Moreover, knocking-down of HMGA2 with small interfering RNA in U937 cells significantly increased expression of TET3 mRNA and demethylated the p16 promoter, suggesting that HMGA2 expression may contribute to methylation of the p16 promoter. In conclusion, deregulated expression of HMGA2 due to downregulation of let-7 miRNAs, which may lead to some epigenetic modifications such as methylation of the p16 promoter, is a possible therapeutic target of HDACi in MPNs. DisclosuresIkeda:Novartis Pharmaceuticals: Other.

P53 Suppression Mediates Blasts Survival, Proliferation and CD34 Maintenance in Pediatric Acute Myeloid Leukemia

Aberrations in the DNA damage responses contributes to the uncontrolled proliferation and therapy resistance in many cancers. Acute myeloid leukemia (AML) presents enhanced DNA damage as compared to MDS characterized by induced levels γ-2HAX and phosphorylated ATM that correlated with AML blast percentages (Boehrer et al. Oncogene 2009). The low incidence of p53 mutations (7% in adult AML, The Cancer Genome Atlas Research Network, N Engl J Med 2013) in AML indicates downstream suppression of DNA damage control system proteins, as was proposed previously by overexpression of MDM2 and BCL-2 (Wojcik et al. Neoplasma 2005). However, the role of p53 and downstream effectors regulating the DNA damage pathway in CD34+ AML blasts is still relatively unclear.Surprisingly, using RPPA analysis, we found a common significant increase in p53 phosphorylation at serine 15 in pediatric AML as compared to CD34+ NBM (AML n=31 and NBM n=10, Mann-Whitney U, P = 0.003). Therefore, we challenged to target p53 using a shRNA approach. In the sh-p53 model, primary pediatric AML samples transduced with sh-p53 showed a significant increase in AML proliferation over time as compared to scrambled control (paired-samples t-test, P = 0.005). The overall AML cell viability was significantly improved in sh-p53 AML cultures (paired-samples t-test P = 0.001). DNA cell cycle analysis revealed that sh-p53 AML cultures contained significant increased percentages of cells in S and G2/M phases of the cell cycle as compared to their scrambled controls (paired samples t-test, P = 0.015). The CD34+ leukemic population was maintained in p53 knockdown cultures and thereby outcompeted scrambled control cultures. Knockdown of p53 in primary pediatric AML samples resulted in a higher proliferation rate, repressed differentiation and prolonged survival of AML blasts, especially in MLL-rearranged AML. Interestingly, we previously showed that MLL-rearranged AML samples express significantly higher BCL-2 peptide phosphorylation as compared to NBM (Kampen et al. Leukemia 2014). Growth factors are extrinsic cues that can regulate BCL-2 expression (Pidgeon et al. Br J Cancer 2001). Growth factors like VEGF, have been shown to be involved in leukemia pathogenesis in the bone marrow where leukemic stem cells are preserved in tightly controlled niches (Kampen et al. Cell Mol Life Sci 2013). Accordingly, we found that FGF and VEGFC stimulation of CD34+ AML blasts both significantly accelerated BCL-2 mRNA expression by 28-fold and 2-fold, indicating that indeed the niche is important for inhibiting pro-apoptotic p53-dependent signals further downstream the pathway, which protects AML CD34+ blasts (Student’s t-test, both P < 0.001), where p53 and p21 mRNA expression remained unchanged. The availability of VEGFR-2 and FGFR1 previously highlighted their therapeutic targeting potential in AML (Kampen et al. Leukemia 2006, Kentsis et al, Nature Med 2012). This study presents the effect of p53 suppression on AML blast proliferation, survival and maintenance in pediatric AML and indicates the importance of exploring extrinsic factors that contribute to DNA damage control system suppression in vivo for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Metabolic Engineering of Saccharomyces cerevisiae for Isoprenoid Production

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of either therapeutic or industrial interest. However, heterologous protein productivity is often low due to limitations of the selected host strain (1). In the case of the α-amylase, overproduction by S. cerevisiae may lead to an intracellular accumulation of reactive oxygen species (ROS) as a consequence of the formation of disulfide bonds during protein folding in the endoplasmic reticulum, and therefore causing a significant decrease in the cell performance which ultimately affects the protein titer and productivity (2). In order to reduce the internal ROS levels, we describe a strategy based on the overexpression of the Hap1 transcription factor, known to be responsible for both the activation of a set of genes involved in ROS detoxification and the oxidative stress response, as well as genes related to the respiratory metabolism (3). This strategy resulted to have a positive effect in the protein production by increasing the final titer compared to the control strains, while keeping the ROS levels constant during batch cultivations. 1. Hou J, Osterlund T, Liu Z, Petranovic D, Nielsen J. 2013. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 97:3559-3568. 2. Liu Z, Osterlund T, Hou J, Petranovic D, Nielsen J. 2013. Anaerobic alpha- amylase production and secretion with fumarate as the final electron acceptor in Saccharomyces cerevisiae. Appl Environ Microbiol 79:2962- 2967. 3. Zhang L, Hach A. 1999. Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator. Cell Mol Life Sci 56:415-426.

Ultrastructure of skin from Refsum disease with emphasis on epidermal lamellar bodies and stratum corneum barrier lipid organization.

Classic Refsum disease (RD) is a rare, autosomal recessively-inherited disorder of peroxisome metabolism due to a defect in the initial step in the alpha oxidation of phytanic acid (PA), a C16 saturated fatty acid with four methyl side groups, which accumulates in plasma and lipid enriched tissues (please see van den Brink and Wanders, Cell Mol Life Sci 63:1752-1765, 2006). It has been proposed that the disease complex in RD is in part due to the high affinity of phytanic acid for retinoid X receptors and peroxisome proliferator-activated receptors. Structurally, epidermal hyperplasia, increased numbers of cornified cell layers, presence of cells with lipid droplets in stratum basale and reduction of granular layer to a single layer have been reported by Blanchet-Bardon et al. (The ichthyoses, SP Medical & Scientific Books, New York, pp 65-69, 1978). However, lamellar body (LB) density and secretion were reportedly normal. We recently examined biopsies from four unrelated patients, using both OsO4 and RuO4 post-fixation to evaluate the barrier lipid structural organization. Although lamellar body density appeared normal, individual organelles often had distorted shape, or had non-lamellar domains interspersed with lamellar structures. Some of the organelles seemed to lack lamellar contents altogether, showing instead uniformly electron-dense contents. In addition, we also observed mitochondrial abnormalities in the nucleated epidermis. Stratum granulosum-stratum corneum junctions also showed co-existence of non-lamellar and lamellar domains, indicative of lipid phase separation. Also, partial detachment or complete absence of corneocyte lipid envelopes (CLE) was seen in the stratum corneum of all RD patients. In conclusion, abnormal LB contents, resulting in defective lamellar bilayers, as well as reduced CLEs, likely lead to impaired barrier function in RD.

AB0939 Canakinumab in Clinical Practice. Effectiveness in A Cryopyrin-Associated Periodic Syndrome Single Center Cohort

BackgroundCryopyrin-associated periodic syndrome (CAPS) comprises a group of rare, but severe, autoinflammatory diseases, characterized by urticaria, periodic fever, central nervous system inflammation, arthropathy, and increased risk of amyloidosis. In a...

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407-4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434-1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.

Vinculin‐p130Cas interaction is critical for focal adhesion dynamics and mechano‐transduction

Adherent cells, when mechanically stressed, show a wide range of responses including large-scale changes in their mechanical behaviour and gene expression pattern. This is in part facilitated by activating the focal adhesion (FA) protein p130Cas through force-induced conformational changes that lead to the phosphorylation by src family kinases. Janostiak et al. [Janostiak et al. Cell Mol Life Sci (2013) DOI 10.1007/s00018-013-1450-x] have reported that the phosphorylation site Y12 on the SH3 domain of p130Cas modulates the binding with vinculin, a prominent mechano-coupling protein in FAs. Tension changes in FAs (due to the anchorage of the SH3 domain and C-terminal) bring about an extension of the substrate domain of p130Cas by unmasking the phosphorylation sites. These observations demonstrate that vinculin is an important modulator of the p130Cas-mediated mechano-transduction pathway in cells. The central aim should be now to test that vinculin is critical for p130Cas incorporation into the focal adhesion complex and for transmitting forces to the p130Cas molecule.